Metrics details Meibomian glands secrete lipid-rich meibum Aging-related Meibomian gland shrinkage may result in part from stem cell exhaustion and is associated with evaporative dry eye disease a common condition lacking effective treatment The identities and niche of Meibomian gland stem cells and the signals controlling their activity are poorly defined we identify markers for stem cell populations that maintain distinct regions of the gland and uncover Hedgehog (Hh) signaling as a key regulator of stem cell proliferation we show that human Meibomian gland carcinoma exhibits increased Hh signaling Aged glands display decreased Hh and EGF signaling and loss of collagen I in niche fibroblasts indicating that alterations in both glandular epithelial cells and their surrounding microenvironment contribute to age-related degeneration These findings suggest new approaches to treat aging-associated Meibomian gland loss but evidence that these LRCs directly contribute to MG homeostasis is lacking Lineage tracing with a constitutive Krox20/Egr2-Cre mouse line shows that EGR2+ cells give rise to the MG during development lineage tracing experiments with an inducible Egr2-Cre line to determine whether EGR2+ cells self-renew and give rise to differentiating progeny in adult MGs have not been performed specific markers for adult MG stem cells have yet to be identified it is unknown whether Hh signaling regulates adult MG homeostasis and MG stem cell activity in vivo We used lineage tracing and ex vivo live imaging assays to test the contributions of cells expressing putative stem cell markers to MG homeostasis These data identified multiple stem cell populations that can self-renew and produce differentiated progeny to maintain the MG duct and acinus We found that loss of the Hh receptor Smoothened (SMO) in MG epithelial cells caused decreased proliferation and MG dropout while forced expression of an activated form of the Hh pathway effector GLI2 (GLI2ΔN) promoted expansion of MG stem cells at the expense of differentiated acini GLI2 and other stem cell markers were expressed in human MG and showed elevated levels in MG carcinoma (MGC) suggesting that MGC involves expansion of MG stem cells Aged versus young mouse MGs displayed fewer acinar basal cells and surrounding dermal cells expressing downstream Hh pathway genes aged MG exhibited lower levels of EGF signaling identifying additional mechanisms that could contribute to MG degeneration in aging our data uncover stem cell populations and molecular mechanisms that sustain the adult MG and are altered in aging and in human disease Specific markers for MG stem cells are poorly defined As MGs share many characteristics with HF-associated SGs we interrogated the snRNA-seq data for known SG stem cell markers and validated the results using RNAscope assays on independent biological samples of adult MG The expression of SG stem cell markers in the acinar basal layer and ductule suggested that these markers may also label stem cells within the MG suggesting that Gli2-expressing ductular cells can contribute to the central duct Slc1a3-CreERT2-marked cells were present in the acinar basal layer (Fig. 2f, white arrowhead) and in meibocytes (Fig. 2f Slc1a3-CreERT2 marks acinar stem cells but not stem cells that contribute to ductules or central duct lineage tracing with Gli2-CreERT2 revealed the presence of descendants of Gli2-expressing ductular cells in the central duct and this is likely the major mechanism for their replenishment a–f Progressive expansion of acinar basal cell clusters (black arrows) in GLI2ΔN-expressing MG after 2 (a f) days of doxycycline treatment at 8 weeks of age g–l Progressive expansion of GLI2ΔN+/KRT5+ cells (yellow arrows) and reduction of PLIN2+ meibocytes (white arrows) in Gli2ΔNKrt5rtTA MG acini after 2 (g N = 3 control mice lacking Krt5-rtTA or tetO-GLI2ΔN (2 males and 1 female) and n = 3 Gli2ΔNKrt5-rtTA mice (2 males and 1 female) were analyzed at each time point in (a–l); representative samples are shown m–p Hyperproliferation of GLI2ΔN+ cells in Gli2ΔNKrt5-rtTA MG acini (m r Quantification of acinar basal cell (q) and ductal basal cell (r) proliferation Samples from n = 5 control mice (3 males and 2 females) lacking Krt5-rtTA or tetO-GLI2ΔN and samples from n = 5 Gli2ΔNKrt5-rtTA mice (3 males and 2 females) were analyzed in (m–r) r) was calculated with unpaired two-tailed Student’s t-test At least 100 acinar cells and 110 ductal cells were analyzed from each animal t Overgrowth of Ptch1-deficient acinar basal cells (yellow arrows) in KRT14-CreERT2 Ptch1fl/fl mice 12 weeks after tamoxifen treatment at 8 weeks of age v Expansion of KRT5+ cells in Ptch1-deficient acini (red arrows) Independent biological samples from n = 3 male KRT14-CreERT2 Ptch1fl/fl mice n = 2 age-matched male controls and n = 2 age-matched female controls were analyzed in (s–v) or through deletion of endogenous epithelial Ptch1 a Scheme for bulk RNA-seq of laser-captured MG samples b GO enrichment analysis of bulk RNA-seq data from GLI2ΔNKrt5rtTA and littermate control MGs 4 days after induction at 8 weeks of age showing the top 10 enriched pathways c Volcano plot showing genes upregulated or downregulated at 4 days Independent biological samples from n = 3 GLI2ΔNKrt5rtTA mice (2 male and 1 female) and independent biological samples from n = 3 littermate control mice lacking Krt5-rtTA or tetO-GLI2ΔN (2 males and 1 female) were analyzed; differentially expressed genes were defined as padj<0.001 and Log2FC < -0.5 or Log2FC > 0.5 FDR calculation was performed by DESeq2 v1.20.0 with the Benjamini-Hochberg procedure d–g RNAscope showing upregulation of Gli1 and Ccnd1 in GLI2ΔNKrt5rtTA MGs i IF data showing decreased PPARγ and PLIN2 expression in GLI2ΔNKrt5rtTA acini (j-m) RNAscope showing expanded Lrig1 and Lgr6 expression in GLI2ΔNKrt5rtTA MGs Independent samples from 3 Gli2ΔNKrt5-rtTA mice (2 males and 1 female) and 3 littermate controls of genotypes tetO-GLI2ΔN or Krt5-rtTA (2 males and 1 female) were used for RNAscope and IF; all mice were doxycycline-treated for 4 days (n-s) GLI2ΔNKrt5-rtTA Rosa26mTmG mice carrying inducible Cre alleles driven by Lrig1 (n s) promoters were tamoxifen-induced at P42 to induce Cre activity and placed on oral doxycycline at P72 to induce GLI2ΔN expression mGFP expression (red signal) and GLI2 expression (green signal) were analyzed by IF at P74 (n GLI2ΔN-expressing cells in the acinar basal layer positive for Lrig1 (n) or Axin2 (r) (yellow arrows) give rise to clones that contribute to MG overgrowth (o n = 3 (1 male and 2 females) samples were analyzed per line per time point These observations indicate that GLI2ΔN-expressing MG stem cells contribute to MG basal expansion b In normal human MG GLI2 protein localizes to acinar basal cells (a yellow arrows) and differentiating meibocytes (a white arrows) but is absent from fully differentiated meibocytes (a light blue arrows); in MGC samples GLI2 is broadly expressed (b) GLI1 mRNA is weakly expressed in some acinar basal cells (c yellow arrows); GLI1+ cells are widely present in MGCs (d) e–h LRIG1+ and LGR6+ cells are primarily present in the acinar basal layer of normal human MGs (e yellow arrows); MGC displays expansion of LRIG1+ and LGR6+ cells (f j Ki-67+ cells localize to the normal human acinar basal layer (i l PLIN2-/KRT14+ cells are restricted to the acinar basal layer in normal human MG (k white arrows) but are distributed more broadly in MGC tissue (l 7 normal human MG and 10 human MGC samples were analyzed; representative data are shown These data indicate that human MGC is characterized by the expansion of poorly differentiated and proliferative tumor cells that express MG stem cell markers this mechanism could potentially account for the observed decrease in the percentages of cells expressing Hh-regulated genes rather than altered expression levels of these proteins may account for increased association of GLI2 with acetylated lysine in aged MGs Taken together these observations suggest that the decreased percentages of cells exhibiting Hh activity in aged MG and the surrounding stroma may result in part from increased levels of GLI2 acetylation that mitigate its transcriptional activity in subsets of acinar and stromal cells b CellChat analysis predicts decreased EGF signaling in aged MGs c CellChat analysis predicts HBEGF signaling between dermal cells and MG ductular cells (green) at 8 weeks and its absence at 21 months d Violin plots of snRNA-seq data show decreased percentages of Hbegf-expressing cells in the indicated cell populations in aged MG e RNAscope shows reduced Hbegf expression levels per cell and fewer acinar basal cells (yellow arrows) and surrounding dermal cells (white arrows) expressing Hbegf in aged MG f IHC shows that p-ERK1/2 levels are decreased but total ERK1/2 levels are similar in the acini of aged compared with young MG White dashed lines in (e) and black dashed lines in (f) outline MG acini Samples from n = 3 8-week and n = 3 21-month-old male C57BL/6 J mice were used for RNAscope and IHC Taken together, our analyses of aged versus young MGs revealed that MG aging is associated with multiple potential mechanisms, including decreased Hh and EGF signaling, altered function of peripheral neurons, a disrupted dermal microenvironment, and inflammation (Fig. 10). Hh signaling is reduced in aging due to inhibited GLI2 transcriptional activity resulting in lower levels of stem cell proliferation peripheral innervation and collagen fibril density are reduced in aged compared with young MGs Little is known about the molecular identities the signaling pathways that control their activity in homeostasis and aging Significant findings of the current study include the identification of markers for specific populations of stem cells that maintain distinct regions of the MG; evidence that ductules harbor stem cells for both duct and acinar structures; a key role for Hh signaling in MG stem cell proliferation; decreased Hh and EGF signaling in aged compared with young MGs suggesting possible therapeutic approaches to enhance MG proliferation in aging; and critical alterations in the MG stromal niche during aging including in collagen expression and nerve density Prior data showed that distinct basal cell populations replenish the central MG ducts and the acini but specific markers for these stem cell sub-populations had not been identified We found that basal stem cells marked by Slc1a3-CreERT2 exclusively contribute to the acinus; by contrast lineage tracing analyses revealed that both duct and acinus were replenished by stem cells marked by Lrig1-CreERT2 Further studies will be needed to identify specific markers for stem cell populations that exclusively renew MG ductules and ducts experimental evidence for this concept has been lacking Our snRNA-seq analysis revealed that ductules express genes that are characteristic of both the MG duct (Krt17 and Egr2) and the acinus (Pparg and Slc1a3) Velocity analysis of the snRNA-seq data predicted that ductular cells contribute to both the central duct and the acinus we found that ductular cells express Slc1a3 as well as Lrig1 While Slc1a3-CreERT2 did not label ductules likely due to low levels of Slc1a3 promoter activity in ductule cells lineage tracing combined with live imaging of MG explants showed that LRIG1+ cells in the ductule can migrate towards the acinus Gli2 expression is absent in the central duct but ductular cells lineage-traced for Gli2 populate the central duct these results indicate that the ductule harbors stem cells for both duct and acinus Further investigation is required to delineate the mechanisms underlying MGC and the contributions of Hh signaling to this disease the decline in Gli2 mRNA expression may reflect the decrease in Hh pathway activity that we observed in aged MGs it is possible that a specific Gli2+ stem cell population is reduced in aging Further work will be needed to distinguish these mechanisms these data support a model in which decreased Hh signaling in aging results in part from altered HDAC1/2 activity that impacts GLI2 Further research will be needed to determine the precise role of HBEGF-EGFR signaling and the mechanisms controlling its activity Limitations of the current study include that snRNA-seq and aging-related experiments were carried out only in male mice While most validation experiments were performed on both male and female samples lack of inclusion of female samples in the snRNA-seq dataset and in aging-related experiments could mean that sex-specific differences in MG biology and aging were missed Millar; all other experiments with mice were carried out under Icahn School of Medicine at Mount Sinai IACUC protocol #2019-0028 Human eyelid tissues were provided by the Department of Ophthalmology at the University of Pennsylvania as de-identified samples that had been discarded from unrelated surgical procedures Use of the human tissue samples in research was approved by the University of Pennsylvania IRB under protocol #827926 Human eyelid samples were paraffin-embedded and sectioned for histological analysis Eight 8-week-old and eight 21-month-old male mice were euthanized The dorsal portion of the eyelids and the surrounding tissues near the tarsal plates were removed using a scalpel The remaining tarsal plates containing MGs were digested with 0.2% collagenase A (Sigma-Aldrich #10103578001) in PBS for 1 hour at 37 °C The tarsal plates were then rinsed twice with PBS and tissues surrounding the MGs were removed with fine-tip forceps Tissues from four littermate mice were pooled together in PBS for each replicate The MG tissue-containing solutions were then transferred into 1.5 mL centrifuge tubes using Pasteur pipettes The tissue solution was centrifuged at 300 g/min for 5 minutes and the tissue pellet was rinsed once with PBS Isolation of nuclei was performed using the protocol (CG000375 Rev B) from 10x Genomics RNA-seq libraries were prepared based on the 10x Genomics protocol (CG000315 Rev E) using 10000 nuclei per replicate Single-cell separation was performed using the Chromium Next GEM Chip G Single Cell Kit (10x Genomics #PN-1000120) The RNA-seq libraries were sequenced in paired-end mode on an Illumina Novaseq platform with the Novaseq S4 flowcell Cell Ranger Single-Cell Software Suite (v7.0.1 10x Genomics) was used to align and quantify sequencing data against the mm10 mouse reference genome (refdata-gex-mm10-2020-A) Cell clustering and differential gene expression were analyzed using Seurat70 (v4.3.0) high-quality nuclei (defined as nFeature_RNA > 200 & nFeature_RNA < 4500 & nCount_RNA > 500 & <nCount_RNA < 25000 & precent.mt <5) were analyzed 6676 nuclei were analyzed from 8-Week replicate 1 The CellCycleScoring function of Seurat was applied to mitigate the effects of cell cycle heterogeneity in the datasets The SCTransform function of Seurat was used to integrate the four datasets 34 clusters were identified with a resolution of 1 the y-axis was plotted against the log normalized data from the data slot of the Seurat object Seurat employs a global-scaling normalization method “LogNormalize” that normalizes the feature expression (each gene) measurements for each cell by the total expression multiplies this by a scale factor (10,000 by default) FDR calculation was performed by the EdgeR or clusterProfiler using the Benjamini-Hochberg procedure the normalized counts were loaded into CellChat and preprocessed with default parameters To establish the ligand-receptor interaction database we selected secreted signaling pathways and utilized the “projectData” function to project gene expression data onto the mouse protein-protein interaction (PPI) we employed the setting “type=truncatedMean trim = 0.1” to compute the communication probability (computeCommunProb) except for Hh signaling (trim =0.001 was used) Core CellChat functions such as “computeCommunProbPathway” and “identifyCommunicationPatterns” were applied with standard parameters for downstream analysis Individual basal cell datasets for Meibomian and sebaceous glands were further merged and processed for Violin plotting in Seurat (v4.3.0) The 10x Visium Spatial Gene Expression kit (10x Genomics # PN-1000184) was employed to construct the spatial transcriptomic library according to the manufacturer’s instructions A pair of upper and lower eyelids from an 8-week-old male mouse was dissected and mounted onto the assay slide containing four capture areas (6.5 mm×6.5 mm) with spatially barcoded poly T capture probes the captured mRNAs underwent reverse transcription and sample indexing were performed in accordance with the manufacturer’s protocol The resulting cDNA libraries were quantified using TapeStation (Agilent) and Qubit (Invitrogen) and then subjected to sequencing in paired-end mode on a NovaSeq instrument (Illumina) at a depth of 50,000-100,000 reads per capture spot The sequencing data were aligned and quantified using Space Ranger Software Suite (v2.1.0 10x Genomics) against the reference genome Paraffin sections were deparaffinized and rehydrated to PBS Antigen retrieval was performed with antigen unmasking solution (Vector Laboratories # H-3300) Sections were washed with PBST (PBS + 0.1% Tween 20) and blocked with 1% BSA for 30 minutes and the sections were incubated overnight at 4 °C sections were subjected to three washes of PBST followed by 1-hour incubation with secondary antibodies The following antibodies were used: Rat monoclonal anti-Ki-67 (eBioscience #13-5689-82; 1:200); Rabbit anti-COL17A1 (Abcam #ab184996 RRID AB_3073438); Rabbit anti-KRT17 (Abcam # ab109725 RRID AB_10889888); Mouse anti-KRT10 (Novus #NBP2-47650 1:200); Rabbit anti-LOR (BioLegend #905103 RRID AB_2734676); Rabbit anti-PPARγ (Cell Signaling Technology #2435 T RRID AB_2166051); Rabbit anti-FASN (Cell Signaling Technology #3180 T RRID AB_2100796); Guinea Pig anti-PLIN2 (Fitzgerald #20R-AP002 AB_1282475); Rabbit anti-PLIN2 (Sigma-Aldrich #393A-1 RRID AB_2313768); Rabbit anti-KRT5 (BioLegend #905504 RRID AB_2616956); Rabbit anti-KRT14 (Thermo Fisher Scientific #MA5-11599 RRID AB_10982092); Rabbit anti-ERK1/2 (Cell Signaling Technology #4695 T RRID AB_390779); Rabbit anti-p-ERK1/2 (Cell Signaling Technology #4370 T RRID AB_2315112); Mouse anti-Myctag (Cell Signaling Technology #2276S RRID AB_331783); Rabbit anti-GLI2 (Novus Biologicals #NB600-874SS RRID AB_10001953); Goat anti-GLI2 (R&D Systems #AF-3635 RRID AB_2111902); Rabbit anti-Acetylated-Lysine (Cell Signaling Technology #9441 s RRID AB_331805); Rabbit anti-HDAC1 (Thermo Fisher Scientific #49-1025 RRID AB_2533875); Rabbit anti-HDAC2 (Thermo Fisher Scientific #51-5100 RRID AB_2533908); Donkey anti-Rabbit IgG Secondary Antibody RRID AB_162543); Donkey anti-Mouse IgG Secondary Antibody Biotinylated (Vector Laboratories #BA-1000 RRID AB_2313606); Streptavidin Fluorescein (Vector Laboratories #SA-5001 RRID AB_2336462); Streptavidin Texas Red (Vector Laboratories #SA-5006 The sections were washed with PBST and mounted with DAPI-containing medium (Invitrogen # P36930) sections were washed with PBST and then incubated with 3% H2O2/PBST for 15 minutes followed by two washes of PBST and 30-minute incubation with biotinylated secondary antibodies Subsequent steps and DAB staining followed the manufacturer’s instructions IF and IHC data were documented using a Leica Microsystems DM5500B microscope equipped with two cameras for fluorescent and bright field images (Leica Microsystems) RNAscope was performed on paraffin sections using the RNAscope Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics #323110) following the user’s guide provided by the manufacturer Probes were all obtained from Advanced Cell Diagnostics The probes used were as follows: Mm-Slc1a3 #430781; Mm-Scd4 #486071; Mm-Lrig1 #310521-C2; Mm-Lgr6# 404961; Mm-Axin2 #400331-C2; Mm-Gli1 #311001-C2; Mm-Ccnd1 #442671; Mm-Smo #318411-C2; Mm-Gli2 #405771; Mm-Shh #314361-C2; Mm-Ihh #1259141-C1; Mm-Ptch1 #402811; Mm-pan-Hh #415051; Mm-Slit3 #542771; Mm-Hbegf #437601; Mm-Col1a1 #319371; Hs-GLI1 #310991-C2; Hs-LRIG1 #407421; Hs-LGR6 #410461 200 μl Tamoxifen (Sigma-Aldrich #T5648) dissolved in corn oil (10 mg/ml) was injected intraperitoneally once for lineage tracing daily intraperitoneal injections were performed on five consecutive days and the eyelids were dissected with scissors Tissues surrounding the tarsal plates were removed using a surgical scalpel The tarsal plates were placed in a 35-mm Lumox-bottom dish (Sarstedt #94.6077.331) and incubated in DMEM/F12(1:1) medium (Gibco #21041-025) supplemented with 5% FBS for 2 hours in an environmental chamber supplied with 5% CO2 at 37 °C before imaging A Leica SP8 laser scanning confocal microscope equipped with Power HyD detectors and an environmental chamber was used to image the cultured explants Images were captured as z stacks at 3 µm intervals and tissues were imaged every 15 min with low laser power (<3%) with a HC PL APO CS2 10x/0.40 DRY objective lens Stacked images were maximum projected with Leica LAS X 3.6.0.20104 software to generate the time-lapse video and video annotations were performed with Fiji software 2.1.0 Doxycycline at 200 µg/ml (Sigma-Aldrich # D9891) together with 5% sucrose was fed to the mice continuously in the drinking water after initiation of its administration The solution was freshly prepared every 5 days for induction periods of over 5 days Genes with padj<0.001 and |Log2FC | >0.5 were selected for GO enrichment analysis which was carried out by clusterProfiler (4.4.4) The results were plotted by ggplot2 (v3.4.0) and volcano plots were generated using EnhancedVolcano (v1.14.0) 5 μm paraffin sections were dewaxed and rehydrated followed by blocking with 5% donkey serum/PBS/0.8% Triton X-100 for 30 minutes and incubation with solutions containing primary antibodies overnight at 4 °C The sections were washed with PBST and incubated in PLA probe anti-rabbit plus or anti-goat plus and PLA probe anti-rabbit minus for 2 hours The subsequent ligation and amplification steps followed the manufacturer’s instructions The sections were washed twice in washing buffer and once in PBS for 20 minutes each before mounting and imaging using a Leica SP8 laser scanning confocal microscope Sample size for experiments involving quantitation was pre-determined using the statsmodels (0.9.0) package in Python (3.7) Statistical analysis and graphical representation were performed using Microsoft Excel 2023 Unpaired or paired two-tailed Student’s t-test was used to calculate statistical significance between two groups of data Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Meibomian gland dysfunction: what have animal models taught us Meibomian gland dysfunction: hyperkeratinization or atrophy Anatomy and histopathology of human meibomian gland The international workshop on meibomian gland dysfunction: report of the subcommittee on anatomy and pathophysiology of the meibomian gland Recent advances in age-related meibomian gland dysfunction (ARMGD) Effects of age and dysfunction on human meibomian glands Meibocyte differentiation and renewal: Insights into novel mechanisms of meibomian gland dysfunction (MGD) Age-related changes in the meibomian gland Renewal of the holocrine meibomian glands by label-retaining Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin Dynamics of Lgr6(+) progenitor cells in the hair follicle Distinct mechanisms for sebaceous gland self-renewal and regeneration provide durability in response to injury A unique lineage gives rise to the meibomian gland Signaling pathways governing stem-cell fate Targeting cancer stem cell pathways for cancer therapy Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells Hedgehog signaling regulates sebaceous gland development Indian hedgehog and beta-catenin signaling: role in the sebaceous lineage of normal and neoplastic mammalian epidermis Expression of Shh and Wnt signaling pathway proteins in eyelid sebaceous gland carcinoma: clinicopathologic study Hedgehog signaling pathway regulates the proliferation and differentiation of rat meibomian gland epithelial cells The effects of age and dysfunction on meibomian gland population dynamics Biomarkers for progenitor and differentiated epithelial cells in the human meibomian gland Neuroendocrinology and neurobiology of sebaceous glands Characterization of the innervation of the meibomian glands in humans Parasympathetic innervation of the meibomian glands in rats Meibomian gland stem/progenitor cells: The hunt for gland renewal Glutamate transporter Slc1a3 mediates inter-niche stem cell activation during skin growth WNT10A mutation causes ectodermal dysplasia by impairing progenitor cell proliferation and KLF4-mediated differentiation Turnover and migration of meibomian gland cells in rats’ eyelids Development and homeostasis of the sebaceous gland Stem and progenitor cells in sebaceous gland development Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation Basal cell carcinomas in mice arise from hair follicle stem cells and multiple epithelial progenitor populations HDAC1/2 control proliferation and survival in adult epidermis and pre‒basal cell carcinoma through p16 and p53 Wakayama Symposium: Peroxisome proliferator-activated receptor-gamma (PPARgamma) and meibomian gland dysfunction Inference and analysis of cell-cell communication using CellChat Ptch1 and Gli regulate Shh signalling dynamics via multiple mechanisms Gli2 acetylation at lysine 757 regulates hedgehog-dependent transcriptional output by preventing its promoter occupancy HDAC1/2 and HDAC3 play distinct roles in controlling adult Meibomian gland homeostasis Epidermal growth factor receptor cell proliferation signaling pathways Effect of growth factors on the proliferation and gene expression of human meibomian gland epithelial cells Clonal growth and differentiation of rabbit meibomian gland epithelium in serum-free culture: differential modulation by EGF and FGF Role of EGF receptor signaling on morphogenesis of eyelid and meibomian glands Transcriptome analysis of aging mouse meibomian glands Neurotransmitter influence on human meibomian gland epithelial cells Age-dependent alterations in mouse exorbital lacrimal gland structure Mechanisms of extraorbital lacrimal gland aging in mice: an integrative analysis of the temporal transcriptome Looking older: fibroblast collapse and therapeutic implications and antioxidants: chronic diseases and aging Clinicopathological study of meibomian carcinoma of eyelids - an experience of two years in a tertiary care center Sonic hedgehog stimulates mouse embryonic stem cell proliferation by cooperation of Ca2+/protein kinase C and epidermal growth factor receptor as well as Gli1 activation Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation Sonic hedgehog induces epidermal growth factor dependent matrix infiltration in HaCaT keratinocytes Changes in mouse sudomotor function and sweat gland innervation with ageing ID1/ID3 mediate the contribution of skin fibroblasts to local nerve regeneration through Itga6 in wound repair Human lung fibroblasts secrete nerve growth factor: effect of inflammatory cytokines and glucocorticoids Novel-smoothened inhibitors for therapeutic targeting of naïve and drug-resistant hedgehog pathway-driven cancers Expansion of hedgehog disrupts mesenchymal identity and induces emphysema phenotype BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties Histone deacetylases 1 and 2 redundantly regulate cardiac morphogenesis Comprehensive Integration of Single-Cell Data A relay velocity model infers cell-dependent RNA velocity edgeR: a Bioconductor package for differential expression analysis of digital gene expression data clusterProfiler 4.0: A universal enrichment tool for interpreting omics data Cytoscape: a software environment for integrated models of biomolecular interaction networks Salmon provides fast and bias-aware quantification of transcript expression Tximeta: reference sequence checksums for provenance identification in RNA-seq Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Download references We thank the Department of Ophthalmology at the University of Pennsylvania PA for de-identified discarded human eyelid tissue; Dr Robert Sebra and the Genomics Core Facility USA for snRNA sequencing and spatial transcriptomics; Dr Steven Prouty for advice on laser capture; Drs Elaine Fuchs and Michael Rendl for KRT14:H2BGFP mice; Dr This work was supported by NIH grants R01AR081322 (SEM); R37AR047709 (SEM); R01EY035337 (CI); R01EY036135 (CI); R01AR065409 (SYW); and a pilot grant from the Mount Sinai Skin Biology and Diseases Resource-based Center (SBDRC) P30AR079200 (XZ) Core facilities used in this study were provided by the Mount Sinai SBDRC P30AR079200 (PI Elena Ezhkova); the University of Michigan SBDRC P30AR075043 (PI Johann Gudjonsson); the University of Michigan Cancer Center P30CA046592 (PI Eric Fearon); and the University of Pennsylvania SBDRC P30AR069589 (PI Present address: Graduate School of Biomedical Sciences These authors contributed equally: Xuming Zhu Johns Hopkins University School of Medicine Perelman School of Medicine at the University of Pennsylvania Department of Cell and Developmental Biology M.X.; Project Administration: S.E.M.; Resources: X.Z. M.X.; Writing - Original Draft Preparation: X.Z.; Writing - Review and Editing: X.Z. The authors declare no competing interests Nature Communications thanks Hua-Tao Xie and the other reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-025-56907-6 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research Damage or removal of calcium-regulating parathyroid glands during endocrine surgery can put children at risk for poor growth and slow mental development Preserving and preventing damage to a child’s calcium-regulating parathyroid glands — a rice-sized organ — during thyroid surgery is important A recent study from researchers at Monroe Carell Jr Children’s Hospital at Vanderbilt shows that a procedure known as parathyroid gland (PG) auto-transplantation or reimplanting pieces of the tiny organ back into the body shows promise as an effective option to restore the glands’ function in children surgical co-director of the Vanderbilt Pediatric Thyroid Nodule and Cancer Program any disease burden associated with permanent hypoparathyroidism resulting from a thyroidectomy or parathyroidectomy.  While the auto-transplantation procedure is not new little research has been published about the technique and outcomes in children the surgeon takes the removed parathyroid gland/glands (often attached to thyroid) cuts it into tiny pieces and transplants the pieces into the neck muscle (either in the sternocleidomastoid or strap muscles) or in the forearm “We found that overall that when we reimplanted the parathyroid glands in the sternocleidomastoid muscle the success rate of avoiding hypocalcemia (low blood calcium) was better possibly because the muscle is highly vascularized making them more likely to receive the blood flow they require to work again,” said Belcher associate professor of Otolaryngology–Head and Neck Surgery at Monroe Carell Monroe Carell has become one of the leading pediatric surgery programs in the country for thyroidectomies in large part due to the multidisciplinary Vanderbilt Pediatric Thyroid Nodule and Cancer Program established at the hospital in 2021 The study “Outcome Analysis of Parathyroid Gland Auto-transplantation in Pediatric Patients: A Retrospective Review,” looked at pediatric patients who underwent parathyroid gland auto-transplantation following total thyroidectomy or parathyroidectomy at Monroe Carell between January 2000 and December 2022 Patients were identified through a comprehensive search of operative records and electronic medical records A total of 14 pediatric patients underwent PG auto-transplantation at Monroe Carell of pediatric patients achieved normalized calcium levels within six months after PG auto-transplantation indicating a high rate of successful engraftment. The remaining two patients Belcher says more research is needed to fully understand the impact of PG auto-transplantation in children “There are certainly confounders in this retrospective research approach for this surgical technique and outcome such as if the children had other functional parathyroid glands that were undisturbed during surgery,” Belcher said “But given the limited information out there in the literature we felt this was an important study to have some guidance on the procedure in the pediatric population.” “It’s important not to draw large conclusions from a small subset of patients,” Belcher added “It’s really important for us to evaluate this as a multi-institutional study — because children needing total thyroidectomies are decently rare — as the multi-institutional approach can accrue a high volume of data on this surgical technique This surgical technique is another tool in the surgeon’s armamentarium to help us give a child every chance to prevent permanent hypocalcemia.” The study was published in the Annals of Otology Other Vanderbilt study authors are Andrea Lopez A Vanderbilt study found that a probe technology that uses near-infrared auto-fluorescent lighting helps positively identify and preserve childrens’ parathyroids during endocrine surgery A team of surgeons and biomedical engineers have shown the use of probe-based near infrared autofluorescence technology helps confirm the identification of parathyroid glands during endocrine surgery The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission All content on this site: Copyright © 2025 Elsevier B.V. Research, Innovation, Press Releases The study found that nearly 9 out of 10 participants achieved what the study defined as successful treatment over five years.  A five-year study on men that had just the cancerous portion of their prostate glands destroyed shows that the procedure averted cancer recurrence in most patients while preserving urinary and sexual function Led by researchers at NYU Grossman School of Medicine the study tracked patient outcomes after primary partial gland cryoablation (PPGCA) a procedure where only the cancerous part of the prostate gland is treated with extreme cold (cryotherapy) to destroy it concentrated on a small area of the prostate gland (focal therapy) is increasingly used in place of more-traditional treatments: surgical removal or irradiation of the whole gland Authors of the current study from NYU Grossman School of Medicine had previously reported that PPGCA avoids incontinence and minimizes sexual dysfunction no study had yet tracked the effectiveness of the combination of partial gland ablation with intense follow-up to watch for Published recently in the journal Urology the study showed that 81 out of 91 participants (89 percent) achieved the main study measure of treatment success over five years a patient has not died specifically from prostate cancer seen his cancer spread outside of the prostate (metastasize) successful round of focal therapy when cancer recurred after the FFF period ended while 15 (16.5 percent) needed to undergo whole-gland treatment in the end “We found that PPGCA can avert the profound consequences that can come with gland removal while still showing excellent results in preventing recurrence.” Advances in magnetic resonance imaging (MRI) enabled the team to reliably identify the sites which in turn determined the best candidates for focal therapy intermediate-risk prostate cancer were found to meet the study inclusion criteria 91 could be evaluated for freedom-from-failure over the entire five years because they had no prostate cancer mortality or need for whole-gland treatment during that time The reason to study only intermediate-risk patients was that this level of disease aggressiveness would otherwise have required whole-gland treatment immediately upon diagnosis Dr. Lepor added that the study results, and his experience in having performed more than 5,000 whole-gland removals (radical prostatectomies) in his career, both argue that 80 percent of men with intermediate-risk disease would choose to undergo focal cryotherapy over prostatectomy if they had the choice the current study was designed with especially intense surveillance of patients over time after PPGCA which the researchers say was as important as the initial ablation study patients underwent prostatic-specific antigen (PSA) tests—which capture levels of protein known rise in the presence of prostate cancer—every 6 months Keeping close tabs on patients through the study may also explain in part why just 3.3 percent of patients in the study dropped out (were lost to follow-up) over five years Lepor’s team employs two full-time research coordinators dedicated to ensuring that men return over time after PPGCA for follow-up PSA tests and MRI “This study represents the largest comprehensive, prospective study of men with intermediate-risk prostate cancer treated with partial gland cryoablation,” said study author James S. Wysock, MD assistant professor in the Department of Urology “We ensured rigorous follow-up over five years with high patient compliance for PSA testing we’ll expand our evaluations to include a broader spectrum of patients—particularly those with lower-risk cancer that’s not appropriate for active surveillance but may not require whole-gland treatment.” study authors from the Department of Urology at NYU Langone were Eli Rapoport many of whom had undergone the study procedure NYU Langone Health is a fully integrated health system that consistently achieves the best patient outcomes through a rigorous focus on quality that has resulted in some of the lowest mortality rates in the nation 1 comprehensive academic medical center in the country for three years in a row News & World Report recently placed nine of its clinical specialties among the top five in the nation NYU Langone offers a comprehensive range of medical services with one high standard of care across seven inpatient locations and more than 300 outpatient locations in the New York area and Florida the system also includes two tuition-free medical schools and a vast research enterprise with over $1 billion in active awards from the National Institutes of Health Experts showcase their latest clinical findings at American Urological Association’s annual meeting He will lead urologic care at NYU Langone Hospital—Brooklyn 2025 (GLOBE NEWSWIRE) -- Actuate Therapeutics (NASDAQ: ACTU) (“Actuate” or the “Company”) a clinical-stage biopharmaceutical company focused on developing therapies for the treatment of high-impact difficult-to-treat cancers through the inhibition of glycogen synthase kinase-3 beta (GSK-3β) today announced that data on elraglusib in advanced salivary gland carcinoma will be presented in a poster session at the American Association for Cancer Research (AACR) Annual Meeting 2025 taking place from April 25th – 30th at McCormick Place Convention Center The abstracts will be published in the online Proceedings of the AACR Poster presentation details:Title: Elraglusib a glycogen synthase kinase 3 beta (GSK-3β) inhibitor plus chemotherapy with or without immunotherapy for advanced salivary gland cancerSession Title: Phase II Clinical Trials 2Session Date and Time: April 29 2:00 PM - 5:00 PMLocation: Poster Section 50Abstract Presentation Number: CT212 "Elraglusib with chemotherapy and immune priming represents a novel sequential therapy approach to treat advanced salivary cancers We were encouraged by the response rate among the non-Adenoid Cystic Carcinoma (ACC) population with nuclear GSK-3β overexpression given this difficult to treat and rare cancer type" Director of the Center for Cancer Therapeutic Innovation (CCTI) the early drug development program at Dana Farber Cancer Institute who led the team conducting this clinical study Wafik S El-Deiry from Legorreta Cancer Center at Brown University will present a poster demonstrating the synergistic effects of elraglusib in combination with ONC206 or ONC212 investigational compounds from Jazz Pharmaceuticals plc Poster presentation details:Title: Elraglusib (9-ING-41) a glycogen synthase kinase-3β inhibitor in combination with imipridones for treatment of solid cancerSession Title: Drug Combination Strategies for Cancer TreatmentSession Date and Time: April 28 9:00 AM - 12:00 PMLocation: Poster Section Poster Section 19Abstract Presentation Number: 1693 Actuate is a clinical-stage biopharmaceutical company focused on developing therapies for the treatment of high-impact targets molecular pathways in cancer that are involved in promoting tumor growth and resistance to conventional cancer drugs such as chemotherapy through the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and DNA Damage Response (DDR) Elraglusib may also mediate anti-tumor immunity through the regulation of multiple immune checkpoints and immune cell function please visit the Company’s website at http://www.actuatetherapeutics.com This press release contains forward-looking statements about us including our and other parties’ clinical trials and development plans The words “anticipate,” “believe,” “continue,” “could,” “estimate,” “expect,” “intend,” “may,” “might,” “ongoing,” “plan,” “potential,” “predict,” “project,” “should,” “target,” “will,” “would,” or the negative of these terms or other comparable terminology are intended to identify forward-looking statements although not all forward-looking statements contain these identifying words other than statements related to present facts or current conditions or of historical facts contained in this press release are forward-looking statements substantial risks and uncertainties which could cause actual results to differ materially from those expressed in them including but not limited to that clinical and preclinical drug development involves a lengthy and expensive process with uncertain timelines and outcomes results of prior preclinical studies and early clinical trials are not necessarily predictive of future results and elraglusib may not achieve positive clinical results or favorable preclinical results or receive regulatory approval on a timely basis if at all; that we may not successfully enroll additional patients or establish or advance plans for further development including through conversations with the FDA or EMA and the standards such bodies may impose for such development; that elraglusib could be associated with side effects adverse events or other properties or safety risks which could delay or preclude regulatory approval including because our financial condition raises substantial doubt as to our ability to continue as a going concern and we will require substantial additional capital to finance our operations and a failure to obtain this necessary capital in the near term on acceptable terms reduce or terminate our development programs commercialization efforts or other operations any forward-looking statements are qualified in their entirety by reference to the factors discussed under the heading “Item 1A Risk Factors” in our Annual Report on Form 10-K for the year ended December 31 Because the risk factors referred to above could cause actual results or outcomes to differ materially from those expressed in any forward-looking statements made by us or on our behalf you should not place undue reliance on any forward-looking statements any forward-looking statement speaks only as of the date on which it is made and it is not possible for us to predict which factors will arise we cannot assess the impact of each factor on our business or the extent to which any factor may cause actual results to differ materially from those contained in any forward-looking statements we do not undertake any obligation to release publicly any revisions to such forward-looking statements to reflect events or circumstances after the date of this press release or to reflect the occurrence of unanticipated events.Investor Contact Mike MoyerManaging DirectorLifeSci Advisors Metrics details Mammary gland development is a complex process involving dynamic interaction between the epithelial and stromal components at different critical stages While epithelial tissue changes are well-documented and transcriptomic analyses to investigate molecular and cellular dynamics in the mammary gland during pre-puberty (Post Natal Day (PND21)) The epithelial area was significantly smaller at PND21 than at PND46 and PND90 with a higher complexity at PND21 compared to PND46 Significant differences in adipocyte number and size were observed between PND21 Transcriptomic analysis revealed that 1563 genes changed significantly between PND21 and PND46 with only 14 genes altered between PND46 and PND90 Enrichment analyses indicated dynamic regulation of pathways related to proliferation 29/43 and 7/43 fatty acids differed significantly between PND21 - PND46 and PND46 - PND90 These results suggest that mammary gland development involves complex interactions between metabolic demands These studies indicated a clear relation between the transcriptional regulation and the lipid compositions of the mammary gland we utilized whole mammary tissue to compare morphological and physiological parameters from pre-puberty to adulthood identifying differences in gene expression profiles using RNA sequencing we evaluated changes in fatty acid composition using lipidomics exploring how lipid dynamics in adipocytes impact mammary gland development and function This integration of lipidomic and transcriptomic analyses provides a deeper understanding of the underlying mechanisms regulating mammary gland development and function emphasizing the crucial role of adipocytes and immune cells in these complex biological processes Morphologic characteristics of mammary gland sampled from rats during pre-puberty (PND21) (A) Representative diagram of mammary gland sampling and mammary gland weight normalized to total rat weight per group (C) Left: representative images of whole mount for each group (scale bar 1 cm) Right: graphic representation of the skeletonized epithelium of the mammary gland obtained from those images using ImageJ (D) Average epithelial area and epithelial complexity) for each group Graphs represent the average of the group with SD (*p < 0.05 Identification of differentially expressed genes using RNA-sequencing analysis between PND21 to PND46 (A) Principal component analysis showing the normal distribution of the groups PND21 (B) Volcano plot illustrating the significantly upregulated (red) and not significant (not sig (grey)) genes in the transition between PND21 and PND46 of rat’s mammary gland (p-adj < 0.05 (C) Volcano plot illustrating the significantly upregulated and downregulated genes in the transition between PND46 and PND90 of rat’s mammary gland (p-adj < 0.05 (D) Gene Ontology enrichment analysis of biological processes for upregulated and downregulated genes during the transition between PND21 and PND46 including Tnfrsf11a (TNF receptor superfamily member 11a) and Cd3e (CD3 epsilon subunit of T-cell receptor complex) showed increased expression during the transition from PND21 to PND46 and PND90 by qPCR (Fig These results suggest increased transcriptional activation of genes/gene networks implicated in the activity of the immune compartment all three genes were significantly lower in PND46 and PND90 compared to PND21 (Fig These results suggest that after pre-puberty an important metabolic reprogramming occurs notably affecting lipid metabolism at the transcriptional level with an overlap observed in the transcriptional profiles of the PND46 and PND90 stages genes from clusters 1 and 2 generally showed a higher expression at PND21 compared with PND46 and PND90 while genes from clusters 3 and 4 showed an opposite pattern Hierarchical cluster analysis by K-Means from the PND21 Rows represent genes clustered using K-means clustering and columns represent samples (n = 4) from PND21 Metascape enrichment network visualizations of the most common genes (B) in cluster 1 related to cellular growth and differentiation dynamics (C) in cluster 2 related to metabolic and proliferative regulation (D) in cluster 3 related to hormonal regulation and stress response and (E) in cluster 4 related to immune response and cellular signaling dynamics Black arrows point to the specific pathways in each cluster that relate to mammary gland development Cluster 2, related to metabolic and proliferative regulation, involved processes such as cell cycle regulation, oxidative phosphorylation, the tricarboxylic acid cycle (TCA cycle), acyl-CoA metabolic process, and pyruvate metabolism, among other cellular metabolic processes (Fig. 3C) Changes in metabolic gene expression during development suggest an important metabolic reprogramming throughout the mammary gland evolution notably between pre-puberty and peri-puberty/adulthood Given our interest in the role of the stromal compartment as they are more closely related to functions generally attributed to the stroma such as lipid metabolism and immune functions Identification of lipid metabolic reprogramming pathways and adipogenesis in the transition between PND21 and PND46 via transcriptome analysis (A) Venn diagram for overlapping significant genes downregulated and upregulated and cluster 2 during the transition between PND21 and PND46 (B) Enrichment pathway analysis using overlapping genes (C) and (D) Gene Set Enrichment Analysis (GSEA) plots for biological pathways enriched in the transition between PND21 to PND46 and PND90 Hallmarks gene sets are shown for (C) adipogenesis and (D) fatty acid metabolism during the transition between PND21 to PND46 and PND46 to PND90 The red color indicates higher expression in heatmaps and the blue color indicates lower expression (E) Graphic representation of total fatty acid across developmental stages Among the downregulated genes related to adipogenesis and genes involved in fatty acid synthesis like Fatty acid synthase (Fasn) and Glycerol-3-phosphate dehydrogenase 1 (Gpd1) (Fig and the expression level were reduced in PND46 and PND90 compared to PND21 as validated by qPCR for specific genes (Fig these results highlight the profound changes in lipid metabolism that notably occur between pre-puberty and peri-puberty The changes of fatty acids composition across developmental stages of the mammary gland (A) Principal component analysis showing the differential distribution of the fatty acids across the groups (B) In the heatmap of 41 fatty acids across developmental stages the concentration of the lipids was clustered and represented in the rows while the samples are represented in the columns from PND21 (C) Correlation of fatty acids showing the different lipid profiles at PND21 and PND90 groups; the red color indicates a negative correlation in the interaction between lipids and the blue color indicates a positive correlation (D) Histogram representation of all categories of fatty acids such as saturated fatty acids and ratio of omega-3 to omega-6 fatty acids there was no significant alteration in the categories of fatty acids Current data suggest modulation at the transcriptional and relative expression level as well as from alterations in lipid levels Adipocyte analysis across developmental stages (A) Representative image of Masson’s Trichrome stained adipose tissue from PND21 and PND90 rats and of the analysis of adipocytes using the Adiposoft plugin from ImageJ (B) Histogram representing the number of adipocytes per cm2 per group and adipocyte size in µm2 RNA-seq analyses indicate significant enrichment for immune response (A) Hallmarks gene sets are shown for an interferon-a response during the transition between PND21 to PND46 and PND46 to PND90 (B) Venn diagram for overlapping significant genes upregulated and cluster 4 during the transition between PND21 and PND46 (C) Metascape enrichment network visualization of the 629 overlapping genes notably for immune response activation (D) Gene Ontology enrichment analysis of molecular function for upregulated and downregulated genes during the transition between PND21 and PND46 we performed a GSEA M8 - cell type signature gene analysis which predicts the enrichment of specific cell types These results confirmed a strong enrichment of immune cell gene signatures such as those linked to CD4-positive T-cells and B cells in PND46 compared to PND21 (Fig cell types such as antigen-presenting cells maintaining the immune response between PND46 and PND90 (Fig These results indicated that the transition from pre-pubertal to peri-pubertal stages is characterized by alterations in transcriptional signatures associated with the aforementioned biological functions Both animal and human mammary gland development follow the same sequence of events the mammary gland experiences successive transitional stages: pre-puberty we measured the gene expression of rat mammary glands across three development stages and its relationship with epithelial development the combination of transcriptomic and lipidomic profiling during mammary gland development has not been reported Although one of the limitations of our study is the fact that whole tissue homogenates were used thus encompassing both epithelial and stromal tissues transcriptomic signatures suggested that important modifications were happening in the stroma it cannot be excluded that it also reflect a change in transcriptional regulation in epithelial cells Further studies are needed to verify this hypothesis Our study found that adipocyte size decreased from pre-puberty to peri-puberty but increased as the gland transitioned to adulthood supporting the premise that epithelial proliferation is linked with a high demand for lipids during the phase of ductal elongation followed by the accumulation of lipids within adipocytes once the growth of the epithelium stabilizes PPAR signaling was decreased from pre-puberty to puberty these findings indicated that there is a significant downregulation of genes involved in lipid synthesis and metabolic processes during the transition from pre-puberty to peri-puberty Our findings highlight the dynamic changes in lipid metabolism during mammary gland development emphasizing the critical role of adipocytes in tissue remodeling and homeostasis The observed shifts in fatty acid profiles and adipocyte morphology underscore the intricate balance between lipid synthesis and epithelial expansion necessary for proper mammary gland maturation The significant enrichment of immune response genes during the transition from pre-puberty to peri-puberty highlights the essential role of immune signaling in mammary gland remodeling and development and regulatory genes underscores the intricate balance of immune processes necessary for effective mammary tissue maturation and development during critical transitional periods this study provides a comprehensive view of the morphological and metabolic changes in the mammary gland during pre-puberty The results highlight the complexity of mammary development and underscore the importance of metabolic and proliferative regulation and lipidomic analyses offers a robust platform for future research to understand the underlying mechanisms of mammary development which may also have implications for studying diseases related to the mammary gland Rats were weighed and euthanized by CO2 followed by cervical dislocation at different time points: pre-puberty (PND21) and two pairs of mammary glands (thoracic and inguinal) were taken and weighed and fixed in Carnoy`s solution (6:3:1 of 100% ethanol and glacial acetic acid) overnight at room temperature (RT) Samples were rehydrated again through serial ethanol gradient from 70 to 0% in water and stained in carmine alum (2% carmine 5% potassium aluminum sulfate in water) overnight at RT Samples were dehydrated through serial ethanol baths and cleared in xylene samples were mounted with Permount (FisherChemical Images were captured using a Canon PowerShot G9x digital camera on a transilluminator (Henning Graphics LR299343) and a measurement scale was used to compare sample sizes The raw sequencing data quality was checked using FastQC v0.12.0 before being trimmed using TrimGalore v0.6.10. The quality was then reverified with FastQC and aggregated with MultiQC v1.2. The trimmed RNA sequences with high-quality reads were aligned to the Rattus norvegicus Rnor_6.0 reference transcriptome (https://may2021.archive.ensembl.org/Rattus_norvegicus) using Kallisto v0.46.1 pseudo-aligner sequencing data were deposited in NCBI under the accession number PRJNA1157723 Amplification curves were next analyzed using the comparative cycle threshold method (ΔΔCt method) The fatty acids profile was realized by capillary gas chromatography using a temperature gradient on an HP5890 gas chromatograph (Hewlett Packard Canada) equipped with an HP-88 capillary column (100 m x 0.25 mm i.d x 0.20 μm film thickness; Agilent Technologies) coupled with a flame ionization detector (FID) Helium was used as carrier gas (split ratio 1:50) Fatty acids were identified according to their retention time using the following standard mixtures as a basis for absolute quantification: the FAME 37 mix (Supelco Inc. the GLC-411 fatty acid mix (NuChek Prep Inc the methylated fatty acids C22:5 w6 (Larodan AB the methylated fatty acids C22:5 w3 (Supelco Inc. a mixture of trans-FA containing: C18:2 w6 cis/trans (Supelco Inc Bellefonte a mixture of cis/trans C18:3 w3 (Supelco Inc Bellefonte and the following Nucheck fatty acids: C14:1 trans-9 Results were expressed as percent of total fatty acids and mgFA/g of mammary gland tissues All the statistical analyses were done using GraphPad Prism 9 and the R-Studio environment The normality of the distribution was verified using Shapiro-Wills or Kolmogorov-Smirnov normality test Data that satisfied assumptions of normality and homoscedasticity were analyzed using Anova and student’s t-test Kruskal-Wallis and Mann-Whitney U-test non-parametric tests were used Pearson’s correlation was used to measure the degree of relationship between variables and changes of Log2 fold of 1.5 were considered significant p-value < 0.05 was considered as a significant difference Visualization techniques such as volcano plots, complex heatmaps, principal component analysis (PCA), boxplots, bar plots, and Dot plots were performed using an R environment. 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Res. 49, 316–325. https://doi.org/10.1161/01.res.49.2.316 (1981) Direct transesterification of all classes of lipids in a one-step reaction Download references The authors are thankful to Fabien Joao and Rhizlane ElOmri for their technical support and EAW from the National Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2020-05726 DT is supported by scholarships from the Réseau Québecois en reproduction and Fondation Armand-Frappier Institut National de la Recherche Scientifique INRS Centre Armand-Frappier Santé Biotechnologie Centre de recherche du CHU de Québec-Université Laval Department of Microbiology-Infectious Diseases and Immunology Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Below is the link to the electronic supplementary material Download citation DOI: https://doi.org/10.1038/s41598-025-97532-z Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology Metrics details The murine mammary gland is sustained by distinct pools of stem cells that are limited in space and time the specific identities of the bipotent and unipotent mammary stem cells remain unclear we investigated spatial heterogeneity of the mammary gland at the single-cell transcriptional level We found that mammary basal cells exhibited spatially distinct populations and characteristics which can be further divided based on the expression of CD34 and CD200 markers CD34−CD200+ basal cells enriched at the nipple region demonstrated strong long-term self-renewal ability and possessed the highest stem cell frequency while CD34+CD200− basal cells enriched in the terminal end buds (TEBs) showed reduced stem cell potency Through lineage tracing experiments based on their signature genes we discovered that Bcl11b+ cells were enriched in the CD34−CD200+ population and exhibited bipotency even in the postnatal mammary gland with an increasing contribution to mammary epithelia observed during long-term tracing and after multiple rounds of pregnancies predominantly revealed their unipotent nature and significant contribution during alveologenesis the Bcl11b+ cells displayed a slow response to pregnancy but contributed to long-term mammary homeostasis in contrast to the rapid response observed in Sema3a+ cells Bcl11b progenies survived much better than Sema3a progenies during involution stage thereby exhibiting increased coverage in the mammary gland after multiple rounds of pregnancies depletion of Bcl11b in Krt14+ mammary basal cells resulted in reduced bipotency of mammary stem cells and impaired their long-term contribution to the mammary gland our study identifies distinct bipotent and unipotent populations of mammary basal cells with different dynamic properties that play critical roles in maintaining postnatal mammary homeostasis These findings are crucial for advancing our understanding of breast health and breast cancer research It remains highly controversial how adult mammary gland is maintained by what types of stem cells and the identities of the long-lived bipotent mammary stem cells and unipotent mammary stem cells in the mammary gland are poorly understood It is also unresolved whether the various cellular states of the same stem cell population or the distinct stem cell populations resulted in the variable tracing outcomes under physiological condition it is imperative to comprehensively investigate the heterogeneity of basal cells and their contributions to mammary homeostasis we characterized the spatial distribution of mammary cells at the single cell level and found that mammary basal cells can be categorized into three distinct populations based on the expression of CD34 and CD200 The CD34−CD200+ quiescent cells exhibited the highest efficiency in mammary reconstitution Lineage tracing assays demonstrated that both bipotent and unipotent mammary stem cells co-existed in postnatal mammary gland The bipotent cells exhibited a slow but long-lasting contribution while the unipotent cells displayed a fast but limited contribution Loss of long lived bipotent mammary stem cells in adult mouse impairs architectural and functional integrity of the mammary gland our findings suggest that both bipotent and unipotent mammary stem cells play crucial roles in maintaining normal mammary gland physiology a Schematic workflow for scRNA-Seq in b−f from mammary glands of 5-week-old K14-Cre/R26-mTmG mice were processed into single-cell suspensions and subjected to 3’ UTR single-cell SMART-seq c t-SNE plots show the clustering of mammary cells from various locations Distinct mammary populations are displayed in different colors in b The anatomical locations of cells are shown in different colors in c indicating that mammary epithelial cells from different locations cluster well into basal and ER+ luminal populations (in red circles) while still exhibiting heterogeneity within basal and stromal cells based on spatial distribution d Signature gene expression in mammary epithelial cells e Signature gene expression in fibroblasts (Col1a1 Col3a1 and Pdgfra) and myeloid cells (Cd14 f Proportion of cells in different cell cycle phases for basal cells in TEB suggesting a robust cell fate dynamics associated with locations Taken together, these findings suggest that basal cells are spatially heterogeneous with molecular features (Supplementary Figs. S2a, b, 3a–g, i) associated with distinct cell states/fates This spatial feature provides valuable insights into the molecular identity and function of the postulated inactive stem cells and proliferative basal progenitor cells a FACS plots showing three distinct basal subpopulations characterized by surface markers CD200 and CD34 (data were collected from BD FACSAria Fusion The negative gate of CD34 and CD200 was set according to the luminal cells) b Representative FACS plots showing the expression of CD200 and CD34 in basal or luminal cells of TEB duct and nipple (data collected from CytoFLEX) c Statistical analysis of percentages of different basal subpopulations categorized by CD200 and CD34 FACS staining in TEB Statistical analysis was performed using two-tailed unpaired t-test Representative outgrowth images of 3 repeated wells on day 4 and day 7 for each subpopulation are shown e Colony sizes in d were measured and plotted in the bar graph f Extreme limiting dilution analysis (ELDA) plot of the repopulating ability of FACS-sorted CD200−CD34+ CD200+CD34low or CD200+CD34− basal subpopulations under transplantation g Representative images of reconstituted mammary gland derived from the secondary transplants of CD200−CD34+ CD200+CD34low or CD200+CD34− basal subpopulations h–j Statistical analysis of the secondary transplant in g The relative ratio of area covered by reconstituted mammary gland in the de-epithelialized fat pads are shown in h and the absolute size of recipient fat pads and mammary outgrowths are shown respectively in i it suggests that the nipple region has a higher frequency of stem cells than other regions the features of CD200−CD34+ basal cells enriched in TEB fit better with the concept of progenitor cells with fast proliferation but lower reconstitution capacity a Schematic diagram of doxycycline-inducible Bcl11b-rtTA/TetO-Cre/R26-tdTomato model Bcl11b-rtTA mice were crossed with TetO-Cre mice and R26-tdTomato reporter mice to generate doxycycline inducible reporter mouse model for Bcl11b+ cell lineage tracing b Schematic strategy for lineage tracing Bcl11b+ cells from puberty stage Bcl11b-rtTA/TetO-Cre/tdTomato mice were administered with 2 mg/mouse doxycycline (100 µL 20 mg/mL in PBS) intraperitoneally for three consecutive days at 4 weeks of age The mammary glands were harvested for tdTomato labeling analysis 2 days after pulsing c Representative FACS plots showing the percentage of tdTomato-positive cells in epithelial population (left panel) and the proportion of basal or luminal subpopulation in tdTomato+ epithelial cells (right panel) 2 days after doxycycline induction at puberty d Representative immunofluorescence images showing the co-staining of Bcl11b tdTomato and basal cell marker αSMA in nipple and duct in mammary glands 2 days after doxycycline induction at puberty (left panel) And bar chart showing the labeling efficiency of Bcl11b+ cells by tdTomato in nipple and duct of mouse mammary glands (right panel) Statistical analysis was performed using two-tailed unpaired t test e Representative FACS plots showing the tdTomato-labeling percentage in basal cells (Lin−Epcamlow CD49f+) of TEB duct or nipple 2 days after doxycycline induction from puberty stage f Bar graph showing the statistical analysis of the percentage of tdTomato+ cells in basal or luminal compartments from TEB duct and nipple 2 days after doxycycline induction from 4-week-old mice by FACS as in e Bcl11b-rtTA/TetO-Cre/ tdTomato mice with PBS treatment were control group (CTR) while that with doxycycline treatment were experimented group (EXP) g Schematic diagram showing the lineage tracing strategy of Bcl11b+ cells from embryonic day 15.5 in h−j h Representative whole-mount images of tdTomato-labeled mammary glands in 8-week-old mice traced from embryonic stage i Representative FACS plots display the percentage of tdTomato-positive cells in epithelial population (middle panel) and the proportion of basal or luminal subpopulation in tdToamto+ epithelial cells (right panel) from 8-week-old mice traced from embryonic stage j Bcl11b-lineage tracing from the embryonic stage (E15.5 d) to adulthood (P8w) Bcl11b+ epithelial cells were traced from embryonic day 15.5 (E15.5 d) When these offspring reached 8 weeks of age (P8w) their mammary glands were harvested for tdTomato-labeling analysis j2 Bar graphs showing the percentage of tdTomato-labeled cells in luminal or basal population (j1) and the percentage of luminal or basal cells among the total tdTomato-labeled epithelial cells (j2) k Schematic diagram showing the lineage-tracing strategy of Bcl11b+ cells for induction in pubertal mice at 4 weeks of age with various chase periods as indicated (2 days l Representative FACS plots depict the distribution of tdTomato-traced cells in epithelial gate n Bar charts showing the composition of tdTomato-labeled epithelial cells (m) the percentage of tdTomato-labeled epithelial cells in total epithelial cells (n) in Bcl11b-lineage tracing at designated tracing times after pulsing at puberty n = 5 (20-week chase) and n = 6 (1-year chase) o FACS plots showing the contribution of Bcl11b-traced cells to various mammary populations 20 weeks after pulsing at puberty Sca-1 was used as a marker of the ESR1+ luminal cells p Representative immunofluorescence image showing tdTomato-labeled clone containing basal cell (magenta arrowhead) ESR1+ luminal cell (white arrowhead) and ESR1− luminal cell (green arrowhead) 20 weeks after pulsing at puberty These data collectively demonstrate that Bcl11b-positive cells are rare mammary stem cells that are difficult to label but have rigorous multipotency even in the postnatal mammary gland a Real-time PCR analysis showing mRNA level of Sema3a in basal b Schematic diagram of tamoxifen-inducible Sema3a-CreERT2/R26R-tdTomato mouse model Sema3a-CreERT2 mice were crossed to Rosa26-tdTomato mice to generate tamoxifen inducible reporter mice for Sema3a+ cell lineage-tracing c Schematic diagram illustrating the lineage-tracing strategy for Sema3a+ cells from puberty for experiments in d−i Sema3a-CreERT2/R26-tdTomato mice were treated with 5 mg tamoxifen (TAM) intraperitoneally for only once at 5-week-old Mammary glands of various chase periods (2 days 20 weeks) were harvested for tdTomato labeling analysis d Representative FACS plots showing percentage of tdTomato-labeled basal or luminal cells 2 days after pulsing on postnatal day 35 (upper panel) Real-time PCR analysis showing mRNA level of Sema3a in tdTomato-labeled and unlabeled basal cells (lower panel) e Representative images taken by fluorescence stereomicroscope display tdTomato expression in TEB (upper panel) and duct (lower panel) 2 days after tamoxifen induction at puberty f Representative tissue clearing images showing immunofluorescence staining of tdTomato+ cells with basal markers Krt14 and Trp63 in TEB (upper panel) or duct/nipple (lower panel) 2 days after tamoxifen induction The selected regions by the yellow boxes were magnified in the right panel g Representative images showing 3D immunofluorescence staining of tdTomato and luminal marker Krt8 in tissue cleared mammary gland at different time points after tamoxifen induction at puberty h Representative FACS plots showing percentage of tdTomato-labeled basal or luminal cells at the indicated time points after pulsing at puberty i The upper bar graphs showing the percentage of tdTomato-positive cells in basal or luminal compartment at different time points after pulsing at puberty meanwhile the lower panel showing the relative basal and luminal percentage in tdTomato-positive mammary epithelial cells a Schematic diagram showing the lineage-tracing strategy of Bcl11b+ cells from puberty for experiments in b−i +) female mice were administered with 2 mg doxycycline intraperitoneally for three consecutive days at 4-week-old and then mated during adult stage for multi-pregnancy Mammary glands at various time points as indicated during multiple pregnancies (1st Preg17.5 d 3rd Preg17.5 d) were harvested for tdTomato labeling analysis b Representative fluorescence images showing tdTomato-positive clones in mammary glands of Bcl11b-rtTA/TetO-Cre/tdTomato mice at the indicated time points during multiple pregnancies after pulsing at puberty c Representative FACS plot showing the composition of tdTomato-positive cells in two types of individual clones (bi-lineage in the left panel and uni-lineage in the right panel) in Bcl11b-lineage tracing during multiple pregnancies (1st to 3rd pregnancy) after pulsing at puberty The individual bright tdTomato+ Bcl11b clones in the mammary fat pads during multiple pregnancies were dissected under the fluorescence stereomicroscope and then were digested into single cells and performed FACS analysis of the composition of basal and luminal lineages d Bar chart showing percentage of bi-lineage and uni-lineage clones at day 17.5 of multiple pregnancies (1st to 3rd Preg17.5 d) in Bcl11b-rtTA/TetO-Cre/tdTomato mice after pulsing at puberty and the clones of each group were collected from at least 3 mice f Bar charts showing the composition of tdTomato-labeled epithelial cells (e) and percentage of tdTomato-labeled cells in basal or luminal populations (f) at the indicated time points during multiple pregnancies in Bcl11b-lineage tracing after pulsing at puberty g Representative images showing milk production by Bcl11b+ cells derived epithelial cells at lactation day 7 in Bcl11b-lineage tracing after pulsing in puberty The selected region by the yellow frame was enlarged in the right panel h Bar chart showing the number of tdTomato-labeled clones as shown in b in all ten fat pads of each mouse at the indicated time points during multiple pregnancies in Bcl11b-lineage tracing after pulsing at puberty i Bar chart showing percentage of tdTomato-labeled cells in all epithelial cells at the indicated times during multiple pregnancies in Bcl11b-lineage tracing after pulsing at puberty j Schematic diagram showing the lineage-tracing strategy of Sema3a+ cells from puberty for experiments in k−q +) female mice were treated with single time 5 mg tamoxifen intraperitoneally at 4-week-old 3rd Preg17.5 d) were harvested for tdTomatoto labeling analysis k Representative fluorescence images of tdTomato-positive clones in mammary gland at pregnancy day 17.5 during the 1st or 2nd pregnancies in Sema3a-lineage tracing after pulsing at puberty The selected regions by the yellow frames were enlarged in the right panel l Representative 3D immunofluorescence image of tissue cleared mammary gland showing the co-staining of tdTomato with basal cell markers Krt14 and Trp63 in mammary glands of Sema3a-CreERT2/tdTomato mice during multiple pregnancies after pulsing at puberty The selected region by the yellow box was magnified in the right panel m Bar graph showing the composition of tdTomato-labeled epithelial cells of individual clones at day 17.5 of the 1st pregnancy from each mouse with Sema3a-lineage tracing at puberty Each dot represents one clone from a mouse whose ear tag number was labeled below the corresponding bars o Bar charts showing percentage of tdTomato-labeled cells in basal or luminal populations (n) and basal or luminal proportion in all tdTomato-labeled epithelial cells (o) at the indicated time points during multiple pregnancies in Sema3a-lineage tracing from puberty p Bar chart showing the number of tdTomato-labeled clones in all ten fat pads of each mouse at the indicated time points during multiple pregnancies in Sema3a-lineage tracing after pulsing at puberty q Bar chart showing the percentage of tdTomato-labeled cells in all epithelial cells at the indicated time points during multiple pregnancies in Sema3a-lineage tracing after pulsing at puberty All above statistical analysis was performed using two-tailed unpaired t-test These data suggest that there are different mammary stem cell populations involved in the development of pregnant mammary gland And the relative contributions of various mammary stem cells to the gland are dynamically changing during multiple pregnancies a Schematic diagram showing the tracing strategy of Bcl11b+ cells during multiple pregnancies and involutions c Bar chart showing the percentage of tdTomato-labeled cells in basal or luminal populations (b) or percentage of tdTomato-labeled cells in the whole mammary epithelial population (c) at the indicated time points during multiple pregnancies and involutions in Bcl11b-lineage tracing after pulsing at puberty d Representative immunofluorescence images showing the apoptotic cells indicated by cleaved caspase3 staining during involutions for Bcl11b-traced (tdTomato+) and -untraced (tdTomato-) cells during mammary involution e Bar chart showing the quantification of apoptotic cells in Bcl11b-traced (tdTomato+) and -untraced (tdTomato-) cells during mammary involution f Bar chart showing percentage of tdTomato-labeled cells in basal or luminal populations at the indicated time points during multiple pregnancies and involutions in Sema3a-lineage tracing after pulsing at puberty g Representative images of tdTomato-positive clones in mammary glands at day 12.5 15.5 or 17.5 of the 1st pregnancy (1st Preg12.5 d day 7 of the 1st lactation (Lac7d) and involution day 2 (Invo2d) in Bcl11b-lineage tracing after pulsing at puberty h Bar chart showing the number of tdTomato-positive clones as shown in g in all ten fat pads of each mouse at the indicated time points of the 1st reproductive cycle in Bcl11b-lineage tracing after pulsing at puberty j Bar charts showing percentage of tdTomato-labeled cells in total epithelial population (i) and basal and luminal proportion in all tdTomato-labeled epithelial cells (j) at the indicated time points during the 1st reproductive cycle in Bcl11b-lineage tracing after pulsing at puberty ‘Invo 3 m’ is designated as 90 days after involution k Bar chart showing the tdTomato-labeled clone number in all ten fat pads of each mouse at the indicated time points during the 1st pregnancy in Sema3a-lineage tracing after pulsing at puberty l–n Bar charts showing percentage of tdTomato-labeled cells in the whole mammary epithelial population (l) percentage of tdTomato-labeled cells in basal or luminal subpopulations (m) and basal or luminal proportion in all tdTomato-labeled epithelial cells (n) at the indicated time points during the 1st pregnancy in Sema3a-lineage tracing after pulsing at puberty All above statistical analysis was performed using two-tailed unpaired t test These data collectively indicate that different mammary stem cells respond to the pregnancy cues in different modes Bcl11b+ mammary stem cells are more like a slow responder but persist for longer time and gradually replenish the mammary gland while the Sema3a+ mammary stem cells respond more rapidly to pregnancy but have restricted contributions to the gland in long term a The upper panel shows schematic diagram of doxycycline-inducible Krt14-rtTA/TetO-Cre/R26-mTmG model Krt14-rtTA mice were crossed with TetO-Cre mice and R26R-tdTomato reporter mice to generate doxycycline inducible reporter mouse model for lineage tracing Krt14+ cell The lower panel shows schematic strategy for lineage tracing studies of Krt14+ cells from puberty stage for experiments in b−d Krt14-rtTA/TetO-Cre/R26-mTmG mice were administered with 0.1 mg or 2 mg doxycycline intraperitoneally only once at postnatal 5-week-old (P5w) The mammary glands were harvested for GFP labeling analysis 1 year after pulsing b Representative FACS plots showing the labeling efficiency of mammary basal cells after doxycycline treatment at a low (0.1 mg) or high (2 mg) dose for 2 days c Representative images showing immunofluorescence co-staining of GFP and luminal marker Krt8/18 in mammary glands of Krt14 tracing 1 year after pubertal pulsing with a single low dose of 0.1 mg (upper panel) or a high dose of 2 mg (lower panel) Yellow arrowheads indicate the GFP-traced cells not co-localized or co-localized with Krt8/18 labeling respectively with low or high doxycycline induction d Bar chart showing the similar percentage of tracing-reporter labeled cells in the whole mammary epithelial population 1 year after doxycycline pulsing at puberty tdTomato reporter for Bcl11b-lineage tracing (2 mg doxycycline) and GFP reporter for Krt14-lineage tracing (0.1 mg doxycycline) e Bar chart showing the composition of the Bcl11b-traced and Krt14-traced epithelia 1 year after pubertal pulsing in Bcl11b- and Krt14-lineage tracing f Bar chart showing the composition of Krt14 traced epithelial cells at day 17.5 of the 3rd pregnancy after pubertal pulsing at a high dose of doxycycline 2 mg per mouse g Fluorescence image showing the Krt14-traced positive clones in the mammary gland at day 17.5 of the 3rd pregnancy after pubertal pulsing at a high dose of doxycycline 2 mg per mouse i Schematic diagram showing the lineage tracing strategy for Krt14-positive cells from puberty in the presence of WT or KO of Bcl11b for experiments in j−s Krt14-rtTA/TetO-Cre/mTmG/Bcl11bwt/wt or Krt14-rtTA/TetO-Cre/mTmG/Bcl11bflox/flox mice at 5 weeks of age were pulsed by a high dose of doxycycline (2 mg) and then mated during adult stage for multi-pregnancy Mammary glands chased for various periods of time indicated during multiple pregnancies (1st Preg17.5 d 3rd Preg17.5 d) were harvested for analysis of GFP-labeled cells j Representative fluorescence images of GFP-positive clones in mammary glands at day 17.5 of the 3rd pregnancy in control or Bcl11b KO group after pulsing at puberty k Representative FACS plots showing percentage of GFP-labeled cells in basal or luminal compartments in control or Bcl11b KO group at day 17.5 of the 3rd pregnancy after pulsing at puberty l Bar chart showing the number of GFP-positive clones in all ten fat pads of each mouse in control or Bcl11b KO group at day 17.5 of the 3rd pregnancy after pulsing at puberty m–o Bar charts showing percentage of GFP-labeled cells in the whole epithelial population (m) percentage of GFP-labeled cells in basal or luminal subpopulations (n) and basal or luminal proportion in all GFP-labeled epithelial cells (o) in control or Bcl11b-KO group at day 17.5 of the 3rd pregnancy after pulsing at puberty p Representative images of GFP-positive clones with immunofluorescence staining of GFP and luminal marker Krt8/18 in mammary glands of control or Bcl11b-KO group based on Krt14-lineage tracing at day 17.5 of the 3rd pregnancy after pulsing at puberty The selected regions by the yellow boxes were split for different channels q Quantification of the uni-lineage or bi-lineage GFP clones judged by immunostaining as shown in p from control or Bcl11b-KO group at day 17.5 of the 3rd pregnancy after pulsing at puberty n = 3; for the counted clones of each mouse r Representative whole-mount carmine staining images of the thoracic mammary glands from control (Krt14-rtTA/TetO-Cre/mTmG/Bcl11bwt/wt) and Bcl11b-KO (Krt14-rtTA/TetO-Cre/mTmG/Bcl11bflox/flox) group at day 17.5 of the 1st pregnancy after a high dose of doxycycline (2 mg) induction at puberty The selected regions by the black frames were enlarged in the right panel s Quantification of the blank area without epithelial cells stained by carmine in the thoracic fat pads of control and Bcl11b-KO group displayed in r t Schematic diagram showing the existence of both bipotent and unipotent basal stem cells in pubertal mammary glands The Bcl11b+ bipotent stem cells are enriched in a CD200+CD34− basal subpopulation and locate densely in nipple while sparsely in duct and TEB And the Sema3a+ unipotent basal stem cells are enriched in a CD200−CD34+ basal subpopulation and locate specifically in TEB To assess the physiological significance of Bcl11b+ cells in mammary homeostasis, we analyzed the morphology of mammary gland during pregnancy after Bcl11b-KO. Loss of Bcl11b resulted in a sparser mammary tree with increased blank area on the fat pad compared with the control (Fig. 7r, s) these data demonstrate that mammary glands in adult and reproduction cycles are maintained by both bipotent and unipotent mammary stem cells which contribute to the integrity of mammary tissue in distinct modes This information helps to resolve the long-standing question that whether bipotent mammary stem cell exist in adult mammary gland It will contribute to building the postnatal mammary hierarchy in the field Our genetic tracing tools will facilitate the future cancer of origin research for various types of breast cancers Our data supported a model that adult mammary gland is sustained by various stem cell populations including Bcl11b+ bipotent stem cells and Sema3a+ unipotent stem cells these two stem cell populations may not be the only stem cells in the stem cell pool both Bcl11b+ and Sema3a+ cells are long-term survived stem cells; there should be at least short-term stem/progenitor cells or differentiated cells in the basal population that gradually diminish during the long-term tracing because we clearly observed a significant decrease of basal coverage in Krt14 tracing experiment during the first pregnancy The fate of these putative cell populations remains unclear and needs further investigated to find their markers As to whether basal cell specification depends on Sema3a+ stem cells we speculate that Sema3a+ cells are not the only unipotent mammary stem cells The unipotent mammary stem cells might be a very heterogeneous population with distinct roles we can comprehensively characterize the unipotent mammary stem cells population to reveal more mechanistic insights on mammary homeostasis Our study was mainly conducted with mouse models and it is still not clear whether it holds true in human breast tissues The quiescent bipotent stem cells that we traced with Bcl11b mouse model were not complete for all the Bcl11b+ cells therefore we might have missed those ‘deep quiescent’ cells in our tracing experiment we were not able to trace every cell population of interest due to the low frequency of these cells and low labeling efficiency we were not able to efficiently detect their expression in scRNA-seq analysis and not able to sort them out and directly conduct functional and characteristic analysis including their plasticity dynamics with aging The mammary stem cell population might be very heterogenous and there might be other bipotent and unipotent mammary stem cells that we did not have enough resources to cover it will be interesting to further dissect the details of the lineage hierarchy the inherent association between different cell populations and their significance in cancer initiation in both murine and human background All animal procedures reported herein were performed following the institutional guidelines and approved by the Institutional Animal Care and Use Committee at Westlake University (IACUC Protocol #19-001-CS) Rosa26-tdTomato (stock number 007909) mice were purchased from Jackson Laboratory And the Bcl11b-IRES-rtTA (or Bcl11b-rtTA) mice were made by Shanghai Model Organisms Center The frozen sperms of Sema3a-CreERT2 mouse (stock number T000-620) were purchased from GemParmatech Co the ages of the female mice used were indicated in the text And all the mice used for the Bcl11b-lineage tracing assay had a genotype of Bcl11b-rtTA (+ the mice had a genotype of Sema3a-CreERT2(+) tdTomato (+ +) with or without a homogeneous Bcl11b (loxp All the mice were housed in specific pathogen-free conditions and bred in the laboratory animal resources center of Westlake University Mice were administered with autoclaved food and water 2 mg doxycycline (100 µL 20 mg/mL doxycycline in sterile PBS Sangon Biotech) was injected intraperitoneally into the TetO-Cre/Bcl11b-rtTA/tdTomato mice once every day for three consecutive days starting at week 4 a single dose of 2 mg doxycycline (20 mg/mL) was injected intraperitoneally into the TetO-Cre/Krt14-rtTA/mTmG mice at week 5 For the Sema3a-lineage tracing experiments a single dose of 5 mg Tamoxifen (500 µL of 10 mg/mL tamoxifen in corn oil Sigma) was injected intraperitoneally into the Sema3a-CreERT2/tdTomato mice at week 5 To evaluate the instantaneous labeling efficiency the mammary glands were sampled 2 days after the last dose of induction and subjected to flow cytometry analysis To quantify the tracing progenies during different stages of mammary gland development 20 weeks and 1 year after doxycycline/tamoxifen induction To quantify the tracing outcomes during various stages of reproduction cycles the drug-treated mice were mated in adulthood and then sampled on the 17.5th day during the first We also did complete involution between pregnancies 1st Involution means the involution between the first and second pregnancy for 30 days and ‘Invo long’ indicates involution for more than 90 days The other involutions between pregnancies unless specially stated were all 30 days And ‘1st Lac7’ indicates lactation for 7 days we dissected the individual bright tdTomato+ Bcl11b clones in the mammary fat pads during multiple pregnancies (1st 2nd and 3rd Preg17.5 d) under the fluorescence stereomicroscope and then digested them into single cells and performed the clonal analysis of the composition of basal and luminal lineage by FACS Mammary glands were manually and mechanically minced into 1-mm size and digested with 0.5 mg/mL collagenase type 3 and 50 U/mL hyaluronidase (Stem Cell Technology) for 2 h with gentle pipetting every 30 min Digested mammary homogenate was spun down at 1500 rpm in Thermo Centrifuge (Thermo Scientific) followed by 5 mL ACK lysing buffer (Beyotime) treatment for 5 min on ice to remove erythrocytes mammary cells were digested by 5 mL 0.25% Trypsin-EDTA (GIBCO) for 3−5 min followed by brief DNase I (Worthington) digestion Dissociated mammary cells were filtered by 40-μm strainer to obtain single cell suspension mammary single cells were stained with CD45 (Biolegend) with appropriate conjugated fluorophores for 30 min on ice Then cells were washed and resuspended in HBSS + 2%FBS + 1%P/S + DAPI (1 μg/mL) at a density of 10 million/mL Stained samples were analyzed and sorted on BD FACSAriaTM Fusion (BD Bioscience) with a 100-μm nozzle To address the spatial heterogeneity of mammary gland duct and TEB fragments were dissected from the mammary glands of 5–6-week-old Krt14-cre/mTmG or wild-type (WT) C57BL/6 mice under the dissection microscope The tissue fragments were then digested with collagenase type3 (0.5 mL 3000 U/mL) at 37 ˚C for ~1.5 h until most of the large tissue pieces disappear Wash tissues with HBSS (containing 1% P/S) at least twice Add 300 μL TrypLE Express to digest the Nipples Add 1 mL HBSS (containing 2% FBS + 1% P/S) to neutralize TrypLE Wash at least once with HBSS (containing 1% P/S) Resuspend cells in HBSS (containing 2% FBS + 1% P/S + DAPI (1 μg/mL)) and then stain the cells with appropriate combination of antibodies 30 μL of growth factor reduced Matrigel (BD Bioscience) was added into the 96-well round-bottom plate and then solidified at 37 ˚C for 10 min CD200+CD34low and CD200+CD34− basal cells were resuspended in 200 μL culture media (DMEM/F12 + 2% FBS + 1% P/S + B27 + 10 mM HEPES) supplemented with EGF (10 ng/mL Plate was maintained in 37 ˚C incubator with 5% CO2 for 1−2 weeks mammary outgrowths obtained from the first transplantation were harvested and processed into a single-cell suspension Single-cell suspensions containing 4000 mammary cells from each mammary outgrowth were passaged to recipient mice for secondary transplantation 50–500 primary mammary cells or cultured epithelial cells of various populations were directly sorted into 400 μL Trizol (Life Technologies) RNA was extracted according to the manufacturer’s instruction with addition of ultrapure glycogen (Life Technologies) as carrier RNA was reverse transcribed to cDNA using SuperScript III First Strand Synthesis Kit (Life Technologies) according to the manufacturer’s instructions cDNA was preamplified 15–20 cycles according to the cell number using SybrGreen master mix (Applied Biosystems) and target gene primer pool designed by IDT (Integrated DNA Technologies) Preamplified cDNA was then subjected to the real-time PCR for specific gene target according to manufacturer’s instructions using Jena qTOWER384G The data were normalized to the expression of β-actin Then the value of one reference sample was set to 1 to facilitate inter-sample comparisons Reverse GTACAGGACTCTTTGCTCATCG; Igfbp2: Forward GAACATCTCTACTCCCTGCAC Paraffin sections and immunofluorescence For paraffin sections mammary gland was dissected and immediately fixed by 4% PFA for 2 h at 4 ˚C followed by PBS washing Dehydrated tissue was infiltrated by xylene solution and embedded with paraffin Tissue block was sectioned to 5 μm using Rotary Microtome Leica RM2255 (LEICA) paraffin section was de-paraffinized using xylene and rehydrated with gradient (100% 0%) ethanol solution and subjected to immunofluorescence staining Antigen was retrieved in citrate buffer (10 mM Sodium Citrate pH 6.0) boiled for 15 min in microwave at low-to-medium power Then sections were blocked with TBS + 2% BSA + 5% Donkey serum + 0.1%Triton X-100 for 1 h at RT and then stained with primary antibody including Rat-anti CD34(BD Biosciences) Rabbit anti-Mycn (Cell Signaling Technology) followed by 3× washing by TBST and secondary antibody staining Donkey anti-Rat Mouse or Goat 1:200 (Jackson ImmunoResearch) at RT for 1 h After 3× TBST washing and brief DAPI staining (1 μg/mL) sections were mounted with Fluoromount Aqueous Mounting Medium (Sigma) Seal coverslips with nail polish to prevent drying and movement under microscope The mammary glands were dissected and immediately fixed by 4% PFA at 4 ˚C overnight Then wash with 0.1%TBST (0.1% Triton X-100 in PBS) 3× 1 h Then stain DAPI (1 μg/mL) overnight in 1% TBST Block with 3% NCS (new born calf serum) in 1% TBST overnight 4 ˚C Stained with primary antibody in 1% TBST + 3% NCS + 0.03% NaN3 overnight at 4 ˚C Then take pictures of the tissue with a confocal microscope (FV3000 Data were processed and analyzed by Imaris software version 9.6 We define tdTomato-positive clone as a cluster of cells with bright tdTomato fluorescence with a clear boundary from the peripheral cells To count the number of clones formed during Bcl11b- and Sema3a-lineage tracing the mammary gland is regarded as an ellipse and its longest axis is divided into three equal parts The region closest to the nipple is called the proximal region the region farthest from the nipple is called the distal region and the region between them is called the middle region The areas of the three regions are denoted as S1 After initially counting the number of clones in each region the clone counts are normalized based on the different areas of the three regions In terms of the specific methods for clone type analysis The first approach involves directly using ophthalmic microsurgical scissors under a fluorescence stereo microscope to collect larger visibly tdTomato-positive clones from the mammary glands of pregnant mice or mice at other indicated time points after pubertal pulsing while minimizing the collection of surrounding tissue Each clone is then placed in a separate 1.5-mL centrifuge tube and digested into a single-cell suspension using enzymatic digestion The clone composition of basal and luminal lineage is analyzed using flow cytometry with relevant antibodies The second method is to analyze the composition of each individual clone regardless of size by performing immunofluorescence staining of basal and luminal markers for mammary sections of pregnant mice or virgin mice at designated time points after pubertal pulsing we observe and quantify the composition of basal and luminal cells in different tdTomato-positive clones in mice the 96-well plate was first incubated in 72 ˚C for 3 min and then transferred to ice immediately 2.85 μL of RT mixture containing 40 U SuperScript II reverse transcriptase (Invitrogen) and 1.75 μM template switch oligo (TSO) primer was added into the lysate The reverse transcription was conducted at 25 ˚C for 5 min 7.5 μL of PCR mixture containing 6.25 μL 2× KAPA HiFi HotStart ReadyMix (KK2602) 300 nM ISPCR oligo (AAGCAGTGGTATCAACGCAGAGT) and 1 μM 3’ Anchored oligo (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC) were added to each reaction The sample was amplified with initial denaturation at 95 ˚C for 3 min followed by 10–16 cycles of 98 ˚C for 20 s and 72 ˚C for 5 min; and finally 72 ˚C for 5 min Then the PCR products with different barcodes were pooled together and purified with DNA Clean & Concentrator-5 once (Zymo Research) eluted in 50 μL of H2O following 0.8× XP beads (Beckman the cDNAs were amplified with biotinylated index primer (/Biotin/CAAGCAGAAGACGGCATACGAGATindexGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC) and ISPCR oligo for an additional four to five cycles following purification with 0.8× Ampure XP beads again The biotinylated cDNAs were sonicated (COVARIS #SIAUH006) into ~300-bp fragments Dynabeads MyOne Streptavidin C1 Beads (Thermo Fisher Scientific) were used following manufacturer’s instructions Libraries were prepared using KAPA Hyper Prep Kits (KK8505) with end repair and adapter ligation by using NEB U-shaped adapter libraries were amplified 7−8 cycles followed by purification with 0.8x Ampure XP beads twice and eluted in 30 μL of H2O Quality was checked by Fragment Analyzer-12/96 (GENE-QC006) the libraries were sequenced on a Novaseq platform to generate 150-bp paired-end reads (sequenced by Novogene) Adaptors of raw reads were trimmed by TrimGalore The information of cell barcode and UMI were recognized and extracted from reads1 by using UMI-tools and added to reads2 Those reads2 were mapped to reference genome by STAR with default parameter except for outFilterMultimapNmax = 1 Then we used featureCount to assign mapped reads and used UMI-tools to produce a count matrix We kept the cells whose barcodes exist in the barcode library We got the final dataset containing 1419 cells with a median of 36,842 UMIs and 3198 genes so we filtered out the cells with ERCC of more than 5% We also filtered out the poor quality cells with UMI numbers higher than 120,000 or less than 5000 and unique gene counts over 6000 or less than 200 R package Seurat v3.6 was used to normalize and scale data and downstream including dimensionality reduction We regressed out the ERCC percentage effect We used the top 2000 high variable features to do dimensionality reduction and used the first 14 principles components to run RunTSNE We used R package clusterProfiler to do the pathway enrichment analysis We enriched the significantly changed pathway with FDR < 0.05 The expression of some marker genes was plotted by Seurat or ggplot2 R package we successfully identified the Bcl11b regulon regulated by the Bcl11b’s transfac_pro__M05956 motif By computing the activity of this regulon in our dataset we observed results consistent with our previous computational methods Graphad Prism 9 was used for statistical analysis Data were compared between two groups of samples using the unpaired Sample size for each experiment was determined based on a minimum of n = 3 independent devices for each experimental group Image J and Photoshop were used for image analysis And Adobe Illustrator CS6 was used for image layout and editing The data that support the findings of this study are available from the corresponding author upon reasonable request Source data are provided in the manuscript Singel-cell data were deposited in GEO with the access number GSE251933 Z Hormonal and local control of mammary branching morphogenesis WT Mammary development in the embryo and adult: a journey of morphogenesis and commitment JJ Key stages of mammary gland development: molecular mechanisms involved in the formation of the embryonic mammary gland JE Stem cells and the differentiation hierarchy in mammary gland development JE Keeping abreast of the mammary epithelial hierarchy and breast tumorigenesis J Mammary stem cells and the differentiation hierarchy: current status and perspectives R Developmental stage and time dictate the fate of Wnt/beta-catenin-responsive stem cells in the mammary gland R Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture Identification of multipotent mammary stem cells by protein C receptor expression Identification of quiescent and spatially restricted mammary stem cells that are hormone responsive Developmental stage-specific contribution of LGR5(+) cells to basal and luminal epithelial lineages in the postnatal mammary gland GM Lgr5 is a marker for fetal mammary stem cells but is not essential for stem cell activity or tumorigenesis Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche A Quiescent Bcl11b high stem cell population is required for maintenance of the mammary gland Molecular hierarchy of mammary differentiation yields refined markers of mammary stem cells Distinct stem cells contribute to mammary gland development and maintenance DS Identification of stem cell units in the terminal end bud and duct of the mouse mammary gland Z Candidate regulators of mammary branching morphogenesis identified by genome-wide transcript analysis Identity and dynamics of mammary stem cells during branching morphogenesis Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny Heterotypic cell-cell communication regulates glandular stem cell multipotency Reconstruction of dynamic mammary mini gland in vitro for normal physiology and oncogenesis W Lineage-biased stem cells maintain estrogen-receptor-positive and -negative mouse mammary luminal lineages Lineage-restricted mammary stem cells sustain the development and regeneration of the estrogen receptor positive lineage JE In situ identification of bipotent stem cells in the mammary gland Aging-associated alterations in mammary epithelia and stroma revealed by single-cell RNA sequencing Single cell transcriptome atlas of mouse mammary epithelial cells across development MJ Integrating single-cell RNA-sequencing and functional assays to decipher mammary cell states and lineage hierarchies Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling Differentiation dynamics of mammary epithelial cells revealed by single-cell RNA sequencing S An improved CUT&RUN method for regulation network reconstruction of low abundance transcription factor a marker for hematopoietic progenitor and stem cells I High expression of CD200 and CD200R1 distinguishes stem and progenitor cell populations within mammary repopulating units Characterization and isolation of stem cell-enriched human hair follicle bulge cells CD157 marks tissue-resident endothelial stem cells with homeostatic and regenerative properties Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells Genetic targeting of Purkinje fibres by Sema3a-CreERT2 LR s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity Dual role of COUP-TF-interacting protein 2 in epidermal homeostasis and permeability barrier formation Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer Progressive senescence programs induce intrinsic vulnerability to aging-related female breast cancer Early lineage segregation of multipotent embryonic mammary gland progenitors SCENIC: Single-cell regulatory network inference and clustering Download references Daijin Wu at the Westlake Animal Facility for mouse husbandry Jinhui Li and Yanan Tang at the Flow Cytometry Facility of Westlake University for assistance in FACS sorting Yan Wang for the generous gift of -tdTomato reporter mice Pei Cai and Lei Yuan for valuable discussions and suggestions Fang Xiao at the Microscopy Core Facility of Westlake University for tissue immunofluorescence imaging We thank Zhenzhen Yu at the General Equipment & Autoclave Service Core Facility of Westlake University for experimental equipment management This work was supported by National Key Research and Development Program of China (2024YFA1107400) the National Natural Science Foundation of China (32170803 This work was also supported by the Westlake Education Foundation These authors contributed equally: Zuobao Lin Westlake Laboratory of Life Sciences and Biomedicine Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province conceived the study and experimental design; Z.L. performed certain immunofluorescence analysis Download citation DOI: https://doi.org/10.1038/s41421-025-00794-0 Metrics details Development of breast cancer is linked to altered regulation of mammary gland developmental processes A better understanding of normal mammary gland development can thus reveal possible mechanisms of how normal cells are re-programmed to become malignant E2Fs 1-4 are part of the E2F transcription factor family with varied roles in mammary development but little is known about the role of E2F5 A combination of scRNAseq and predictive signature tools demonstrated the presence of E2F5 in the mammary gland and showed changes in predicted activity during the various phases of mammary gland development Testing the hypothesis that E2F5 regulates mammary function we generated a mammary-specific E2F5 knockout mouse model resulting in modest mammary gland development changes after a prolonged latency the E2F5 conditional knockout mice developed highly metastatic mammary tumors Whole genome sequencing revealed significant intertumor heterogeneity RNAseq and protein analysis identified altered levels of Cyclin D1 suggesting that E2F5 conditional knockout mammary glands and tumors may be dependent on Cyclin D1 Transplantation of the tumors revealed metastases to lymph nodes that were enriched through serial transplantation in immune competent recipients we propose that loss of E2F5 leads to altered regulation of Cyclin D1 which facilitates the development of metastatic mammary tumors after long latency this study demonstrates that conditional loss of E2F5 in the mammary gland leads to tumor formation revealing its role as a transcription factor regulating a network of genes that normally result in a tumor suppressor function Here we have hypothesized that E2F5 plays an essential role in mammary gland development we generated mice with a conditional ablation of E2F5 in the mammary gland In these mice we observed alterations to development and after a long latency the transplantable tumors from these mice develop lymph node metastases a trait not widely reported for other genetically engineered mouse models of breast cancer Using a mouse mammary scRNAseq dataset that was split to various functional stages (A) including nulliparous (Null P) pregnancy day 14.5 (Preg D14.5) lactation day 6 (Lact D6) and involution day 11 (Inv D11) the level of E2F1 (B) and E2F5 (C) expression was plotted Using a separate scRNAseq dataset that was not sorted for epithelial cells (D) expression of E2F5 was examined across cell populations in the involuting mammary gland fibroblasts and alveoli with elevated signal in red and lower signaling in green (E) we overlaid E2F1 and E2F5 expression with elevated expression in green HMECs were infected with increasing multiplicity of infection (MOI) for an adenovirus expressing GFP or E2F5 A western blot demonstrated increasing levels of E2F5 with increasing MOI (G) Generation of a signature for E2F5 activation revealed genes up (orange/red) and down (blue) regulated The activity of the signature was predicted in several human breast cancer cell lines and was plotted against the observed levels in a western blot for E2F5 revealing correlation between the two (H) A mammary gland developmental dataset (Stein et al. 2004) was limited to the E2F5 signature genes and was clustered revealing that genes regulated by E2F5 stratified mammary developmental stages (I) A gene targeting strategy to flank exons 2 and 3 of E2F5 with loxP sites was employed with genotyping primers shown (A) With the introduction of Cre Recombinase under the mammary epithelial specific control of the MMTV promoter/enhancer exons 2 and 3 are lost resulting in a tissue specific knockout Examining ductal extension through wholemounts at 4 weeks we examined 20 control mice (E2F5flox/flox) (B) and 14 E2F5 CKO mice (C) This delay was quantified revealing a consistent outgrowth delay virgin mammary glands were assessed by both wholemount and histology Relative to the MMTV-Cre controls (E) with their somewhat spiked ductal appearance the E2F5 CKO mammary glands resembled a lactating mammary gland with alveoli engulfing the entire fat pad (F) Regular palpation of the mammary glands revealed the onset of tumor formation in E2F5 CKO virgin and multiparous mice The resulting tumors were histologically diverse with numerous patterns noted a pulmonary section reveals numerous metastatic lesions at both low and high power (F) normal lung (center panel) and EMT (right panel) Applying the E2F5 signature to human breast cancer stratified the patients to high/low quartiles These results were consistent with the mouse model as low E2F5 activity was associated with worse survival (G) these data suggest that E2F5 expression and activity normally has a protective role in human breast cancer Whole genome sequencing on five E2F5 CKO tumors were generated An example of a Circos plot generated for a single tumor provides a bird eye view of the genetic alteration identified (A) Starting from the outermost region of each plot an ideogram with labelled mouse chromosomes is first followed by SNVs at the next innermost ring and high (red) predicted impact as defined by Mutect2 annotation The next innermost ring contains all predicted CNVs as defined by the consensus of Delly and Lumpy; deletions are colored blue and duplications are colored red the height of CNVs are scaled relative to the CNV with the largest amount of evidence supporting it as determined by Lumpy annotation (e.g a duplication with 40 pieces of evidence will only be half the height of a deletion with 80 pieces of evidence) The width of each CNV is determined by the start and stop positions determined by the consensus of Delly and Lumpy on the length of the genome Duplications point outward while deletions point inward starting from a shared midpoint The innermost ring contains predicted high impact translocations and high to moderate impact inversions by the consensus of Delly and Lumpy calls Translocation color matches the ideogram color of one of the two chromosomes involved in the event For all CNV and SNVs found in at least 3 tumors an oncoprint style plot was generated with SNV/CNV per tumor shown above and the SNV/CNV per gene shown at right only the high confidence calls are presented (B) Copy number alterations found in the tumors are illustrated with each column representing a chromosome location (C) Red indicates amplification while blue indicates deletion A closer examination at the CNVs located on Chromosome 6 revealed some shared events between the tumors (D) The boxed region in (D) is expanded for the three tumors containing an amplification at that point in panel E The COSMIC SBS mutation signatures enriched among the tumors were identified and are presented in panel F Implantation of E2F5 CKO tumors into FVB MMTV-Cre recipients resulted in mammary and axillary tumor formation The strategy for serial transplantation of E2F5 CKO axillary tumors into the abdominal mammary gland to enrichlymphatic metastasis is shown (A) the primary tumor has been excised (abdominal gland bottom of panel) but a tumor has also formed in the region of the axial lymph node (top) (B) Labels include ALNT (axial lymph node tumor) LV (enlarged lymphatic vessel) and the remnants of the excised primary tumor (PT) in the abdominal gland Cross section of the lymph node reveals both lymph (left side) tissue and metastatic (right side) cells (C) Staining with a pan-cytokeratin antibody reveals nests of metastatic cells throughout the lymph node (D) Cross section and staining of the enlarged vessel from (B) for podoplanin reveals a positively staining lymphatic vessel containing counterstained tumor cells (E) A summary for four transplanted lines shows the number of rounds of transplantation and where the optimal lymph node enrichment point was observed with the percent of tumor bearing mice with lymph node metastasis included for each (F) Examining human breast cancer with known lymph node status for the E2F5 activation gene signature revealed that metastatic tumors had lower E2F5 activity (G) the samples with the lowest levels of E2F5 were most likely to have lymph node metastasis (P < 0.0001 Together these data indicate that loss of E2F5 is associated with tumors that metastasize to the lymph node in both our conditional knockout mouse model as well as in human breast cancer Together our data and the literature suggest that E2F5 functions to regulate development in a context dependent manner and other E2Fs may compensate for loss of E2F5 and mitigate the developmental abnormalities Given the functional redundancy and shared binding motif between E2F family members it is likely that E2F5 can also regulate Cyclin D1 expression we propose that loss of E2F5 in the mammary gland leads to deregulation of Cyclin D1 contributing to tumor development and progression Given the wide range of SNV and CNV alterations we suggest that this occurs in a complex mutational environment with numerous other pathways Although there is evidence suggesting that E2F5 may be directly Cyclin D1 expression it is also possible that disruption of E2F5 leads to dysregulation of other targets that can result in Cyclin D1 expression To confirm the role of Cyclin D1 in E2F5 CKO mice tumorigenesis our future studies will characterize the effects of loss of Cyclin D1 in the E2F5 CKO model which revealed a unique mutational spectrum the E2F5 CKO tumors share the Cyclin family pattern with Neu tumors This suggests some similarity to the Neu tumors but the mutational analysis also provides key differences this model of enriched lymph node tumors is unique and can be a tool to examine the mechanisms driving lymph node metastasis it should be noted that this transplantation technique includes a 1 x 1x 1 mm fragment of the tumor which includes cells from the microenvironment including other cell types such as fibroblasts endothelial cells and a variety of immune cells and this may alter the properties and signaling of the cancer cells We theorize that like E2F8 and other E2F family members E2F5 may behave as an oncogene or tumor suppressor depending on context this context may be dependent upon tumor type and on when the dysregulation of E2F5 occurs Since the majority of available transgenic mouse models develop tumors rapidly it does not allow time for age-related changes to occur which has an average tumor latency of 18–21 months may be a favorable model to elucidate the role of aging in breast cancer development and progression the E2F5 CKO syngeneic transplantation model can be a significant resource to studying the mechanism of lymphatic metastasis To analyze E2F5 expression in a single cell RNA-seq mammary development dataset, the following website was used: https://marionilab.cruk.cam.ac.uk/mammaryGland/. To analyze E2F5 expression in various mammary cell types, the following website was used: https://bis.zju.edu.cn/MCA/ The following published microarray datasets were used for analysis: terminal end bud and duct (GSE2988) and mammary gland developmental stages (GSE12247) Newly generated RNAseq and WGS data has been deposited at the NIH NLM under BioProject # PRJNA887715 Human Mammary Epithelial cells were infected with adenovirus expressing E2F5 or GFP Cells were collected eighteen hours after infection Total RNA was extracted using Qiagen RNeasy Mini kit and submitted to Michigan State University Genomics Core facility for gene expression analysis using Affymetrix Human Genome U133 chip Robust Multichip Average (RMA) algorithm was used to normalized microarray dataset Significance Analysis of Microarray was used to identify differentially expressed genes in HMEC-E2F5 E2F5 activation signature was generated based on previously described bayesian approach [40] The E2F5 activation signature was applied to publicly available human breast cancer cell line data set GSE3156 E2F5 expression was evaluated in two cell lines with the highest predicted E2F5 activity and two cell lines with the lowest predicted E2F5 activity via Western blot The subset of E2F5 regulated genes enriched in each mammary developmental stage were characterized by gene ontology using https://maayanlab.cloud/Enrichr/ (Kuleshov et al Mice were monitored weekly for tumor development The endpoint for primary tumor was 2000 mm3 abdominal mammary fat pads were excised and placed on glass slides The slides were incubated in acetone for 24 h rehydrated through an ethanol progression and stained in Harris’ Modified Hematoxylin for 24 h The slides were destained in acidified ethanol Slides were dehydrated and mounted with permount the distance from the nipple to the leading edge of the epithelium and the distance from the nipple to the midpoint of the thoracic lymph node were measured Samples for histology were fixed in 10% formalin and submitted to Michigan State University Pathology lab Tabular data from tumor VCF files were sorted and processed using a custom Python script using Python 3.8.8 All unaltered VCF files are available in supplementary Circos plots were generated using Circos/0.69-6 [79] All SNVs found in either FVB background were filtered out from the tumor data Only SNVs with a somatic p-value <= 0.05 were included in all downstream analyses as determined by VarScan CNVs and inversions found in the FVB/NJ background that are on the same chromosome and have a difference of less than 100 bps in length from either FVB background event are excluded from analysis all events on either FVB background that are within 1 kb of either the donor or receiver position on each chromosome are dropped These steps were accomplished using a custom python script The mouse GRCm38 (mm10) reference assembly was used for trinucleotide context when calling SBS signatures The resulting weights were plotted using matplotlib in Python Human mammary epithelial cells were cultured in Mammary Epithelial Cell Basal Medium (ATCC PCS-600-030) supplemented with rH-insulin (5 ug/mL) extractP (0.4%) and hydrocortisone hemisuccinate (100 ng/ml) Tumor cell lines were validated for tumorigenic potential by implantation into recipient (MMTV-Cre) lines where they were noted to form tumors The following primers were used (5’ to 3’): CCND1 forward Primer efficiency was 90–110% for all primers used Delta-delta CT method was used for fold change analysis 3 control and 3 conditional knockout samples were used and experiments were done in triplicate samples were homogenized using mortar and pestle in liquid nitrogen Sample were lysed in RIPA lysis buffer (Thermo Scientific 89900) with proteinase inhibitor (Thermo Scientific A32963) for 1 h on ice with constant agitation Protein was quantitated using BCA Protein Assay Kit (Thermo Scientific 3225) and then boiled at 100 °C for 5 min Samples were loaded onto an 8–12% polyacrylamide gel Separated protein was transferred onto an Immobilon- FL PVDF membrane (Millipore Sigma PFL00010) Membranes were blocked in 5% milk in TBS with 0.1% Tween-20 (TBS-Tween) for 1 h and then incubated in primary antibody overnight at 4 °C Following three washes in TBS-Tween the membrane was incubated in the appropriate antibody at a dilution 1:10,000 in 5% milk in TBS-Tween for 1 h at room temperature Membranes were washed 3x in TBS-Tween and imaged on LI-COR Odyssey imaging system The following antibodies were used: 1:100 E2F5 (Santa Cruz Biotech sc-374268) 1:1000 Grb2 (Cell Signaling Technology 3976) 1:4000 Vinculin (Cell Signaling Technology 13901) 1:2000 Cyclin D1 (Thermo Scientific 516356.) 1:2000 Cyclin D3 (Thermo Scientific PA5-97551) and 1:1000 Cyclin D2 (Proteintech 10934-1-AP) Image studio version 5.5 was used for analysis and quantification of the Western blot E2F5 CKO mammary tumors were harvested and stored in DMEM with 20% FBS and 10% DMSO at −80 °C before long term storage in liquid nitrogen vapor phase Tumors were thawed and orthotopically implanted into the abdominal mammary gland of 6-to-10-week old MMTV Cre female mice Mice were palpated 2x a week for mammary tumor formation samples were harvested for further analysis Graphpad was used to create the survival plot in patients with high vs low E2F5 activation Broad Institute’s Morpheus was used to generate the heatmap demonstrating E2F5 activation in human breast cancer patient and its lymph node metastasis status all statistical comparisons are performed with an unpaired students two-tailed Data is freely available and bioinformatic data has been deposited at BioProject # PRJNA887715 Involution of the mouse mammary gland is associated with an immune cascade and an acute-phase response Gene expression profiling of mammary gland development reveals putative roles for death receptors and immune mediators in post-lactational regression The regulation of E2F by pRB-family proteins Conserved functions of the pRB and E2F families Signaling networks that link cell proliferation and cell fate The E2F family: specific functions and overlapping interests Expression of transcription factor E2F1 induces quiescent cells to enter S phase E2F2 and CREB cooperatively regulate transcriptional activity of cell cycle genes Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A An E2F1-dependent gene expression program that determines the balance between proliferation and cell death E2f3b plays an essential role in myogenic differentiation through isoform-specific gene regulation E2f1-3 switch from activators in progenitor cells to repressors in differentiating cells E2F-1 functions in mice to promote apoptosis and suppress proliferation Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation leading to the development of autoimmunity E2f3 is critical for normal cellular proliferation Loss of E2F4 activity leads to abnormal development of multiple cellular lineages nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting Patterns of cell signaling pathway activation that characterize mammary development Compensation and specificity of function within the E2F family Transcription factor compensation during mammary gland development in E2F knockout mice Prediction and Genetic Demonstration of a Role for Activator E2Fs in Myc-Induced Tumors Increased metastasis with loss of E2F2 in Myc-driven tumors The E2F transcription factors regulate tumor development and metastasis in a mouse model of metastatic breast cancer HER2/Neu tumorigenesis and metastasis is regulated by E2F activator transcription factors Conserved E2F mediated metastasis in mouse models of breast cancer and HER2 positive patients Distinctions in the specificity of E2F function revealed by gene expression signatures Myc requires distinct E2F activities to induce S phase and apoptosis E2F2 suppresses Myc-induced proliferation and tumorigenesis Inactivation of E2f1 enhances tumorigenesis in a Myc transgenic model E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control Differential activities of E2F family members: unique functions in regulating transcription Comparative analysis of E2F family member oncogenic activity A potential oncogenic role of the commonly observed E2F5 overexpression in hepatocellular carcinoma Human E2F5 gene is oncogenic in primary rodent cells and is amplified in human breast tumors Overexpression of E2F-5 correlates with a pathological basal phenotype and a worse clinical outcome Mapping the Mouse Cell Atlas by Microwell-Seq Oncogenic pathway signatures in human cancers as a guide to targeted therapies Candidate regulators of mammary branching morphogenesis identified by genome-wide transcript analysis Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis Targeted disruption of ErbB2/Neu in the mammary epithelium results in impaired ductal outgrowth Histological subtypes of mouse mammary tumors reveal conserved relationships to human cancers Survival analysis across the entire transcriptome identifies biomarkers with the highest prognostic power in breast cancer Enrichr: a comprehensive gene set enrichment analysis web server 2016 update Genes that mediate breast cancer metastasis to the brain A multigenic program mediating breast cancer metastasis to bone Genes that mediate breast cancer metastasis to lung Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway A pathway-based classification of human breast cancer E2f5 is a versatile transcriptional activator required for spermatogenesis and multiciliated cell differentiation in zebrafish The E2F5 repressor is an activator of E6/E7 transcription and of the S-phase entry in HPV18-associated cells A combinatorial mechanism for determining the specificity of E2F activation and repression Expression and amplification of cyclin genes in human breast cancer Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice Use of MMTV-Wnt-1 transgenic mice for studying the genetic basis of breast cancer Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease Specific protection against breast cancers by cyclin D1 ablation Cyclin D3 compensates for the loss of cyclin D1 during ErbB2-induced mammary tumor initiation and progression Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition of cyclin D1 promoter activity through E2F and Sp1 Integrated analyses of murine breast cancer models reveal critical parallels with human disease Studying Lymphatic Metastasis in Breast Cancer: Current Models MicroRNA-154 inhibits growth and invasion of breast cancer cells through targeting E2F5 Synergistic functions of E2F7 and E2F8 are critical to suppress stress-induced skin cancer E2f8 mediates tumor suppression in postnatal liver development Park SA, Platt J, Lee JW, López-Giráldez F, Herbst RS, Koo JS. E2F8 as a Novel Therapeutic Target for Lung Cancer. J Natl Cancer Inst. 2015;107.https://doi.org/10.1093/jnci/djv151 E2F8 contributes to human hepatocellular carcinoma via regulating cell proliferation Mouse Models of Breast Cancer Share Amplification and Deletion Events with Human Breast Cancer Trimmomatic: a flexible trimmer for Illumina sequence data Fast and accurate short read alignment with Burrows-Wheeler transform The Sequence Alignment/Map format and SAMtools The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing DELLY: structural variant discovery by integrated paired-end and split-read analysis LUMPY: a probabilistic framework for structural variant discovery A program for annotating and predicting the effects of single nucleotide polymorphisms SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3 Circos: an information aesthetic for comparative genomics CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing DeconstructSigs: delineating mutational processes in single tumors distinguishes DNA repair deficiencies and patterns of carcinoma evolution RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome STRING: known and predicted protein-protein associations integrated and transferred across organisms A meta-analysis of gene expression-based biomarkers predicting outcome after tamoxifen treatment in breast cancer Download references This work was supported by a grant from the Elsa U Pardee Foundation and NICHD 1R01HD104606-01 to ERA and Aitch Foundation scholarships to BT and JPR J-RJ was involved in methodology and investigation RK and JH were involved in analyzing data and investigation JN was involved in conceptualization and ERA was involved in conceptualization Download citation DOI: https://doi.org/10.1038/s41388-024-03172-4 Metrics details Model organisms are vital for biomedical research and drug testing but face high costs While newer techniques like organoids and assembloids have shown improvements they still remain inadequate in addressing all research needs we present a new method for maintaining the prostate gland of the earthworm Eudrilus eugeniae ex vivo and examine its potential for regeneration and drug screening We successfully maintained the earthworm prostate gland in cell culture media for over 200 days with observed beating behavior confirming its viability Apoptotic staining and histological analysis show no significant changes indicating that the prostate gland remains stable significant overexpression of H3 and H2AX on the 10th and 50th days suggests stem cell proliferation and differentiation Alkaline phosphatase expression analysis confirmed that the stem cell niche is localized to the anterior region the posterior region of the prostate gland demonstrated significant regenerative capacity with complete regeneration occurring within 45 days following amputation treatment with valproic acid enhanced posterior regeneration leading to full restoration within 12 days This study confirms the feasibility of maintaining the prostate gland of earthworms in an ex vivo setting providing a valuable model for studying regeneration and conducting drug screening there have been remarkable advancements and innovations in basic science and medical research greatly enhancing our understanding of various biological aspects and disease progression Animal model systems have played a significant role in these contributions we still face challenges in comprehending and finding solutions for complex multifactorial diseases like cancer It’s crucial to recognize that these models also have their own limitations and drawbacks which leads us to question their appropriateness and accessibility in the modelling process and potential alternative model system that allows us to test potential drugs and ultimately minimizes the time for drug entry into preclinical trials when compared to other ex vivo models Achieving this level of biomimicry requires sophisticated techniques in tissue engineering An ex vivo prostate model offers a unique platform for high-throughput drug screening providing a more accurate and ethical alternative to animal models It allows for the direct assessment of drug efficacy and toxicity in a controlled environment that closely mimics human physiology This has the potential to accelerate the development of new therapies for prostate diseases and reduce the time and cost associated with drug discovery we developed a method to maintain ex vivo prostate organs from the invertebrate earthworm Eudrilus eugeniae and used it as a model to study regeneration and drug screening (A) The mature worm consists of the (1) pre-clitellum (B) The prostate gland is located in-between the 18th and 23rd segments The prostate gland processing for ex vivo maintenance The dissected glands are collected in distilled water then rinsed with 1X PBS and an antibiotic solution the glands are placed in L15 media containing 10% FBS They are then carefully maintained under sterile conditions in an incubator at 23 °C Stereo microscopic observation of ex vivo organ at different time periods. (A) 10th day of ex vivo organ (B) 45th day of ex vivo organ (C) 115th day of ex vivo organ (D) 157th day of ex vivo organ (E) 175th day of ex vivo organ (F) 200th day of ex vivo organ prostate (Note: Scale bar may not be consistent across images). (A) Alkaline phosphatase signal at anterior tip (stained blue) (B) signal at the anterior duct tissue of prostate (stained blue) Ex vivoprostate gland posterior regeneration the healing process commences with epithelial tissue beginning to cover the wound forming a protective layer around the wound the amputated part is completely regenerated The dotted arrow and circle highlight the amputation site and the observed changes (B) Tissue growth at the tip of the duct region in the control group S5) and their presence on the outer tissue layer of the prostate increased significantly particularly in the amputated region (Suppl Comparison of the amputated regions reveals the presence of bud-like structures at the 25th DPA confirming their ability not only for wound healing but also for ex vivo regeneration (Suppl The increasing cell count reflects the progression of regeneration and is directly proportional to the regenerative process Formation of mini organ like structure (A) Cells attaching and the presence of grouped cells (indicated by arrow) with their fluorescent nature (B) Grouped cells exhibiting enhanced fluorescence intensity (C) Formation of tube-like structures between grouped cells (D) Assembling of two different organoids (pink arrow) through a tube-like structure (blue arrow) (E) Base of the tube-like structure (pink arrow) with cells attaching onto it (blue arrow) (F) Fluorescent nature of the tube-like base and the attached cells (G) Progression towards the formation of a mini prostate organ (I) Similar flagella-like structure observed between the outer cells of the mini organ and the initial cells attached to culture plate from the real ex vivo organ Stereo microscopic observation of valproic acid on ex vivo prostate regeneration Significant changes were observed in the valproic acid treated wound region at 48 h with complete regeneration occurring within 12 days when compare to control Incorporating heat-inactivated coelomic fluid from earthworms into culture media could potentially complement FBS by providing earthworm-specific nutrients that may enhance the ex vivo maintenance of earthworm organs further detailed investigation is needed to fully understand the molecular and metabolic stability of ex vivo organ While detailed molecular studies are still needed to understand the underlying mechanisms this ex vivo model provides a valuable foundation for advancing our understanding of the cellular processes governing earthworm prostate regeneration and may also offer insights into mammalian prostate regeneration further investigation into the detailed molecular mechanisms is necessary This functional ex vivo whole organ model not only helps assess the regenerative potential of drugs but also offers a platform for screening treatments for other conditions any potential drug should also be tested in higher animal models before being considered for clinical applications This study outlines a method for developing a comprehensive ex vivo organ model The ex vivo functional prostate organ demonstrates remarkable regenerative potential this study represents the first confirmation of the existence of organ-specific or pluripotent stem cells within the earthworm prostate and demonstrated the cellular mechanism of the regeneration This breakthrough opens avenues for in-depth insights into stem cell behavior and the mechanisms of organ regeneration our approach offers significant advancements and proves invaluable in establishing models for prostate-associated diseases and drug screening This ex vivo organ model system holds a higher degree of reliability compared to existing in vitro models effectively bridging the gap between cell cultures and animal models for the generation of new knowledge eugeniae earthworms (weighing approximately 650 mg) are taken and washed with water twice to remove the soil The worms are then anesthetized using ice-cold water for dissection They are inverted and placed on a dissection board Amputation is performed at the 17th and 30th segments of the worms These amputated segments are cross-cut in a longitudinal manner starting from the middle of the ventral side towards the dorsal side This allows for the visualization and removal of two prostate glands which are connected to other organs and tissues of the worm and attached to the earthworm skin tissue at the 17th segment The connective tissue is carefully amputated using a sterile surgical blade ensuring the prostate gland remains undisturbed Once the prostate glands are successfully removed they are immediately transferred to ice-cold water and washed thoroughly for 2 min twice in a cell culture Petri dish they are transferred to a new petri dish and washed with ice-cold 1X PBS for 5 min all procedures should be performed under sterile conditions) the glands are washed twice with ice-cold 0.1% antibiotics solution (consisting of Ampicillin they are washed with 0% L15 medium for 30 s and carefully transferred to L15 cell culture media containing 10% FBS The petri dish is then placed in an incubator at a temperature of 23 °C (Earthworm body temperature is influenced by the external environment we maintained the earthworm organ at the same temperature used for the earthworm culture to ensure consistency with their natural environmental conditions) the organ can be kept alive and maintained without contamination for an extended period The processed glands are maintained in L15 media The ex vivo organs are monitored twice daily using an inverted EVOS light microscope (ThermoFisher MA) and stereo fluorescent microscope (Leica MZ16FA) The survival and stability of the organs are recorded as images and videos The ex-vivo prostate glands are washed with distilled water 1 µl of H33342 Bis-benzamine is added to 1 ml of 1X PBS and mixed well this mixture is added to a 30 mm petri dish and the prostate gland is placed in it and incubated in dark conditions for 10 min 1 µl of Merocyanine 520 is added in the same dark conditions and incubated for 10 min The color intensity of the cells is observed under an inverted fluorescence microscope The Merocyanine 520-stained gland cells appear in red color while Bis-benzamine H33342 appears in blue color The glands are stained using Hematoxylin and Eosin (H&E staining) following a standard protocol the glands are fixed in 10% formalin for 24 h they are dehydrated using a gradient increase in isopropyl alcohol (from 60 to 100%) the glands are embedded in paraffin and sliced into 6 μm thin sections These sections of the prostate gland are placed on glass slides The sections are deparaffinized using xylenes and then stained with hematoxylin and eosin The DPX-mounted slides are examined under a light microscope The prostate gland is taken and homogenized using 2X protein sample buffer protein sample is resolved on a 12% SDS-PAGE gel The resolved proteins on the gel are transferred to a PVDF membrane The membranes are incubated with different primary antibodies H2AX (stem cell proliferation and differentiation) and β-actin (reference protein) This is followed by incubation with a horseradish peroxidase (HRP) conjugated secondary antibody (Abcam) at a dilution of 1:10,000 The membrane is then thoroughly washed for X-ray film development which is a chemiluminescent substrate used for detecting HRP is added to the well-washed blotted PVDF membrane and incubated for 1 min The membranes are then exposed to transfer the protein bands onto an X-ray film placed inside a cassette the X-ray films are dipped a few times in a developer solution and washed with water They are then dipped in a fixer solution and washed with water again and the observed protein bands are documented Once the glands are dissected from the earthworm they are maintained in an ex-vivo condition for approximately 48 h to allow for adaptation The glands are then divided into two groups the posterior lobe tip of the glands is amputated using a sterile razor blade while the control group does not undergo this procedure the amputated glands are washed with 0% L15 media for 10 s the glands are maintained in 10% FBS containing L15 medium and the glands are observed for the following days using microscope and documented The glands are first washed with ice-cold 1X PBST (Phosphate Buffered Saline with Tween-20) They are then fixed with ice-cold methanol for 15 min the glands are washed twice with 1X PBST and incubated with a KCL solution for 15 min they are washed again with 1X PBST and incubated with a permeabilization solution for 15 min the glands are stained with BCIP/NBT (5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium) (Sigma Aldrich -Cat the glands are washed three times with 1X PBST they are washed with an acid solution consisting of acetic acid The glands can then be observed under a microscope Once the organs have adapted to the ex vivo condition the glands are amputated to assess the regeneration potential in the presence of valproic acid the glands are maintained in 10% L15 media containing 500 µg of valproic acid per 2 ml and fresh media containing 500 µg of valproic acid is added each time All experiments were repeated multiple times to ensure reliability Statistical analysis was performed using SAS with a p-value of < 0.05 considered statistically significant (*p < 0.05) Additional analyses were conducted using GraphPad Prism 8.0 software (San Diego Data is provided within the manuscript or supplementary information files Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery Zebrafish as an animal model for biomedical research The mouse ascending: perspectives for human-disease models Screening out irrelevant cell-based models of disease The genome of Schmidtea mediterranea and the evolution of core cellular mechanisms Prospectively isolated tetraspanin + neoblasts are adult pluripotent stem cells underlying planaria regeneration Creation of bladder assembloids mimicking tissue regeneration and cancer Human brain organoids assemble functionally integrated bilateral optic vesicles Human organoids: model systems for human biology and medicine Genome and single-cell RNA-sequencing of the earthworm Eisenia andrei identifies cellular mechanisms underlying regeneration A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity Assembly of functionally integrated human forebrain spheroids A human 3D neural assembloid model for SARS-CoV-2 infection In vitro maintenance of ovaries and ovarian cells from Solenopsis invicta (Hymenoptera: Formicidae) Regenerating tissue from the cockroach leg: nutrient media for maintenance in vitro Some effects of X-irradiation on embryos of the cockroach Blaberus craniifer Organ culture of the Xenopus tadpole intestine Explant culture of adult zebrafish hearts for epicardial regeneration studies Ex vivo functional whole organ in biomedical research: a review ex vivo and in vivo models in prostate cancer research Endocrinology of the aging prostate: current concepts Development and Evaluation of an evidence-based Educational Toolkit on the Knowledge and Practice of Primary care Clinicians’ Regarding Social Determinants of Health that Impact Prostate Health in Black men (A Quality improvement Project Eco-taxonomic profile of an iconic vermicomposter — the ‘African nightcrawler’ earthworm Autofluorescence in BrdU-positive cells and augmentation of regeneration kinetics by Riboflavin Comparative analysis of the survival and regeneration potential of juvenile and matured earthworm Understanding the role of the clitellum in the regeneration events of the earthworm Eudrilus eugeniae Exploring the effect of UV-C radiation on earthworm and understanding its genomic integrity in the context of H2AX expression Stem cell quiescence: the challenging path to activation 3 maintains adult haematopoietic stem cell homeostasis by enforcing chromatin adaptability Multiple facets of histone variant H2AX: a DNA double-strand-break marker with several biological functions Ongoing repair of migration-coupled DNA damage allows planarian adult stem cells to reach wound sites High basal γH2AX levels sustain self-renewal of mouse embryonic and induced pluripotent stem cells Alternative to FBS in animal cell culture-an overview and future perspective Myocardial transfection of hypoxia-inducible factor-1α and co-transplantation of mesenchymal stem cells enhance cardiac repair in rats with experimental myocardial infarction Paracrine mechanisms of mesenchymal stem cells in tissue repair Mesenchymal Stem Cells: Methods Protocols 123–146 (2016) Effect of TGF-β1/SDF-1/CXCR4 signal on BM-MSCs homing in rat heart of ischemia/perfusion injury PDGF-C and PDGF-D signaling in vascular diseases and animal models Importance and regulation of adult stem cell migration Isolation and functional characterization of murine prostate stem cells Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt A stepwise model system for limb regeneration Tumor-derived spheroids: relevance to cancer stem cells and clinical applications The earthworm eudrilus eugeniae: a model organism for regenerative biology Regenerative potential of prostate luminal cells revealed by single-cell analysis Proximal location of mouse prostate epithelial stem cells: a model of prostatic homeostasis Hedgehog signalling in prostate regeneration Integrated single-cell analysis defines the epigenetic basis of castration-resistant prostate luminal cells CD133 does not enrich for the stem cell activity in vivo in adult mouse prostates Anatomy and histology of the human and murine prostate Prostasomes—their effects on human male reproduction and fertility On the Reproductive processes of earthworms: part I The process of Copulation and Exchange of sperms in Eutyphoeus waltoni Mich XVI.—The prostate glands of the earthworms of the Family Megascolecidæ Comparative biology of mouse versus human cells: modelling human cancer in mice Cerebral organoids model human brain development and microcephaly Ngly1–/– rats develop neurodegenerative phenotypes and pathological abnormalities in their peripheral and central nervous systems European Parliament and of the Council Directive on the protection of animals used for scientific purposes2010/63/EU Ex vivo prostate cancer explant organ culture model system for targeted drug development in prostate cancer Valproic acid enhances axonal regeneration and recovery of motor function after sciatic nerve axotomy in adult rats Valproic acid protects neurons and promotes neuronal regeneration after brachial plexus avulsion Assessment of the effect of valproic acid on regeneration in eisenia fetida Suppressive effects of valproic acid on caudal fin regeneration in adult zebrafish Challenges and opportunities for disposal of floral waste in developing countries by using composting method physical activity and hormones: results of a randomized controlled trial Download references Authors thank ‘International Research Centre (IRC) of Sathyabama Institute of Science and Technology Chennai’ for proving all the support to carry out the research work This work was supported by Young Scientist Fellowship (No 12014/78/2020-HR) from DHR/ICMR -New Delhi Molecular Biology and Stem Cell Research Lab Centre for Molecular and Nanomedical Sciences Sathyabama Institute of Science & Technology Kamarajan Rajagopalan & Johnson Retnaraj Samuel Selvan Christyraj The ethical statement is not required for earthworms all research is conducted with appropriate precautions to prevent unnecessary harm to animals Download citation DOI: https://doi.org/10.1038/s41598-025-87039-y Sign up for the Nature Briefing newsletter — what matters in science Metrics details The aim of this study was to investigate the relationship between tear function changes and meibomian gland dysfunction in various phenotypes of polycystic ovary syndrome (PCOS) 4 separate case groups were formed for 4 different phenotypes of PCOS which are variants of clinical presentation A total of 160 women were included in the study 32 women for each group and 32 healthy women as control group Oxford score for corneal and conjunctival involvement lower lid meibomian drop-out grades by non-contact meibography and meibomian gland disease (MGD) distortion and shortening scores were compared between all groups When the PCOS group was compared with the control group phenotype A showed the most significant differences in NIBUT (p = 0.003) Although the mean Schirmer 1 and Schirmer 2 tests value were lower in the PCOS group compared to the control group no significant difference was detected in any phenotype (p = 0.911 A statistically significant weak negative correlation was observed between NIBUT and OSDI values (r = − 0.206 There was a statistically significant positive and very weak correlation between OSDI and BMI (r = 0.079 Tear film instability attributed to MGD was found to be significantly different in all PCOS patients compared to healthy subjects regardless of phenotype and this difference was statistically higher in phenotype A the aim of this study was to compare Schirmer 1 and 2 test results meibomian gland dysfunction (MGD) and ocular surface disease index (OSDI) scores between individuals with PCOS phenotypes with different hormonal variations and healthy controls Correlations between these parameters and body mass index (BMI) and serum estradiol and free testosterone levels were also evaluated A single ovary meeting these criteria was considered sufficient for the diagnosis of PCOS All PCOS definitions were made by the same gynecologist (B.G.) and all information included belongs to newly diagnosed patients phenotyping was performed and four different phenotypes were identified: A Phenotype A met all three criteria (menstrual irregularity phenotype B was seen with menstrual irregularity and hyperandrogenism without ultrasound evidence phenotype C with hyperandrogenism and PCO on ultrasound and phenotype D with only menstrual irregularity and PCO on ultrasound without hyperandrogenism Patients were categorized into one of these four groups according to phenotypic criteria a total of 5 groups were included in our study 4 study groups for PCOS phenotypes and 1 control group it was concluded that reliable findings could be obtained with 32 subjects in the control group and 128 subjects in the PCOS group with a medium effect size of d = 0.77 for the independent two-sample t test All patients were between the ages of 18–45 After the diagnosis was made in the Gynecology and Obstetrics Clinic the patients underwent the necessary ultrasound and laboratory evaluations Venous blood samples were collected from women during the early follicular phase of their menstrual cycle (days 3–5) after spontaneous menstruation or the onset of progesterone-induced bleeding Blood samples were collected from antecubital veins between 8:00 and 9:00 a.m after a 12-h fast for precise measurement of total and free testosterone including age and personal medical history in order to increase the reliability of our study single and same-sided eye of each patient was included in the study in accordance with the literature Clinical ocular examinations were performed by the same physician (blinded to PCOS phenotypes (F.S.) in the Faculty of Ophthalmology Recep Tayyip Erdogan University All participants underwent the same detailed ophthalmologic examination as follows: visual acuity assessment intraocular pressure measurement and fundoscopic evaluation Tear and meibomian gland (MG) evaluations were performed in the following order: non-invaziv tear break-up time (NIBUT) meibomian gland morphologic droup-out scoring Oxford scoring and meibomian expressibility grading The study excluded patients with congenital adrenal hyperplasia or antiandrogens during the previous six months Participants were ineligible for the study if they had any of the following conditions: corneal or ocular surface abnormalities; a history of glaucoma; ocular surgery; ocular trauma; refractive error greater than two diopters; infectious keratoconjunctivitis; inflammatory ocular surface diseases; meibomian gland dysfunction caused by Demodex infestation; corneal scars; concurrent use of topical ophthalmic medications; topical ophthalmic steroids taken within four weeks of the study; systemic medications affecting tear production; aqueous tear insufficiency; or a history of contact lens use The collected data were analysed with the IBM SPSS program Compliance with a normal distribution within and between groups was analysed by the Kolmogorov‒Smirnov test and the Shapiro‒Wilk test The Mann‒Whitney U test was used to compare nonnormally distributed data according to binary groups Normally distributed data were analysed via one-way analysis of variance and nonnormally distributed data were analysed via the Kruskal‒Wallis H test Fisher’s exact test was used to investigate categorical data according to groups and multiple comparisons were analysed by the Z test with Bonferroni correction The relationships between quantitative characteristics that were not normally distributed were analysed with Spearman’s rho correlation coefficient The results of the analyses are presented as the mean ± standard deviation median (minimum—maximum) for quantitative data and frequency (percentage) for categorical data A significance level of p < 0.05 was considered statistically significant (a) Normal meibomian glands with no distortion nor dropout.PCOS-Phenotype D (b) Grade 1 with dilatation and tortuosity of the MG.PCOS-Phenotype C (c) Grade 2: dropout of MG along with gland distortion (d) Grade 3: MG dose not traverse the total tarsal with mottling of details When the relationships between demographic and clinical characteristics were analysed in the patient group, a positive correlation was found between OSDI values and BMI. The relationships between other demographic and clinical characteristics were not statistically significant (p > 0.05). These results are summarized in Table 4 A statistically significant correlation was found between BMI and OSDI among demographic and clinical characteristics according to the MGD value in the patient group, especially in group 2, which expressed moderate MGD (p < 0.01). These results are summarized in Table 5 This is the first study in the literature to evaluate the relationship between meibomian glands and PCOS subphenotypes The main results of this study indicated that meibomian glands are affected in all subtypes of PCOS we found a higher OSDI score in patients with PCOS phenotype A in our study reported that all women had hyperandrogenism and hyperinsulinaemia and they reported that the OSDI score was significantly higher than that of the control group These authors argued that both the brush cytology score and the serum ALB concentration secreted by tears significantly increased which may be related to the peak estrogen concentration in the follicular phase suggesting that dry eye is an inflammation-related pathology the current body of literature predominantly supports the notion that PCOS primarily correlates with dysfunction of the meibomian glands rather than the lacrimal glands underscoring the need for further investigation into the implications of hormonal changes on lacrimal gland health within this population we found no difference in Schirmer’s results showing lacrimal gland function BMI was positively correlated with OSDI scores and MGD and these results were most prominent in Phenotype A including a small sample size and the inability to evaluate the long-term effects of PCOS on the ocular surface due to the relatively young age of the study population We suggest that further investigations with larger cohorts encompassing diverse age groups and long-term follow-up periods would enhance our understanding of this subject Another limitation of our study is that patients were included in the study after diagnosis and were directed to an eye check-up This prevents standardization of patients in terms of how long the disease has been present or how long they have been exposed to hyperandrogenemia due to the potential for possible effects on the eye The effects of hyperandrogenemia may depend on the duration as well as the level We believe that this limitation can be the subject of other studies PCOS is a common hormonal disorder among women of reproductive age that significantly affects ocular surface and meibomian gland function This effect is particularly evident in phenotype A which closely resembles the classic PCOS manifestation observed in both laboratory and clinical settings This makes it crucial for healthcare providers to recognise the link between dry eye symptoms and PCOS This allows for a more holistic treatment strategy that addresses both the endocrine and ocular manifestations of the syndrome clinicians can significantly improve the quality of life of women suffering from PCOS and ensure that all aspects of their health are considered The datasets used and/or analysed during the current study available from the corresponding author on reasonable request Ehrmann, D. 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Evaluation of meibomian gland dysfunction in polycystic ovary syndrome and obesity. Curr. Eye Res. 42(5), 661–665. https://doi.org/10.1080/02713683.2016.1233985 (2017) Download references The authors would like to thank Mine Kaya for the statistical analysis of the study Jerremy Huggsfor editing the English of the article This study has been supported by the Recep Tayyip Erdogan University Development Foundation (Grant Number: 02024012003197) İstanbul Gaziosmanpasa Training and Research Hospital F.S have constructed/constructed the main idea and hypothesis of the study and BG and developed the theory and arranged/edited the material and method section has evaluated the data in the Results section all authors discussed the entire study and approved the final version The study protocol was approved by the local human research ethics committee from Recep Tayyip Erdogan University Protocol number and date 2020/242 and 17.12.2020 All patients included in this study gave their informed consent which adhered to the tenets of the Declaration of Helsinki and written informed consent was obtained from all subjects Consent was also obtained from our institution Download citation DOI: https://doi.org/10.1038/s41598-025-94890-6 Metrics details To develop an atrophic Meibomian Gland Dysfunction (MGD) animal model via liquid nitrogen cryotherapy the eyelid edges of C57 mice exposure to liquid nitrogen for 30 s Morphology of MG and ocular surface were assessed using stereomicroscopy and a slit lamp microscope at multiple time points post-injury Acinar loss and atrophy were observed from day 7 and decreased proliferation in acinar cells Corneal epithelial defects appeared after day 14 Liquid nitrogen induced selective damage to meibomian acinar cells providing a relevant model for atrophic MGD research Animal models of primary meibomian gland atrophy remain missing It is widely acknowledged that thermal therapy and massage have therapeutic effects on meibomian glands it is questioned whether cryogenic freezing can cause similar damage to the meibomian glands as it does to the skin and whether such damage can simulate the pathophysiological process of atrophic meibomian gland dysfunction (MGD) What molecular mechanism changes can temperature or hypothermia induce in the meibomian glands The following experiments were conducted to investigate these questions we introduce a murine model of meibomian gland dysfunction (MGD) that is induced by a targeted application of liquid nitrogen to the eyelid margin This model is characterized by significant loss of meibomian gland acini and ductal structures as well as decreasing secretion of meibomian glands The development of this model is expected to advance our understanding of the pathophysiological alterations within meibomian glands subjected to cold-induced injury it provides a platform for the exploration of novel therapeutic approaches for MGD and the potential for regenerative medicine strategies targeting the meibomian glands MG loss and morphological changes after liquid nitrogen (A) Representative stereoscopic microscope images show MG loss and morphological changes of MGs after liquid nitrogen The red rectangle represents the area of MG loss (B) The area of meibomian gland loss gradually increases after liquid nitrogen freezes the meibomian margin Oil Red O staining indicates that after liquid nitrogen cryopreservation, the staining intensity of the meibomian gland acinar cells has become lighter. The morphology of the meibomian gland acini exhibited varying degrees of atrophy with extended observation time. The Oil Red O staining within the ducts shows incomplete filling (as shown in Fig. 4A) Histological changes and lipid synthesis of MGs after liquid nitrogen injury of the eyelid margin (A) Oil red O staining showing the meibomian gland ducts are partially filling 21 and 28 post liquid nitrogen injury (circle) Some oil red O-labeled droplets indicated atrophy acini (black star) C) The expression of PPARγ in immunofluorescence significantly decreases on the 7th and 28th days following liquid nitrogen cryopreservation E) Western blot analysis shows the decline in PPARγ after liquid nitrogen treatment The expression reduction was most significant at 7 days and 21days Inflammation of MGs after liquid nitrogen injury of eyelid margin IL-18 were upregulated in the MG of mice post liquid nitrogen injury D) Immunofluorescence staining of TNFα was upregulated in the MG post liquid nitrogen injury E) Immunofluorescence staining of K14 was downregulated in the meibomian gland acinar cells post liquid nitrogen injury We performed CD68 + iNOS double-labeling fluorescence staining to assess inflammatory cell infiltration in the meibomian glands of different groups Observations revealed that CD68-positive cells were present at all stages following liquid nitrogen-induced eyelid margin injury with iNOS-positive cells being more prominent in the early stages but decreasing at later stages (as shown in supplementary Figure) Cell proliferation and apoptosis in MGs after liquid nitrogen injury B) Immunofluorescence staining of Ki67 and positive cell counting indicates a decreased in positive cells after liquid nitrogen injury with labelled cells rarely detected at day 28 D) TUNEL staining and positive cell counting shows abundant positive cells around MGs post liquid nitrogen injury Scale bars represent 50 μm (n = 3) The TUNEL staining indicates that apoptosis is consistently present following liquid nitrogen injury, showing an upward trend from the 7th to the 14th day of observation. From day 14 to day 28, apoptosis can be essentially maintained at a relatively high level (as shown in Fig. 6C keratinization of MGs after liquid nitrogen injury of eyelid margin (A) Immunofluorescence staining of K10 revealing hyper-keratinization and thickening of the MG duct at days 21 and 28 post liquid nitrogen injury (B) Gene expression levels of Sprr1β was upregulated in the MG of mice post liquid nitrogen injury We have delineated a mouse model of atrophic meibomian Gland Dysfunction (MGD) we found that liquid nitrogen can specifically disrupt the cell membranes of acinar cells in the meibomian glands in the acute post-injury phase (within 24 h) without damaging adjacent tissues such as the orbicularis oculi muscle or the cornea there is a significant decrease in lipid content following injury the meibomian glands have shown varying degrees of atrophy and loss the eyelid margins have become thickened and rounded the cornea exhibited significant fluorescence staining Since the use of the liquid nitrogen freezing method to create a meibomian gland dysfunction (MGD) model is being applied for the first time there are no ophthalmology-specific references to explain the underlying mechanisms The inspiration for this model comes from dermatology where the mechanisms of similar models have been better studied Based on the existing literature and the current experimental results the possible mechanisms can be summarized as follows: These models often exhibit dysplasia of the meibomian glands a gene knockout mouse that not only alters the function of the meibomian glands but also causes significant morphological changes in the meibomian glands Although these gene knockout models can explain the effects of the gene on the development and function of meibomian glands at the molecular or pathway level from some perspectives most of them cannot mimic the pathophysiological process of patients with meibomian gland dysfunction in clinical practice because the vast majority of patients have meibomian gland dysplasia or even abnormal and the changes in meibomian gland morphology and function are also caused by various acquired factors and these gene knockout models do not explain how acquired factors affect the gene there are still limitations to the study of pathogenic mechanisms gene knockout mice are the knockout of whole body cells and it is impossible to specifically knock out one or several genes of meibomian gland tissue which will inevitably cause damage to the ocular surface and other tissues and organs of the body to varying degrees these side injuries are not desirable to us and are not conducive to observing the pathophysiological changes that should occur on the ocular surface under MGD genetic selection in knockout mice is also limited I have to admit that the knockout genes that exist at present do have an impact on meibomian gland function but there is no definite conclusion that these genes must be key and necessary influencing factors and a large part of the genes that affect meibomian gland function may not be able to construct such transgenic mice because those mice cannot survive after the gene is deleted so that their effect on meibomian glands cannot be observed and explored through animal experimental models This also limits the understanding of the mechanisms of meibomian gland dysfunction at the genetic level although the elderly animal model well simulates some ocular surface changes in patients with age-related MGD in clinical practice and not all elderly animals will suffer from MGD and the low mold rate is a factor that has to be considered and TNFα in this model accumulate and increase over time particularly noticeable by the 21st and 28th days which we believe contributes to the further deterioration of acinar cell function The apoptosis of acinar cells becomes increasingly evident and the expression of the basal cell marker K14 also gradually decreases This suggests that in the advanced stages of atrophic MGD the self-renewal capacity of the acini has declined possibly due to issues with the multipotency of basal cells the specific mechanisms still require further experimental research to be revealed which will also be the direction of future research for our group these models do not elucidate the mechanisms and processes by which adult meibomian glands self-renew This indicates that the meibomian glands indeed possess regenerative potential and this model can also serve as an animal model for subsequent studies tracking the localization of meibomian gland stem/progenitor cells and the mechanisms underlying acinar regeneration A21206) and Alexa Fluor 594conjugated IgG (A11058) were from Invitrogen (Eugene 40,6-diamidino- 2-phenylindole (DAPI; H-1200) Eight-week-old male C57/BL6 mouses were from Changchun-bio (China) Experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and with approval of the Animal Ethical Committee of Harbin medical University Animals were given free access to standard rodent chow and water and kept in a standard pathogen-free environment at 25 °C ± 1 °C and alternating 12 h light-dark cycles (from 8:00 AM to 8:00 PM) we administered liquid nitrogen cryotherapy to the eyelid margins of mouse in the treatment group This procedure was carried out under general anesthesia induced by inhalation of 4% isoflurane (Sinopharm Chemical Reagent Co. followed by topical application of 0.4% oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical Japan) for local anesthesia of the eyelid margins Liquid nitrogen was placed in a thermos flask and then the temperature was conducted using a specially crafted pure copper device to apply cryotherapy to both the upper and lower eyelid margins The temperature of the device that touches the eyelids can be maintained at minus 110 degrees Celsius Schematic illustration of the procedure to perform local Liquid nitrogen cryotherapy on mouse MG orifices (B) About 13 min after filling with liquid nitrogen the temperature of the unit tends to stabilize at -100 degrees Celsius ± 15 degrees Celsius the patency of the meibomian gland (MG) orifices was evaluated utilizing a slit-lamp microscope The state of the orifices was documented with a digital camera and the quantity of obstructed orifices out of a total of ten MG orifices located centrally in the upper eyelid was enumerated Orifices were categorized as obstructed if they exhibited opacity and swelling To appraise the integrity of the corneal epithelium one microliter of a 1% solution of liquid sodium fluorescein (Jingmingxin Co. China) was instilled into the conjunctival sac The corneal epithelium’s uptake of fluorescein was subsequently observed after a 90-second interval employing a slit-lamp microscope equipped with a cobalt blue filter A consistent ophthalmologist captured all images using an identical camera and settings throughout the examination process Post-injury collection of mouse eyelids was conducted at specific time points: 0 These eyelid samples were then embedded either in Optimal Cutting Temperature (OCT) medium or paraffin were prepared with three sections per slide resulting in a total of 3 animals per experimental group Hematoxylin and eosin (H&E) staining was applied to the paraffin-embedded sections to assess tissue morphology immunofluorescent staining and Oil Red O staining were conducted on the OCT-embedded frozen sections to further analyze cellular and lipid characteristics The accumulation of lipids in the meibomian glands (MGs) was determined by evaluating the intensity of the Oil Red O stain The frozen sections of the eyelids were initially fixed in a 5% paraformaldehyde solution for a duration of 10 min followed by a wash in phosphate-buffered saline (PBS) for 5 min the sections were stained with a freshly prepared Oil Red O solution for 25 min the sections were rinsed with PBS for another 5 min the sections were mounted using a 90% glycerol solution and examined under a light microscope to visualize the lipid deposits For the immunofluorescent staining procedure were treated by being fixed in cold acetone at -20 degrees Celsius for 10 min This was followed by a series of three washes with phosphate-buffered saline (PBS) The sections were then permeabilized with a 0.3% Triton X-100 solution for 30 min and rinsed again with PBS in triplicate the sections were blocked to reduce non-specific binding by incubating them with 2% bovine serum albumin (BSA) in PBS for 60 min at ambient temperature which were then incubated for 16 h at 4 degrees Celsius negative control sections were treated with a non-relevant isotype control antibody in place of the primary antibody Following the incubation with primary antibodies the sections were washed extensively with PBS for 10 min per wash The sections were then incubated with the secondary antibodies either Alexa Fluor 488-conjugated IgG (diluted 1:250) or Alexa Fluor 594-conjugated IgG (diluted 1:250) for 1 h at room temperature in a dark environment to protect the fluorescent labels from light-induced bleaching the sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei and then examined using a fluorescence microscope to capture images of the stained cells we employed the TUNEL assay kit from Elabscience One-step TUNEL In Situ Apoptosis Kit (E-CK-A320) paraffin-embedded tissue sections underwent rehydration and were then treated with a Proteinase-K solution in Tris/HCL buffer at a pH of 7.4 for a duration of 30 min at a controlled temperature of 37 °C the sections were thoroughly rinsed with Phosphate-Buffered Saline (PBS) repeating the process three times for 5 min each to ensure complete removal of the Proteinase-K 50 µl of the TUNEL reaction cocktail was applied to the sections which were then incubated in the dark at 37 °C for a period of 1 h the sections were again subjected to three rounds of washing with PBS for 5 min each to eliminate any unbound reaction components the sections were subsequently counterstained with 4’,6-diamidino-2-phenylindole (DAPI) a fluorescent stain that binds specifically to DNA the sections were mounted onto slides for preservation and clarity the stained sections were examined and documented using a high-resolution microscope (model DM2500 from Leica Microsystems) capturing images that reveal the presence of apoptotic cells through the TUNEL assay Isolated meibomian gland (MG) samples were collected using a cold lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors (Catalog No Protein content was determined utilizing a BCA Protein Assay Kit (Catalog No Each experimental set comprised three replicates with each replicate being a composite of MGs harvested from both ocular glands of an individual mouse A standardized quantity of protein lysate (20 µg) from each sample was loaded and resolved on 10% tricine gels followed by transfer to a polyvinylidene fluoride (PVDF) membrane The membranes were then blocked with 5% bovine serum albumin (BSA) for 60 min Incubated with primary antibodies specific for PPAR-γ (dilution 1:1000) or β-actin (dilution 1:8000) after cropping at 4 °C overnight After three rinses with Tris-buffered saline with 0.05% Tween 20 for 10-minute intervals the membranes were treated with horseradish peroxidase (HRP)-tagged secondary antibodies directed against either mouse or rabbit IgG an HRP-tagged anti-mouse antibody was applied as a loading control Protein expression was visualized using an enhanced chemiluminescence substrate and documented 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Surf. 29, 497–507. https://doi.org/10.1016/j.jtos.2023.07.004 (2023) Download references National Natural Science Foundation of China (Grant No 82301173); The Natural Science Foundation of Heilongjiang Province LH2020H039); Heilongjiang Provincial Department of Education Heilongjiang Provincial Higher Education Fundamental Research Project (2021-KYYWF-0226); Heilongjiang Provincial key research and development plan guidance project (GZ20220125); The Science Fund for Excellent Young Scholars of First Affiliated Hospital of Harbin Medical University (Grant No.2024YQ04) The First Affiliated Hospital of Harbin Medical University the contributions of the authors were as follows:- Shu Wang Yulin Lin were involved in the conception and design of the research.- Shu Wang Jingfan Gao and Jia Lin conducted the experiments and initial analysis.- Xin Jin were responsible for the statistical analysis and interpretation of the results.- Hong Zhang contributed to the acquisition of funding and supervision of the project.- Shu Wang drafted the initial manuscript and revised it critically for important intellectual content.- All authors read and approved the final manuscript I can confirm that our study has been conducted in accordance with the ARRIVE guidelines (Animal Research: Reporting In Vivo Experiments) Download citation DOI: https://doi.org/10.1038/s41598-024-84742-0 Metrics details Previous studies have confirmed that methylation regulates gene transcription in the hypothalamus-pituitary-gonadal axis during puberty initiation but little is known about the regulation of DNA methylation on gene expression in the pineal gland To screen pineal gland candidate genes related to the onset of goat puberty and regulated by genome methylation we collected pineal glands from prepubertal and pubertal female goats determined the DNA methylation profile by whole genome bisulfite sequencing and the transcriptome by RNA sequencing on Illumina HiSeqTM2500 We analyzed differentially expressed genes between the Pre group and Pub group using the DESeq2 software (version 1.20.0) and applied the Benjamini and Hochberg method for adjusting P-values Genes with a P-value less than 0.05 and an absolute log2 fold change greater than 0 were considered differentially expressed genes Results showed that there was no significant difference in the whole-genome methylation level of the pineal gland between prepubertal and pubertal goats but the methylation pattern changed significantly indicating that genomic DNA methylation of the pineal gland might play a role in regulating the initiation of goat puberty Changes in DNA methylation patterns affected some pineal gland transcriptomes while the transcriptional level of most genes remained unaffected by DNA methylation differences Genes regulated by DNA methylation regulates genes primarily involved in metabolic processes and signaling pathways related to thermogenesis Methylation significantly regulated the expression of genes such as ATP5F1D and DCC showed the most notable fold changes which may indicate their involvement in the onset of puberty which can shorten the generation interval and improve the reproductive capacity The age of pubertal onset is an important indicator reflecting the reproductive performance of goats the basic molecular mechanisms that regulate the onset of puberty in goats are still unclear in order to elucidate the mechanism of pubertal initiation it is necessary to investigate the expression regulation of genes involved in regulating the onset of puberty These genes deserve further study to elucidate their roles in the onset of puberty in female mammals The objective of this study is to profile gene expression and genome-scale DNA methylation of the hypothalamus in prepubertal and pubertal goats using RNA sequencing (RNA-seq) and whole genome bisulfite sequencing (WGBS) The results will provide useful insights into the epigenetic mechanisms involved in the onset of puberty in goats The animal experimental protocols and procedures for this study were approved by the Animal Care and Use Committee of Anhui Agricultural University and complied with the EU Directive 2010/63/EU for animal experiments We raised twelve healthy prepubertal female Wanlin White goats (WWGs) in a semi-enclosed shed We fed them with a 1:1 mixture of fattening goat concentrate (Jiangsu Mudi Feeds Co Q/321324JSMD002) and corn in the morning and evening and give them dried peanuts seedlings at midday each goat consumed about 0.15 kg of feed per meal The amount of feed for each goat was gradually increased as body weight All the goats had free access to water and natural light and ovarian histological sections of prepuberta; and pubertal goats a1) is the vulva of the prepubertal goat; (a2) is the vulva of the adolescent goat; (a3) is the ovary of the prepubertal goat; (a4) is the ovary of the pubertal goat (b) histological sections of the prepubertal ovaries had a large number of primordial follicles (PrF) primary follicles (PF) and secondary follicles (SF) (b1 The ovaries of pubertal goats had a large number of tertiary follicles (TF) and mature follicles (MF) (b3 We collected pineal glands from six prepubescent WWGs (aged 2.5–3.0 months and six pubescent WWGs (aged 4.0–4.5 months To ensure that each sample has sufficient quantity for DNA methylation and transcriptome sequencing analysis we pooled pineal gland samples from the same period in pairs ultimately obtained 3 prepubertal samples and 3 pubertal samples We took out half of each sample from these three prepubertal and three pubertal samples for RNA sequencing we pooled the remaining prepubertal and pubertal samples together then aligned the clean reads with the reference genome We used StringTie for new gene prediction and rMATS software for alternative splice (AS) analysis of RNA-Seq data We classified AS events and evaluated the differences in AS events between different samples The significance threshold for filtering differentially spliced events was set at FDR less than 0.05 We used GATK software for variant calling and utilized SnpEff for annotating information on each variant GO terms with a corrected P < 0.05 were considered significantly enriched by differentially expressed genes The cluster Profiler R package was also used to analyze the statistical enrichment of differentially expressed genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways after mixing 5.2 μg of genomic DNA with 26 ng of λDNA we broke it into 200–300 bp fragments by ultrasonication we performed end-repair on the sheared DNA fragments and ligated sequencing adapters that had been methylated at all cytosines we treated the DNA fragments twice with the EZ DNA Methylation-GoldTM Kit (Zymo Research) to convert them to disulfite and amplified the products by PCR we then performed paired-end sequencing of the DNA library on Illumina HiSeq 2500 sequencing instrument at Novogene (Beijing A corrected P < 0.05 was considered significant for GO term and pathway enrichment analysis Transcriptional changes of goats’ pineal gland during the onset of puberty (a) To detect relatedness between sample individuals Pearson’s correlation coefficient (r2) was used as theevaluation index (b) Principal component (PC) analysis of the transcriptomes for two stages of pineal glands Pineal glands from the same stage are shown in the same color PC1 (38.84%) and PC2 (19.64%) represent the top two dimensions of detected genes among the prepubertal and pubertal pineal glands (a) Volcano plot of the DEGs between prepuberty and puberty The red spots indicate genes that were upregulated in puberty the green spots indicate downregulated genes and the blue spots indicate genes that were not differentially expressed (b) Hierarchical clustering of DEGs of prepubertal and pubertal goats’ pineal glands indicates the normalization level of gene expression (c) Significant functional pathways revealed from up- or downregulated genes in comparisons between prepubertal and pubertal goats’ pineal glands (d) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs between prepuberty and puberty Enrichment analysis of DEGs during goat development (a) Venn chart of differentially expressed genes between prepuberty and puberty (b) Gene Ontology of prepubertal-specifically expressed genes (c) Scatter plot of prepubertal-specifically expressed genes with significant enrichment (d) Gene Ontology of pubertal-specifically expressed genes (e) Scatter plot of pubertal-specifically expressed genes with significant enrichment DNA methylation levels across genomic elements in prepubertal and pubertal goats (a) The average ratio of DNA methylation types in prepubertal and pubertal genomes of goats and orange colors represent methylated mCG (b) Violin plot for the overall distribution of methylation levels for different methylation types: CG The abscissa represents the different samples the ordinate represents the level of methylation of the samples the width of each violin represents the density of the point at that methylation level and the boxplot shows the methylation levels in each violin (c) Circos map of genome-wide differences in methylation levels and overall differences between prepuberty and puberty The three circles from the outside to the inside represent the methylation level of puberty and the methylation level of prepuberty Internal scales: DNA methylation level represents methylation level and DNA methylation difference among samples represents heatmap (d) DNA methylation levels across genomic elements in prepubertal and pubertal goats The orange and green colors represent prepuberty and puberty The abscissa represents different genomic elements: CGI The left ordinate represents the methylation levels of CG contexts and the right ordinate represents the methylation levels of the CHG/CHH context Enrichment analysis of CG-type differentially methylated genes (a) The number of differentially methylated regions between different methylation types Histograms show the distribution numbers of differentially methylated regions (DMRs) in different genomic elements in the CG (b) Heatmap cluster analysis of DMRs between prepuberty and puberty highly methylated loci are displayed in red and sparsely methylated loci are displayed in blue (c) Counts of differentially methylated genes (DMGs) enriched for the top 30 Gene Ontology (GO) terms The abscissa represents the number of DMGs and the ordinate shows the GO pathway terms The ordinate represents the enriched pathways and the abscissa represents the Rich factor of the corresponding pathways; the size of the spots represents the number of genes related to DMRs enriched in each pathway while the color of the spot represents the corrected P value for each pathway The Rich factors indicate the ratio of the number of DMGs mapped to a certain pathway to the total number of genes mapped to this pathway A greater Rich factor means greater enrichment Analysis of overlapping genes between DMGs and DEGs (a) GO enrichment histogram of overlapping DMRs and DEGS genes in the promoter region (b) GO enrichment histogram of overlapping DMRs and DEGS genes in the promoter region (c) Construction of the network of DMGs related to puberty initiation Analysis of the interaction between DMGs related to puberty initiation using STRING software according to the interplay index (confidence > 0.9) (d) Scatter diagram of DMR differential methylation and differential expression gene levels Previous studies have analyzed DNA methylation in the hypothalamus whole-genome methylation analysis of the pineal gland has not been reported before This study is the first to systematically compare the genome-wide DNA methylation profile and transcriptome changes of the prepubertal and pubertal goat pineal gland we analyzed the differential expression of genes and pathways related to puberty in the goat pineal gland between the pre and pub stages by transcriptome analysis We also studied the whole-genome DNA methylation spectrum of goat pineal glands by WGBS to clarify the relationship between transcription and DNA methylation at different developmental stages we obtained 270 million clean reads per sample Approximately 1.8% of the cytosine sites were methylated which was similar to the results found in other species and tissues the methylation level of TSSs was the lowest which was consistent with the results found by WGBS in sheep ovaries we identified 30,000 DMRs and 22,161 genes associated with these DMRs The proportion of DMRs in the promoter region with the majority being concentrated in the distal intergenic region The pineal gland is an important neuroendocrine organ and our research enriches metabolic pathways such as metabolism Bioinformatics results show that DMGs related to these regulatory processes are significantly different between prepuberty and puberty suggesting that they may play an important role in the onset of puberty little is known about how DNA methylation affects gene expression and how these genes work together We generated DMG interaction networks to determine whether DMGs play a determinative role in the onset of puberty in goats and DCC Netrin 1 Receptor (DCC) are key nodes This suggests that these genes might play important roles in puberty initiation and the differentiation and regulation of these genes through DNA methylation might be one of the mechanisms that determine the difference in puberty initiation time and reproductive capacity of goats This study also showed that the Lin28B gene in the goat pineal gland was significantly upregulated in puberty compared with prepuberty the reproductive regulation of Lin28B in mammals such as goats and mice are opposite which may be due to the different effects of photoperiod on the reproduction of different animals it is still necessary to further study the mechanism by which the Lin28B gene regulates puberty initiation It was also found that the intracerebroventricular injection of Nesfatin-1 could advance the opening time of the vulva in rats promote the onset of puberty and increase the transcription levels of GnRH Combined with the results of these studies it is suggested that DCC may affect the secretion of GnRH and the expression of related reproductive genes on the hypothalamic-pituitary-ovary axis by promoting the development of GnRH neurons it is still unclear how DNA methylation in the gene affects gene expression and GIP are key regulatory genes in the onset of puberty and that their DNA methylation status may play a functional role in puberty initiation Methylation of these genes may lead to significant differences in puberty initiation and pineal gland neuron development between prepubescent and pubertal goats the epigenetic mechanisms of gene regulation and the genetic regions involved in the onset of puberty and neuronal development require further study we have provided a systematic description of the whole-genome DNA methylation patterns and transcriptomes of goat pineal gland during prepuberty and puberty We have identified several novel and important DMRs These findings provide valuable insights into the genetic and epigenetic mechanisms of economic traits in goats and can be used in marker-assisted selection procedures to promote goat breeding Zhang, B. et al. 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Genet. 39, 457–466. https://doi.org/10.1038/ng1990 (2007) Download references The authors are very grateful to the research technicians for supporting our experiments This work was supported by the National Natural Science Foundation of China funded projects [grant numbers 31972629 32272881]; Science and Technology Major Project of Anhui Province [grant numbers 202103b06020023];Anhui Key Research and Development Program[grant numbers 2023z04020003] Present address: AnhuiWanbei Electricity Group General Hospital Present address: HefeiTiangang Immune Medicine Co. Present address: College of Veterinary Medicine These authors contributed equally: Bochao Zhang and Genbao Zhu Anhui Provincial Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Breeding Linquan County Modern Agriculture Technology Cooperation and Extension Service Center Ya Liu and Fugui Fang designed the experiments Jianlin Wu and Genbao Zhu analyzed the dataand drafted the manuscript Bochao Zhang,Jiankun Cui and Wei Qian collected the samples andperformed the experiments Fuqiang Pan and Qing Yi prepared figures and performed part of the data analysis Yunsheng Li and Ya Liu reviewed the manuscript All authors have read and approved the final manuscript The study is reported in accordance with ARRIVE guidelines Download citation DOI: https://doi.org/10.1038/s41598-024-84559-x In a retrospective study of patients with refractory dry eye who had exhausted conventional treatment and elected to receive intense pulsed light and meibomian gland expression (IPL/MGX) 58% of patients' symptoms improved from as much as 25% to more than 50% after treatment Study results were published in Cornea in 2016 Joanne F. Shen, M.D. director of the dry eye clinic at Mayo Clinic's campus in Phoenix and a research team studied 35 patients treated with IPL/MGX Standard Patient Evaluation of Eye Dryness 2 (SPEED2) symptom survey scores slit-lamp examinations and meibomian gland evaluations at baseline and at each visit before IPL/MGX treatments All patients had a minimum of six months of follow-up after the first treatment and typically received 1 to 4 treatments spaced 4 to 6 weeks apart a paired sample t-test showed a significant (p < 0.0001) decrease in SPEED2: "The combination of IPL and MGX can significantly improve dry eye symptoms — in this retrospective analysis in 89% of patients — and meibomian gland function which in this study improved in 77% of patients in at least one eye," says Dr "The study confirms that IPL treatment for meibomian gland dysfunction can improve dry eye symptoms and is a reasonable option for patients who have not shown improvement with other therapies." each patient underwent Fitzpatrick skin typing and the IPL machine was set to appropriate settings — 1D the eyelids were bilaterally closed and sealed shut with disposable eye shields A generous amount of ultrasonic gel was applied to the treated skin Then patients received about 30 pulses (with slight overlapping applications) from the right preauricular area across the cheeks and nose to the left preauricular area treating up to the inferior boundary of the eye shields Each treatment was followed by MGX with a cotton-tipped applicator and digital pressure to empty meibum from bilateral upper and lower eyelids Patients used preservative-free ketorolac drops twice a day for two days after IPL treatment Slit-lamp examination was performed before each treatment "Patients underwent four monthly examinations and IPL/MGX treatments or until symptoms were resolved to their satisfaction treatments became intolerable or they were unable to continue the treatment protocol," says Dr patients who responded early experienced 5 to 7 days of symptomatic improvement followed by regression until the next treatment patients experienced 1 to 2 weeks of improvement Slow responders did not see improvements until after the second or third treatment most patients had at least three months of sustained improvement 63% of this IPL/MGX-responsive group previously failed to respond to LipiFlow thermal pulsation," says Dr we recommend a single IPL/MGX treatment that varies between patients but most will require repeat single treatments every 3 to 6 months." Use of topical and systemic medications usually can be discontinued after IPL/MGX Since the Dry Eye Assessment and Management (DREAM) study results published in the New England Journal of Medicine in 2018 omega-3 fatty acids are no longer prescribed but many new treatments have come to market such as varenicline nasal spray and perfluorohexyloctane artificial tears "My philosophy is still focused more on restoring the ocular surface and tear film and less with artificial tear substitutes," says Dr There are newer IPL platforms since 2016 that have smaller sized application devices to handle the delicate eyelid area skin Vegunta S, et al. Combination therapy of intense pulsed light therapy and meibomian gland expression (IPL/MGX) can improve dry eye symptoms and meibomian gland function in patients with refractory dry eye: A retrospective analysis Dry Eye Assessment and Management Study Research Group. n−3 fatty acid supplementation for the treatment of dry eye disease Refer a patient to Mayo Clinic. Make a gift that can go twice as far to advance healthcare research. Make a gift that can go twice as far to advance healthcare research. © 1998-2025 Mayo Foundation for Medical Education and Research (MFMER) Metrics details Epidemiological studies associate an increase in breast cancer risk particularly triple-negative breast cancer (TNBC) This is more prevalent in African American women with significantly lower rate of breastfeeding compared to Caucasian women Prolonged breastfeeding leads to gradual involution (GI) whereas short-term or lack of breastfeeding leads to abrupt involution (AI) of the breast Our previous study utilizing a murine model demonstrated precancerous changes a non-obligate precursor of breast cancer in the mammary glands of AI mice Here we investigated mechanisms during early events of AI that prompts precancerous changes in mouse mammary glands Uniparous FVB/N mice were randomized to AI and GI on postpartum day 7 when all pups were removed from AI dams GI dams were allowed to nurse the pups till day 31 Cell death kinetics and gene expression were assessed by TUNEL assay and qPCR respectively Immune cell changes were investigated by flow cytometry cytokine array and multiplex immunofluorescence 3D-organoid cultures were used for in vitro assay of luminal progenitor cells and immunosuppressive myeloid cells infiltration leading to a chronically inflamed microenvironment GI elicits a more controlled immune response and extended cell death AI glands harbored more immunosuppressive myeloid-derived suppressor cells (MDSCs) and CD206 + M2-like macrophages AI glands exhibit an enrichment of CCL9-producing MDSCs and CD206 + M2-like macrophages that promote expansion of ELF5 + /ERα- luminal cells Multiplex imaging of AI glands demonstrated an increase in ELF5 + /WNT5a + luminal cells alongside a reduction in the ELF5 + /ERα + population when involution appeared histologically complete A significantly higher number of CD206 + cells in post involution AI gland attests to a chronically inflamed state induced by AI Our findings reveal significant disparities between AI and GI gland dynamics at the early phase of involution secreted by immune cells at the peak of cell death promotes expansion of Elf5 + /ERα- luminal progenitor cells the putative precursors of TNBC connecting early events of AI with increased breast cancer risk These data imply a significant mechanistic difference between involution following short-term vs Our lab noted similar observations in C57BL/6 mice (unpublished data) None of these changes were observed in gradually involuting dams allowed to nurse for 28–31 days postpartum (PPM) thus providing valuable insight into the mechanism of protective effects of prolonged breastfeeding against breast tumorigenesis The physiological significance of these mechanistic differences and the immune microenvironment could explain the epidemiological link between the length of breastfeeding and breast cancer risk Mice experiments were performed in accordance with a protocol approved by The Ohio State University and Institutional Animals Care and Use Committee strain# 001800) were maintained in-house in barrier cages Eight-week-old virgin mice were paired with males and housed individually once pregnant Only uniparous mice were used in all our studies litter size was standardized to 6 pups/dam Mice were randomized to GI and AI cohorts at 7 days PPM when all 6 pups were removed from the dams of the AI cohort gradual weaning was achieved by the removal of 3 pups each on PPM days 28 and 31 Mammary glands were harvested from the dams on PPM days 7 All experimental animals were humanely euthanized by CO2 inhalation followed by cervical dislocation before necropsy within the Molecular Signatures Database (MSigDB) Pathways with FDR q-value ≤ 0.25 and p-value ≤ 0.05 were considered for further investigation The right inguinal gland from each mouse (n = 3 mice per group per time point) after removal of the lymph node was snap-frozen Frozen mammary glands were disrupted in Trizol (Invitrogen USA) using a mechanical disruptor (Precellys 24 Touch USA) and total RNA was isolated using Norgen total RNA isolation kit (Norgen Biotek Corporation In-column DNase digestion was performed to remove genomic DNA contamination cDNA was synthesized from DNase-treated RNA using a cDNA synthesis kit (Applied Biosystems qRT-PCR analysis was performed in triplicates using a StepOne Real-Time PCR machine (Applied Biosystems Primer sequences are provided in Supplementary Table S1 Whole tissue extracts were prepared from snap-frozen mammary glands in RIPA buffer (50 mM Tris–HCL,150 mM NaCl 1% NP-40) using a tissue disruptor (Precllys Thirty micrograms of protein separated by SDS-PAGE routinely were probed with specific antibodies Primary antibodies used included anti-CD14 (Cell Signaling Technology Images were captured by LI-COR imaging system or on X-ray film using electrochemiluminescence based imaging system Primary antibodies used for IHC and IF were anti-pStat3Y705 (1:100 FFPE sections (4 μm) of mammary glands were subjected to TUNEL staining using the TUNEL Assay Kit Stained slides were imaged using Vectra 3.0 and analyzed using InForm software One thoracic mammary gland from each mouse (n = 6 mice per group per time point) was used for the analysis A minimum of twenty high-power images captured by Vectra 3.0 microscope were selected randomly for analysis Mammary gland sections were stained with F4/80 (Cell Signaling Technology #70076) by IHC Images of stained tissue sections containing at least 50% area composed of adipocytes were captured at 20 × magnification using EVOS Microscope (ThermoFisher Inc) Five images from each mammary gland section were quantified manually using ImageJ software Only structurally intact adipocytes were quantified Crown-like structures (CLS) were identified and quantified by two independent researchers CLS were identified as any adipocyte surrounded at least 50% by macrophages positive for F4/80 stain Mammary glands were harvested and chopped into fine pieces using a tissue chopper (McIlwain Tissue Chopper followed by digestion with 1X collagenase/hyaluronidase mixture in 5 mL of DMEM media supplemented with 10% FBS for 1 h at 37 °C The enzymes were neutralized by adding 9 mL of DMEM media with 10% FBS and filtered through a 70 μm cell strainer The single-cell suspensions were centrifuged at 350Xg for 4 min at 4 °C The cell pellets were suspended in 1 mL of RBC lysis solution (Gibco This was followed by adding of 10 mL phosphate buffer saline (PBS) and centrifuged at 350Xg for 5 min at 4 °C The cell pellets were suspended in Fc blocking buffer as per the manufacturer’s instructions (BioLegend The cells were washed and incubated with a cocktail of fluorophore-conjugated antibodies specific to different populations of inflammatory cells and Zombie Aqua live/dead dye for 30 min on ice Single-color controls were prepared in parallel the samples were acquired on Cytek Northern Lights-3000 and the data was analyzed using FlowJo software single-cell suspensions of mammary glands stained with different antibodies as described above were processed on BD flow activated cell sorter Aria III to collect CD11b + /F4/80 + /CD206 + populations The sorted cell population was subjected to cytokine array analysis (Ray Biotech USA) as per the manufacturer’s instructions an equal number of cells from each group were lysed using cell lysis buffer The array membranes were incubated for 30 min in a blocking buffer followed by incubation with the cell lysates at 4 °C overnight The membranes were next washed and incubated with a biotinylated secondary antibody cocktail and incubated with HRP-Streptavidin solution for 2 h at room temperature Membranes were then washed and exposed to X-ray film The films were scanned to obtain digital images and analyzed for expression intensity using ImageJ software (NIH The expression intensity density was normalized to internal control after subtracting the background For simultaneous antibody detection on FFPE mammary gland tissue sections mIF was carried out utilizing the Opal fluorophore coupled to tyramide signal amplification (TSA) system from Akoya Biosciences (Marlborough staining conditions were first adjusted using serial tissue sections that underwent tests for antibody stripping and pH- and temperature-dependent antigen retrieval Opal-antibody pairings were evaluated based on marker abundance and co-expression within cellular compartments to limit the possibility of spectral crosstalk high-expressing biomarkers were coupled with less intense Opals version 2.4.11) was utilized to evaluate biomarker signal-to-background ratio (signal: noise ≥ 10:1) and signal balance (signal intensity of adjacent Opals ≤ 10:1) into other channels The optimized mIF staining workflow consisted of antigen retrieval by incubating tissue sections in ER2 solution (pH 9 USA) for 30 min using the Leica Bond RX autostainer the first primary antibody (CCL9) was incubated for 30 min followed by anti-mouse/rabbit-HRP conjugate labeling and Opal-TSA polymer incubation for 10 min The stripping phase consisted of either ER1 (pH 6 Leica Biosystems) or ER2 solutions incubated at 95–98 °C for 20 min between each antibody staining round After staining with the second antibody (CCR1) the protocol was repeated until all the markers were applied The tissue sections were then counterstained for 5 min with spectral DAPI and mounted with ProLong® Diamond Antifade Mountant (Life Technology The Phenoimager HT multispectral digital slide imaging system (Akoya Biosciences) was used to capture whole-slide scans The mammary gland tissue images were scanned at desired magnification and 0.5 µm/pixel resolution with each region of interest (ROI) measuring 931 µm × 698 µm The entire slide scans were opened in Phenochart (Akoya Biosciences) for manual ROI annotation The margins of tissues were excluded due to the possibility of staining artifacts InFORM® was used to access the annotated ROIs for additional image processing such as spectral unmixing and autofluorescence correction Anti-CD20 antibody was paired with each Opal fluorophore (without DAPI counterstain) as a single plex to create a spectral library for spectral correction yielding a single component image InFORM® was utilized to generate machine-learning algorithms for cell segmentation and binary phenotyping using a set of component images This included instructing inFORM® to distinguish individual cells stratified by inclusion within tissue regions and by cell morphology and marker expression patterns to classify cells into positive or “other” categories normalized counts/exposure time per cytoplasm and/or nucleus) was utilized to compute the thresholds for marker positivity DAPI and ELF5 labeling allowed nuclei to be detected based on intensity and size while CCR1 staining aided in delineating membrane structures to refine borders relative to adjacent cells After optimizing the segmentation and phenotyping algorithm on a small subset of ROIs the semi-optimized algorithm was used in batch analysis on the remaining ROIs All ROIs were evaluated for agreement with machine learning such as harboring false positives due to fluorescence from nearby cells were retrained yielding the optimized algorithm the InFORM® data was merged and quantified using the R programming language in phenoptrReports (version 0.3.2 The cell density for each cell type was estimated for each selected image as the number of positive cells per area (cell counts/mm2) and then normalized to the total nucleated cells The average normalized cell percentage and standard error of all randomly selected images for a specific sample were calculated to reflect population and heterogeneity Images of immune-stained tissue sections from multiple mice were captured using Vectra 3.0 and analyzed using InForm® software Twenty random fields per tissue section were subjected to blinded quantification entire images of tissue sections were analyzed A positive signal threshold for each staining parameter was established prior to quantification the percentage of DAB-positive cells in the epithelium and stroma were assessed the total percent of the positive area was used for quantification Mean ± SD of the quantitative outcomes are shown on the figures Two sample or paired t-test was used for two group comparisons depending on whether the data is independent or correlated Linear mixed effects models were used to compare the outcomes with repeated measurements over time to take account of correlations among observations from the same subject Microarray data was analyzed using Transcriptome Analysis Console (TAC 4.0) and pathway enrichment with the differentially expressed genes was analyzed with the GSEA tool Holm’s procedure was used to adjust for multiple comparisons An adjusted p value < 0.05 was considered as significance The SAS 9.4 and R programs were used for data analysis Histological analysis of mammary glands undergoing gradual vs Schematic diagram showing the workflow of mice subjected to abrupt involution (AI) and gradual involution (GI) followed by different harvest times Representative images of H&E-stained sections of mouse mammary glands subjected to AI or GI and harvested at different days postpartum (PPM) as indicated Higher magnification images of H&E-stained section of GI mammary gland on indicated postpartum days 11 and 12: black arrows indicate large acini and clear arrows indicate epithelial cells Day17: clear arrows indicate epithelial cells curved black arrows indicate a small group of adipocytes; Day 22: clear arrows indicate flattened epithelial cells black arrows indicate the nuclear debris and arrowheads indicate repopulating adipocytes Day 25: arrow heads indicate repopulating adipocytes; black arrows indicate immune cells H&E-stained section of AI mammary gland on day 8.5: Grey arrow indicate lactating acini black arrowheads indicate flattened epithelial cells and black arrows indicate karyorrhectic debris Day 11: Grey arrows indicate collapsed alveoli black arrows indicate immune cells and arrowheads indicate adipocytes Day 12: arrowheads indicate adipose tissues Day17: arrow heads indicate adipose tissues Comparison of total number of adipocytes in AI and GI glands harvested on day 28 PPM Comparison of total number of adipocytes categorized by size in AI vs.GI glands and the number of crown-like structures (Supplementary Figs Apoptotic cells with condensed nuclei started appearing in the lumens of the AI glands as early as day 8.5 PPM while that was noted on and after day 17 PPM in the GI glands TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay revealed a sharp increase in cell death within 36 h of pup withdrawal (AI-day 8.5 PPM) that peaked on day 11 PPM (5.1 ± 0.02% positive cells) followed by an equally sharp decline in cell death and return to near baseline by day 17 PPM (0.42 ± 0.0005% positive cells) (Fig there was a gradual increase in cell death in GI glands after day 12 PPM that peaked on day 25 (3 ± 0.1% positive cells) followed by a gradual decline to baseline by day 31 PPM (0.35 ± 0.0004% positive cells) (Fig this shows that AI glands have significantly increased TUNEL-positive cells compared to the GI group averaged across all time points (p = 0.0005) predominantly due to the acute increase in cell death in AI glands between days 8.5 and 12 DNA damage response is heightened in AI glands compared to GI glands Representative images of FFPE sections of AI and GI mammary glands subjected to TUNEL assay at indicated time points (day PPM) Kinetics of cell death between days 7 and 28 PPM Enrichment plot of HALLMARK_DNA_REPAIR pathway in AI vs GI and AI glands harvested on day 120 PPM and in mammary glands of age-matched virgin mice BCL-xS and cleaved PARP in AI and GI mammary glands at early phase of involution in the GI gland increased 24 kDa cleaved PARP level coincided with peak of cell death that remained high through day 28 PPM our findings demonstrate a striking difference in the rate of cell death and degree of DNA damage between the AI vs the role of these genes during the gradual involution of mouse mammary glands remains unknown we next attempted to address whether there is a mere shift of key events and heightened expression of the APR genes during the peak of gradual involution (day 17–28 PPM) or alternatively if these changes are less pronounced thus contributing to the protective effect against breast cancer risk at all the time points were measured relative to the levels on day 7 PPM (lactating gland) Expression pattern of acute phase response genes is distinct in AI and GI glands Western blot analysis and quantitation of Lii Temporal pattern of pSTAT3Y705 positive cell distribution in AI and GI glands between day 7 and day 28 postpartum Representative images of FFPE sections of AI (day 11 PPM) and GI (day 22 and 25 PPM) mammary glands subjected to immunofluorescence analysis of pStat3Y705 (green) and CD14 (red) DAPI (blue) was used to counterstain the nuclei Bar diagram showing the number of pStat3.Y705 and CD14 double-positive cells per field of view (FOV) in mammary gland sections of AI (day 11 PPM) and GI (day 22 and 25 PPM) this data suggests a minimal role of luminal epithelial cells in the clearance of apoptotic cells during GI as opposed to AI Altogether, based on gene and protein expression analysis of the whole mammary glands, we see diverse and distinct patterns between AI and GI glands, which are summarized in Table 1 Abundance of myeloid-derived suppressor cells and M2-like macrophages in AI glands at the peak of cell death secreting CCL9 Temporal pattern of F4/80 positive cell distribution in AI and GI mammary glands harvested between days 7 and 25 PPM FFPE sections of mammary glands were subjected to IHC using F4/80 antibody Images of the stained slides were captured using Vectra 3.0 and analyzed using InForm software Mammary glands from AI-day 11 and GI-day 25 female mice were processed to identify different subpopulations of myeloid cells by multi-color flow cytometry The representative flow cytometric dot plots and graphs of % B CD11b + Gr1 + MDSCs (out of CD45 + cells); D CD11b + F4/80 + total macrophages (out of CD45 + cells); E MHCII + M1-like macrophages (out of CD45 + CD11b + F4/80 + total macrophages); F CD206 + M2-like macrophages (out of CD45 + CD11b + F4/80 + total macrophages); n = 3 per group CD11b + F4/80 + MHCII-CD206 + M2-like macrophages were sorted from mammary glands of AI-day 11 and GI-day 25 mice The cells were lysed and subjected to cytokine/chemokine array analysis The images show expression of cytokine/chemokine and bar diagrams showing CCL9 expression quantified using ImageJ software Quantitative RT-PCR analysis of Ccl9 expression in mammary glands over time (days 7 to 28 PPM) Quantitative RT-PCR analysis of Tgfβ3expression in mammary glands over time (days 7 to 28 PPM) Tgfβ3 expression is elevated again around day 22 PPM The marked overlap in the biphasic pattern of Ccl9 and Tgfβ3 expression in the AI glands suggests a possible role of TGFβ3 in regulating Ccl9 expression Tgfβ1 and Tgfβ2 expression was undetectable in both AI and GI glands these results show that at the peak of cell death AI glands have more MDSCs and M2-like macrophages compared to GI glands concomitant with elevated levels of CCL9 chemokine CCL9 promotes the expansion of Elf5 positive/ERα negative luminal progenitor cells Effect of murine CCL9 recombinant protein (100 ng/mL) on organoid culture of LP cells sorted from the mammary glands of 6-week-old female virgin mice treated for up to 12 days PBS-treated organoids are referred to as control Red arrowheads indicate filopodia-like structures Quantification of luminal outgrowths (branching) in PBS vs CCL9 treated LP cell-derived organoid on day12 of treatment LP cells isolated from AI and GI glands harvested on day 56 PPM are grown as organoids and imaged after 14 days in culture Quantitative RT-PCR analysis of Elf5 and E Esr1 expressions in organoids either treated with PBS or CCL9 protein for 12 days as described in Fig Multiplex imaging demonstrates a distinct profile of epithelial and immune cells in AI vs Schematic diagram showing the workflow of multiplexing imaging of selected markers in mammary glands of AI and GI mice at different time points n = 3 per group and the number of fields analyzed is n = 12 The representative 10 × merged figures (inset 40 × merged) and bar diagrams showing the normalized % of B ERα expressing ELF5 + LP cells in mammary glands of AI-day 11 vs The representative 10 × merged figures (inset 40 × merged) and bar diagrams showing the normalized E ERα + ELF5 + LP cells in mammary glands of AI-day 28 vs the significant increase in Elf5 + cells in AI glands demonstrates expansion of LP cell population compared to GI glands while ERα negativity of these cells signifies their potential to be precursors of basal-like breast cancer our multiplexed imaging of mammary glands harvested from AI vs GI mice at different time points has revealed the differential abundance of CCL9-producing CD206 + M2-like macrophages and ELF5 + LP cells with diverse functional statuses our findings demonstrate that the duration of breastfeeding (short-term vs prolonged) distinctly influences global remodeling of the post-lactational mammary gland environment This impact extends beyond temporal considerations and includes quantitative and qualitative differences in apoptosis and expression patterns of genes related to DNA damage Key factors involved in differential regulation of abrupt involution in mouse mammary gland and its long-term impact Milk stasis and massive cell death during AI triggers Acute phase response (APR) and cascade of events regulated predominantly by TGFβ3 and STAT3 directly (solid arrows) or indirectly (broken arrows) Schematic summarizing the early events during abrupt vs gradual involution leading to precancerous changes in the AI gland as opposed to remodeling to near pre-pregnancy state in the GI glands The precise mechanism of adipocyte repopulation during GI is beyond the scope of this study though it is a worthy topic for further investigation had late and relatively sustained elevated levels in GI glands This could lead to overactivation of PARP in the AI glands blocking cell death and allowing damaged cells to propagate unlike in GI glands The subtle differences observed during cell death in AI glands could be due to differences in mouse strains and are worthy of further study Chi3L expression is sustained over a longer period of time suggesting extended inflammatory state in AI glands in contrast with GI glands Analysis of APR genes clearly demonstrated a distinct pattern of involution in AI vs GI mammary glands in mice that could contribute to increased breast cancer risk following lack of or short-term breastfeeding Our study reveals similar findings where only ~ 3% of the ELF5 + cells are ERα positive in the GI gland This provides additional evidence that our murine model is a valuable animal model to study breast involution reduced ELF5 + /ERα + cell population in spite of higher ELF5 + cells in the AI gland indicates predominance of ERα-/ ELF5 + cells in the AI-day 28 glands supporting our in vitro data and highlighting the distinct molecular events underlying AI vs Our findings collectively relate AI to increased breast cancer risk and highlight the fact that early events during mammary gland involution could have a profound impact on future breast cancer risk While addressing racial disparity in cancer outcomes should include addressing barriers to breastfeeding in Black women a mechanistic understanding of differences between the effect of prolonged vs a lack of or short-term breastfeeding addressed in this study will lead to options for preventive measures While our work has shown a distinct impact of AI vs increasing the risk of breast cancer with further hits the lack of a breast cancer model that shows development of invasive cancer just by altering the process of involution is a limitation AI shows that AI triggers rapid cell death leading to heightened inflammation and promoting infiltration of myeloid cells GI induces a controlled immune response resulting from prolonged cell death facilitating comprehensive mammary gland remodeling CCL9 secreted by myeloid cells in the AI glands but not in GI glands promotes the expansion of ERα negative luminal progenitor cells thus connecting early events during AI to increased risk of breast cancer From the public health perspective this work is highly significant and impactful Understanding the mechanisms will increase prevention strategies even for those mothers who cannot breastfeed and empowering all mothers and providers with this knowledge of risk reduction will improve the uptake of breastfeeding practices and improve racial disparities in breast cancer mortality No datasets were generated or analysed during the current study Parity and breastfeeding among African-American women: differential effects on breast cancer risk by estrogen receptor status in the Women’s Circle of Health Study Estrogen receptor positive tumors: do reproductive factors explain differences in incidence between black and white women AC: Breast Cancer Facts & Figures 2022–2024 Outcome disparities in African American women with triple negative breast cancer: a comparison of epidemiological and molecular factors between African American and Caucasian women with triple negative breast cancer Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways Genomic signature induced by pregnancy in the human breast Persistent parity-induced changes in growth factors and differentiation in the rodent mammary gland collaborative reanalysis of individual data from 47 epidemiological studies in 30 countries including 50302 women with breast cancer and 96973 women without the disease A collaborative study of the etiology of breast cancer subtypes in African American women: the AMBER consortium Palmer JR, Viscidi E, Troester MA, Hong CC, Schedin P, Bethea TN, Bandera EV, Borges V, McKinnon C, Haiman CA, et al. Parity, lactation, and breast cancer subtypes in African American women: results from the AMBER Consortium. J Nat Cancer Inst. 2014. https://doi.org/10.1093/jnci/dju237 Reproductive history and oral contraceptive use in relation to risk of triple-negative breast cancer and hyperplasia linking lack of breastfeeding with increased risk of breast cancer Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers cPLA2 blockade attenuates S100A7-mediated breast tumorigenicity by inhibiting the immunosuppressive tumor microenvironment Fine-needle aspiration-based patient-derived cancer organoids BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis Conditional deletion of Stat3 in mammary epithelium impairs the acute phase response and modulates immune cell numbers during post-lactational regression Autophagy and unfolded protein response (UPR) regulate mammary gland involution by restraining apoptosis-driven irreversible changes The molecular signatures database (MSigDB) hallmark gene set collection Mammary stem cells and tumor-initiating cells are more resistant to apoptosis and exhibit increased DNA repair activity in response to DNA damage Involution: apoptosis and tissue remodelling that convert the mammary gland from milk factory to a quiescent organ Bax and Bcl-xs are induced at the onset of apoptosis in involuting mammary epithelial cells Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration Stat3 controls lysosomal-mediated cell death in vivo Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization C/EBPdelta is a crucial regulator of pro-apoptotic gene expression during mammary gland involution Anti-inflammatory and antimicrobial roles of secretory leukocyte protease inhibitor Inhibitory activity of YKL-40 in mammary epithelial cell differentiation and polarization induced by lactogenic hormones: a role in mammary tissue involution Matrix metalloproteinases and their expression in mammary gland Diverse myeloid cells are recruited to the developing and inflamed mammary gland The immune environment of the mammary gland fluctuates during post-lactational regression and correlates with tumour growth rate Slit2 inhibits breast cancer metastasis by activating M1-like phagocytic and antifibrotic macrophages CCL9 Induced by TGFbeta signaling in myeloid cells enhances tumor cell survival in the premetastatic organ Mammary gland involution provides a unique model to study the TGF-beta cancer paradox Transforming growth factor beta3 induces cell death during the first stage of mammary gland involution Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages Generation of a functional mammary gland from a single stem cell The Ets transcription factor Elf5 specifies mammary alveolar cell fate Breastfeeding and Breast Cancer Risk Reduction: Implications for Black Mothers breast-feeding and risk of triple negative breast cancer: the Breast Cancer Etiology in Minorities (BEM) study Rac1 controls cell turnover and reversibility of the involution process in postpartum mammary glands Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution Adipocyte hypertrophy and lipid dynamics underlie mammary gland remodeling after lactation BCL-w: apoptotic and non-apoptotic role in health and disease and invasion in cells in triple-negative breast cancer Secretory leukocyte protease inhibitor (SLPI) as a potential target for inhibiting metastasis of triple-negative breast cancers CHI3L1 plays a role in cancer through enhanced production of pro-inflammatory/pro-tumorigenic and angiogenic factors Stem Cells and the Differentiation Hierarchy in Mammary Gland Development The protective role of pregnancy in breast cancer Pregnancy-associated breast cancer: a diagnostic and therapeutic challenge and survival in the Carolina Breast Cancer Study Descriptive analysis of estrogen receptor (ER)-negative the so-called triple-negative phenotype: a population-based study from the California cancer Registry Differences in breast carcinoma characteristics in newly diagnosed African-American and Caucasian patients: a single-institution compilation compared with the National Cancer Institute’s Surveillance Download references We thank Angela Dahlberg of Ohio State University for her editorial help and Dr This research is supported by NIH R01 (CA231857) to B.R & R.K.G. NIH R01 (CA292020) and Department of Defense Breast Cancer Breakthrough (W81XWH1910088) awards to R.K.G. and Department of Defense grant (W81XWH2210019) to A.E.V Research reported in this publication was supported by The Ohio State University Comprehensive Cancer Center and the National Institutes of Health under grant number P30 CA016058 (OSUCCC Genomics core facility Sanjay Mishra and Neelam Shinde have contributed equally to this work The Ohio State University Comprehensive Cancer Center Gautam Sarathy & Bhuvaneswari Ramaswamy Department of Biomedical Informatics and Center for Biostatistics Mi.; Writing-Reviewing & Editing: B.R. All animal studies were performed according to the Institutional Animal Care and Use Committee of the Ohio State University guidelines Download citation DOI: https://doi.org/10.1186/s13058-024-01933-3 It seems as if eye doctors who treat the ocular surface and eyelids have been looking for a safe and effective treatment for the signs and symptoms of meibomian gland disease forever Has there ever been a treatment that successfully passed muster with the FDA and became approved to treat meibomian gland disease (MGD) on label Only TobraDex (tobramycin/dexamethasone ophthalmic suspension I can’t remember if Alcon was able to pull that off My beloved AzaSite (azithromycin ophthalmic solution 1% my visit to Inspire headquarters pre-2010 sparked interest but Inspire didn’t survive long enough to take a chance on a study in patients who are diagnosed with both MGD and grade 2 or higher Demodex Fifteen central glands were assessed in each patient at baseline and days 43 and 85 number of glands excreting oil and percent of glands improved to clear oil Patient symptoms were evaluated using visual analog scale (VAS) scoring Both Xdemvy and its vehicle were very well tolerated with only two adverse events in the Xdemvy cohort No subjects discontinued the trial due to medication-related reasons Statistically significant positive changes were seen in the Xdemvy trial in all areas tested Glands were scored as 0: no excretion; 1: thick opaque; 2: cloudy; or 3: clear (higher score equals better function) Scores improved from 21.9 at baseline to 27.8 at day 43 and 33.2 at day 85 Glands yielding oil went from 7.1 to 10.7 at day 43 and 12.7 out of 15 at day 85 Subjects with more than three glands improving to “clear” were 44.7% at day 43 and 78.9% at day 85 Xdemvy improved objective measurements of meibomian gland function across the board Patients care about how they feel and how they look Pretty much the same story across the board VAS scores symptoms from 0 (none) to 100 (it’s all you can think about) were measured at baseline Fluctuating vision decreased from 46.5 to 22.2 to 13.1 The same thing was seen with itching (47 to 16 to 11.4 75%) and eyelid redness (43.6 to 16.6 to 12.2 Bottom line: Xdemvy appears to be a safe and effective treatment for the signs and symptoms of MGD in the presence of Demodex What does this mean in our dry eye disease (DED) clinics? I think this data give us a clear path to consider Xdemvy when we make a diagnosis of evaporative DED (poor tear function) along with poor gland function and Demodex infestation Depending on your core treatment philosophy you could certainly use Xdemvy as a single agent in this setting I find myself using Xdemvy to go after the underlying cause of the DED while simultaneously treating the tears themselves with a second agent This is all super new; time will tell if one strategy is better than the other it sure looks like we finally have a winner to treat MGD Get the latest news and education delivered to your inbox The email address associated with your Healio account is: If you would like to edit or change the email address that your subscriptions and alerts are sent to You'll receive reminders to complete your saved activities from Healio CME Metrics details has gained heightened attention following recent reports of highly pathogenic avian influenza in dairy cattle and cow-to-human transmission in the USA This review explores general aspects of influenza A virus (IAV) biology and discusses the key considerations for developing vaccines to prevent or curtail IAV infection in the bovine mammary gland and its spread through milk we provide a brief overview of the biology and natural history of influenza A virus (IAV) infections and describe the key mechanisms involved in the interactions between IAV and its mammalian hosts We also highlight the main recent observations on HPAIV infections in dairy cows and discuss the major aspects to be considered for the development of candidate vaccines able to prevent productive IAV replication in the mammary gland and its spread through milk while the RNA termini are associated with the three subunits (PA PB2) of the viral RNA-dependent RNA polymerase Following HA binding to sialic acids (SAs) linked to cellular glycoproteins After fusion of the viral envelope with the endosome membrane where both viral mRNAs transcription and viral genome replication occur providing multiple opportunities for reassortment events The peptide sequence corresponding to the cleavage site is shown on the right (blue rectangle highlights the stretch of basic residues upstream of the cleavage site) Alignment was performed using the “emma” and “shoawalign” tools in EMBOSS and manually edited A few representative sequences from the Eurasian set of H5 sequences (up to year 1996) were retrieved from the Influenza virus resource corresponding to previous HPAIVs in Europe These observations demonstrate that the specific SA present in bovine tissues enable avian or mammalian-origin IAV to infect this species For details refer to60 raw milk and raw milk-based products from affected farms represent a risk for human consumers and other animals Efficient vaccines against bovine mastitis are still lacking85 the extensive trial-and-error efforts over the last decades to set up improved solutions have provided valuable insights into mammary gland immunity This accumulated knowledge could contribute significantly to the design of candidate vaccines aimed at preventing viral proliferation in the bovine mammary gland and its subsequent dispersal via milk These observations indicate that vaccine formulations capable of inducing not only a neutralizing IgG response but also mucosal and cellular immune responses in the mammary gland might represent ideal candidates to limit symptoms and viral spread during HPAIV infections in cattle This finding indicates that the induction of local influenza-specific antibodies might contribute to viral clearance from the udder but further research is necessary to clarify the nature of involved Igs if it can be modulated through vaccination These observations suggest that the capacity to efficiently trigger local CMI mechanisms should be incorporated into the requirements of a candidate vaccine designed to prevent HPAIV infections in the bovine mammary gland an immunization strategy taking advantage of the intramammary route could promote the elimination of HPAIV at the site of entry in case of intramammary transmission and/or prevent productive viral replication in the mammary gland and dispersal in milk the development of an intramammary vaccine candidate against HPAIV necessitates careful consideration in the design of formulations including the selection of appropriate adjuvants to avoid compromising the role of the mammary gland as a milk-producing organ vaccinology and bovine immunology will be essential to achieve this objective Highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus infection in domestic dairy cattle and cats Avian influenza A(H5N1) virus among dairy cattle Outbreak of highly pathogenic avian influenza A(H5N1) viruses in U.S dairy cattle and detection of two human cases - United States Genome-wide analysis of influenza viral RNA and nucleoprotein association Examining the hemagglutinin subtype diversity among wild duck-origin influenza A viruses using ethanol-fixed cloacal swabs and a novel RT-PCR method The evolutionary genetics and emergence of avian influenza viruses in wild birds Pathobiological origins and evolutionary history of highly pathogenic avian influenza viruses Natural history of highly pathogenic avian influenza H5N1 Highly pathogenic avian influenza viruses at the wild-domestic bird interface in Europe: future directions for research and surveillance Role for migratory wild birds in the global spread of avian influenza H5N8 Highly pathogenic avian influenza A virus (HPAIV) H5N1 infection in two European grey seals (Halichoerus grypus) with encephalitis The episodic resurgence of highly pathogenic avian influenza H5 virus Recent changes in patterns of mammal infection with highly pathogenic avian influenza A(H5N1) virus worldwide Highly pathogenic H5N1 avian influenza virus outbreak in cattle: the knowns and unknowns European surveillance network for influenza in pigs: surveillance programs diagnostic tools and Swine influenza virus subtypes identified in 14 European countries from 2010 to 2013 Review of influenza A virus in swine worldwide: a call for increased surveillance and research Equine-Like H3 avian influenza viruses in wild birds Estimates of global seasonal influenza-associated respiratory mortality: a modelling study Zoonotic animal influenza virus and potential mixing vessel hosts Outbreak of influenza A(H7N2) among cats in an animal shelter with cat-to-human transmission-New York City Avian influenza A(H10N7) virus-associated mass deaths among harbor seals Highly pathogenic avian influenza A(H5N1) virus infection in farmed minks Highly pathogenic avian influenza A(H5N1) virus infection on multiple fur farms in the South and Central Ostrobothnia regions of Finland Sialic acid receptors: the key to solving the enigma of zoonotic virus spillover Adaptation of influenza viruses to human airway receptors A human isolate of bovine H5N1 is transmissible and lethal in animal models Assembly of endocytic machinery around individual influenza viruses during viral entry Innate immunity to influenza virus infection The dual role of innate immunity during influenza New fronts emerge in the influenza cytokine storm The pathology of influenza virus infections Immune response in influenza virus infection and modulation of immune injury by viral neuraminidase Apoptosis and pathogenesis of avian influenza A (H5N1) virus in humans Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia Local and systemic cytokine responses during experimental human influenza A virus infection Relation to symptom formation and host defense Severe seasonal influenza in ferrets correlates with reduced interferon and increased IL-6 induction Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals Risk assessment of a highly pathogenic H5N1 influenza virus from mink Pathogenic potential of interferon alphabeta in acute influenza infection Protective role of beta interferon in host defense against influenza A virus Host immune response to influenza A virus infection Human influenza viruses and CD8(+) T cell responses Influenza A in bovine species: a narrative literature review Experimental infection of cattle with highly pathogenic avian influenza virus (H5N1) Influenza A virus receptor identification in the respiratory tract of quail Comparative distribution of human and avian type sialic acid influenza receptors in the pig Avian flu: influenza virus receptors in the human airway Sialic acid receptor specificity in mammary gland of dairy cattle infected with highly pathogenic avian influenza A(H5N1) virus The avian influenza A virus receptor SA-alpha2,3-Gal is expressed in the porcine nasal mucosa sustaining the pig as a mixing vessel for new influenza viruses Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle Evidence of influenza A virus infection in dairy cows with sporadic milk drop syndrome Detection of antibodies to influenza A virus in cattle in association with respiratory disease and reduced milk yield Significant rising antibody titres to influenza A are associated with an acute reduction in milk yield in cattle Cells and cytokines in inflammatory secretions of bovine mammary gland Immune defenses of the mammary gland epithelium of dairy ruminants Innate immune pattern recognition: a cell biological perspective Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells but not Staphylococcus aureus triggers an early increased expression of factors contributing to the innate immune defense in the udder of the cow Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells In depth analysis of genes and pathways of the mammary gland involved in the pathogenesis of bovine Escherichia coli-mastitis Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling Association between bovine leukemia virus proviral load and severity of clinical mastitis Quantifying production losses associated with foot and mouth disease outbreaks on large-scale dairy farms in Rift valley Invited review: the role of contagious disease in udder health The growth and persistence of foot-and-mouth disease virus in the bovine mammary gland Viral infections and bovine mastitis: a review Role of bovine herpesvirus 4 in bacterial bovine mastitis Infection of cattle with parainfluenza 3 virus with special reference to udder infection Presence of viral and bacterial organisms in milk and their association with somatic cell counts with particular reference to the mammary gland Further experiments relating to the propagation of virus in the bovine mammary gland Understanding the transmission of foot-and-mouth disease virus at different scales Bovine leukaemia virus: current epidemiological circumstance and future prospective Emergence and interstate spread of highly pathogenic avian influenza A(H5N1) in dairy cattle Experimental reproduction of viral replication and disease in dairy calves and lactating cows inoculated with highly pathogenic avian influenza H5N1 clade 2.3.4.4b Does pasteurization inactivate bird flu virus in milk Strain-dependent variations in replication of European clade 2.3.4.4b influenza A(H5N1) viruses in bovine cells and thermal inactivation in semi-skimmed or whole milk Characterization of highly pathogenic avian influenza virus in retail dairy products in the US Progress towards the elusive mastitis vaccines Understanding immunity to influenza: implications for future vaccine development Influenza vaccines: successes and continuing challenges Mucosal correlates of protection after influenza viral challenge of vaccinated and unvaccinated healthy volunteers Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection The human antibody response to influenza A virus infection and vaccination The role of cell-mediated immunity against influenza and its implications for vaccine evaluation Bovine natural antibodies in antibody-dependent bactericidal activity against Escherichia coli and salmonella typhimurium and risk of mastitis Immune defences of the mammary gland in dairy ruminants Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S Immune and experimental infection responses of dairy cows vaccinated with the combination of six Staphylococcus aureus proteins that are expressed during bovine intramammary infection and a triple adjuvant Local immunization impacts the response of dairy cows to Escherichia coli mastitis The antibody response in the bovine mammary gland is influenced by the adjuvant and the site of subcutaneous vaccination Cellular immune correlates of protection against symptomatic pandemic influenza Baseline innate and T cell populations are correlates of protection against symptomatic influenza virus infection independent of serology Adaptive cell-mediated immunity in the mammary gland of dairy ruminants Enhancing cross-protection against influenza by heterologous sequential immunization with mRNA LNP and protein nanoparticle vaccines but not a high systemic Th1 immune response is associated with protection against E Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk Identifying and addressing barriers and opportunities for bovine respiratory disease complex vaccination: a consensus paper on practical recommendations for best practise vaccination Alarming situation of emerging H5 and H7 avian influenza and effective control strategies Download references Pascal Rainard for the scientific revision of the manuscript This work was funded by the Agence Nationale de la Recherche (ANR-20-CE20-0023 to R.P.M.) and Carnot France Future Elevage (PIGIMMUNITY to I.C.P.) Sascha Trapp & Ignacio Caballero-Posadas Writing—original draft preparation: R.P.M. and P.G.; Writing—review & editing: R.P.M. Download citation DOI: https://doi.org/10.1038/s41541-025-01063-7 Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research Metrics details Venom is a remarkable innovation found across the animal kingdom yet the evolutionary origins of venom systems in various groups we investigated the organogenesis of the venom apparatus in the common house spider The venom apparatus consists of a pair of secretory glands each connected to an opening at the fang tip by a duct that runs through the chelicerae We performed bulk RNA-seq to identify venom gland-specific markers and assayed their expression using RNA in situ hybridisation experiments on whole-mount time-series These revealed that the gland primordium emerges during embryonic stage 13 at the chelicera tip progresses proximally by the end of embryonic development and extends into the prosoma post-eclosion The initiation of expression of an important toxin component in late postembryos marks the activation of venom-secreting cells Our selected markers also exhibited distinct expression patterns in adult venom glands: sage and the toxin marker were expressed in the secretory epithelium forkhead and sum-1 in the surrounding muscle layer while Distal-less was predominantly expressed at the gland extremities Our study provides the first comprehensive analysis of venom gland morphogenesis in spiders offering key insights into their evolution and development the evolutionary and developmental origins of such key evolutionary innovations Expression values are row scaled log2(TPM + 1) there has not been a systematic investigation of the time-series of the development of the spider venom apparatus these observations do not conclusively clarify whether the venom gland primordium originates distally or proximally in the chelicera we aimed to bridge this knowledge gap by providing a more comprehensive description of the development of the venom apparatus in P tepidariorum using both morphological observations and analyses of expression patterns of selected markers at different time points during embryonic and post-embryonic development stages as well as in adult venom glands The postembryos and first instar stages were further categorised into ‘early’ and ‘late’ stages based on specific developmental features (see below) To identify developing venom gland-specific markers we dissected spiders at four days post-hatching (late first instar stage) and extracted mRNA from the venom glands Due to the minute size and delicate nature of the venom glands We obtained two replicates for the prosoma and opisthosoma The RNA-seq libraries ranged in size from 45 to 61 M reads we found 101 which were expressed in the juvenile venom gland; of these 30 of which were also expressed in the juvenile venom gland as well as ‘TATA-binding protein-associated factor 2N-like’ which were specific in both juvenile and adult glands lacked orthologs in other species according to the OrthoDB annotation The experiments were divided into two multi-HCR sets: set 1 comprising toxin Expression of venom gland markers in adult venom glands (a) Whole-mount dissected venom gland with toxin Top row with ×10 dry objective and scale bar 100 μm rows below correspond to higher magnification (×40 oil) images of the proximal (P) (b) Whole-mount venom gland with fkh and sage expression merged sage and fkh were expressed in distinct patterns in the adult glands (Fig. 4b) was predominantly expressed within the muscular layer surrounding the glandular tissue sage was widely expressed within the secretory tissue After assaying the expression of the selected markers in adult venom glands we proceeded with whole-mount HCR experiments on post-hatched juvenile spiders We mounted the prosoma with either the dorsal or ventral side down but we only detected signals when the spiders were mounted in the latter orientation Expression of venom gland markers in the prosoma of postembryos and first instars and Dll in whole-mount postembryos (PE) early and late stages and first (1st) instars early and late stages The images are taken from the dorsal side with a ×40 oil objective Signals from venom glands are indicated with arrows (c) Schematic of the focal plane used for image acquisition progressing from early postembryos to late first instars we observed a change in marker expression from within the chelicerae towards the dorsal side of the prosoma Expression of venom gland markers in stage 14 embryos (a) Expression of sum-1 and Dll in stage 14.1 and 14.2 embryos sum-1 is expressed in the venom glands (arrows) Dll expression (arrowheads) is observed in the appendages including the chelicerae (b) HCR signals of fkh and sage in stage 14.1 and 14.2 embryos Signal in venom glands marked with an arrow Embryonic expression of venom gland markers in stage 13 embryos (a) Expression of sum-1 and Dll in stage 13.1 and 13.2 embryos Dll is expressed in the legs (arrowheads) and on the dorsal side at the tip of the chelicerae (arrow) and zoom-in) Additional punctiform signals are also observed in the coxae (arrowheads) sum-1 is expressed on the dorsal side of the chelicerae (arrow and zoom-in) as well as in the coxae and leg joints (arrowheads) (b) Expression of fkh and sage in stage 13.1 and 13.2 embryos sage expression corresponds to the venom gland rudiments (arrows) which are now closer to the tip of the chelicerae In stage 13.1 sage and fkh are expressed at the base of the epidermal layer of the chelicerae (arrow) We also assayed the expression of our focal genes at stages 9 and 12, but no venom gland-specific signals were detected (Supplementary Fig. S7, S8) Schematic representation of venom gland marker expression during development fkh expression is weak and therefore represented as dots sage is exclusively expressed in the gland primordium at stage 13 while sum-1 has a broader expression at the distal end of the chelicerae Dll is broadly expressed at the distal end of the chelicerae sum-1 is specifically expressed in the muscle layer around the gland rudiments sage and fkh are also expressed in venom glands The toxin begins to be expressed in the late postembryos In postembryos and first instars the expression pattern is similar to the adult but the venom glands are still close to the chelicerae we describe the emergence and development of the venom apparatus in the common house spider The fangs are not yet apparent in embryos and only become visible in the postembryos. The venom apparatus reaches its final form in the early first instar, coinciding with the spiders leaving the cocoon and starting to build the communal web. At this stage, the fangs are developed, possibly ready to deliver venom (Fig. 2) which exhibited upregulation in the venom glands This suggests that the specificity of sum-1 in the muscle fibres surrounding the glandular tissue may be restricted to spiders it is plausible that a similar network has been recruited in spiders we observed Dll expression in the embryonic chelicerae and the distal region of the other appendages We also found Dll expression in the fangs and in adult venom glands The role of this developmental gene in the adult glands requires further investigation Zhu and colleagues19 advocated the silk gland origin hypothesis based on similarities in the transcriptome profiles of the two organs the absence of key tissues and animal groups in the analysis such as coxal glands or salivary glands of other arachnid lineages may have limited consideration of alternative scenarios The absence of sage signal in the silk gland primordia of P along with the lack of expression in adult silk glands in this and other spider species suggests that there is not a direct evolutionary link between venom and silk glands the expression of Dll and sum-1 in the embryonic coxae could suggest a potential link between the venom glands and these organs we argue against this interpretation because both Dll and sum-1 signals were observed in other parts of late embryos the tissue specific expression of sage in both adult and embryonic venom glands supports the salivary gland hypothesis While the expression patterns of our markers indicate that spider venom glands may have evolved from salivary glands spatial expression patterns of additional transcription factors will be crucial to build a more comprehensive understanding of their evolutionary and developmental origins Additional expression data from other glands and arachnid lineages will also aid in elucidating the evolutionary relationship among these exocrine glands tepidariorum colony was kept at 25 °C and humidity of 60% with a gradual light/dark cycle of 16/8 h Adults were kept individually in plastic vials with a coconut husk substrate and fed twice a week with Musca domestica flies; the cocoons were transferred into Petri dishes and juveniles fed with Drosophila sp tepidariorum spiderlings following egg eclosion specifically focusing on postembryos and first instars Specimens were anaesthetised with CO2 prior to dissection of the chelicerae and venom glands Specimens for histological analysis were fixed in 2.5% glutaraldehyde for 1 h at room temperature and subsequently fixed overnight at 4 °C After several washes with 0.1 M phosphate buffer the sample were postfixed in 2% osmium tetroxide solution for 1 h at room temperature dehydrating through a graded series of acetone and embedding using Spurr Low-Viscosity Embedding kit (SIGMA) The samples were incubated in resin blocks at 60 °C for 48 h Semi-thin sections were obtained with a Leica EM UC7 Ultramicrotome (Leica Microsystem) with a DiATOME diamond knife (Diatome Ltd. Sections were stained with 0.5% toluidine blue Images of histological sections were acquired using Zeiss Axio Imager Z2 (Leica Microsystem) To identify genes expressed in venom glands suitable as markers for in situ HCR experiments At four days post-hatching (first instars) the remaining prosoma and opisthosoma from 25 spiders Given the small size and delicate nature of venom glands they were left attached to the chelicerae to prevent damage we pooled 5–15 individuals in each biological replicate we pooled all the 25 samples and obtained only one replicate Total RNA was isolated using the TRIzol™ Plus RNA Purification kit (ThermoFisher) following the manufacturer’s instructions with an additional on-column DNA purification step seven cDNA libraries were generated using the TruSeq RNA Sample Preparation kit (Illumina) with 150 base pair read length The libraries were subjected to pair-end sequencing on an Illumina NovaSeq at the Genomic Technologies Facility of the University of Lausanne we analysed gene expression from published SRA libraries of adult spiders and those defined in this study for the post-eclosion stages we investigated gene expression patterns in dissected adult venom glands Spiders were anaesthetised using CO2 and the opisthosoma removed opisthosoma and legs were removed where possible to facilitate the penetration of the fixative and probes Dissections were performed in 0.1% PBS-Tween-20 (PBS-T) Embryos and post-hatched spiders were dechorionated with 3% bleach for 3–4 min then rinsed 3 or 4 times with MilliQ water samples were fixed in 1:1 37% formaldehyde:heptane overnight at 4 °C on a rotor at low speed (< 20 rpm) and ice-cold methanol was poured onto the heptane supernatant and mixed vigorously for 1 min The methanol/heptane mix was removed and replaced with 100% methanol and sample stored at − 20 °C We performed two sets of multi-FISH experiments: set 1 included toxin (Alexa-488) and Dll (Alexa-594); set 2 included sage (Alexa-647) and fkh (Alexa-488) For each experiment and developmental stage Image acquisition and analysis: Z-stack images were acquired with a Stellaris 5 White Light Laser (Leica Microsystems) inverted confocal microscope using HC PL APO CS2 10× dry (NA 0.4), 20× dry (NA 0.75), 40× oil immersion (NA 1.30), and 63× oil immersion (NA 1.4) objectives (see Table S1 for full details) and z-stacks were collected using z Galvo with the following settings: z size 1.2 μm Excitation wavelengths were set as following: Alexa 488 at 494 nm were acquired and visualised with the same parameters and three dimensional (3D) reconstructions were performed with Imaris ×64 9.8.2 The RNA-seq libraries have been deposited in the NCBI SRA archive with the accession number PRJNA1087249. The HCR images and the R code to analyse the RNA-seq data have been archived at https://doi.org/10.5281/zenodo.10813380 Additional data are provided as supplementary information files The diversity of venom: The importance of behavior and venom system morphology in understanding its ecology and evolution Animal toxins—Nature’s evolutionary-refined toolkit for basic research and drug discovery and future perspectives in biological and applied animal venom research Complex cocktails: The evolutionary novelty of venoms Venom systems as models for studying the origin and regulation of evolutionary novelties Cupiennius salei and Achaearanea tepidariorum: Spider models for investigating evolution and development Evolutionary crossroads in developmental biology: The spider Parasteatoda tepidariorum The common house spider Parasteatoda tepidariorum Testing adaptive radiation and key innovation hypotheses in spiders Barth, F. G. A spider’s world (Springer, Berlin, 2002). https://doi.org/10.1007/978-3-662-04899-3 The form and function of spider orb webs: Evolution from silk to ecosystems The biology and evolution of spider venoms Biology of spiders (Oxford University Press The embryology of the black widow spider Latrodectus mactans (Fabr.) Embryonic and postembryonic morphogenesis of the visual venom- and silk-gland systems in two species of Peucetia (Araneae: Oxyopidae) Developmental biology of the Brazilian ‘Armed’ spider Phoneutria nigriventer (Keyserling 1891): microanatomical and molecular analysis of the embryonic stages Genomic and transcriptomic analyses support a silk gland origin of spider venom glands Embryonic development and staging of the cobweb spider Parasteatoda tepidariorum C 1841 (syn: Achaearanea tepidariorum; Araneomorphae; Theridiidae) Alternative transcription at venom genes and its role as a complementary mechanism for the generation of venom complexity in the common house spider Convergent evolution of venom gland transcriptomes across Metazoa Fork head and Sage maintain a uniform and patent salivary gland lumen through regulation of two downstream target genes Appendage patterning in the South American bird spider Acanthoscurria geniculata (Araneae: Mygalomorphae) Patterning mechanisms and morphological diversity of spider appendages and their importance for spider evolution Novel Function of distal-less as a gap gene during spider segmentation Preliminary studies on the toxicity of black widow spider eggs Isolation and preliminary characterization of proteinaceous toxins with insecticidal and antibacterial activities from black widow spider (L Transcriptome analysis to understand the toxicity of Latrodectus tredecimguttatus eggs An overview of the basic helix-loop-helix proteins Early genes required for salivary gland fate determination and morphogenesis in Drosophila melanogaster Specification of cell fates within the salivary gland primordium From fate to function: the Drosophila trachea and salivary gland as models for tubulogenesis Genome-wide analysis reveals a major role in cell fate maintenance and an unexpected role in endoreduplication for the Drosophila foxA gene fork head Sarmiento, C. E. Hymenopteran venom apparatus. In Encyclopedia of social insects (ed. Starr, C. K.) 504–512 (Springer, Cham, 2021). https://doi.org/10.1007/978-3-030-28102-1_63 Bmsage is involved in the determination of cell number in the silk gland of Bombyx mori sage controls silk gland development by regulating Dfd in Bombyx mori The embryonic origin of the ampullate silk glands of the spider Cupiennius salei Ultrastructural changes in the nymphal salivary glands of the rabbit tick Fine structure of the salivary glands of unfed male Dermacentor variabilis (Say) (Ixodoidea: Ixodidae) Morpho-functional variety of the coxal glands in cheyletoid mites (Prostigmata) fastp: an ultra-fast all-in-one FASTQ preprocessor Near-optimal probabilistic RNA-seq quantification Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences OrthoDB v10: Sampling the diversity of animal bacterial and viral genomes for evolutionary and functional annotations of orthologs FlyBase: A guided tour of highlighted features The Bgee suite: Integrated curated expression atlas and comparative transcriptomics in animals Immunofluorescent antibody staining of intact Drosophila larvae Download references This work was supported by a Swiss National Science Foundation COST grant IZCOZ0_205460 and by the European Union’s Horizon 2020 Research and Innovation program through Marie Sklodowska-Curie Individual Fellowship (Grant Agreement No We thank Anna Schoenauer for initial exploratory dissections; the personnel of the Genomic Technologies Facility for the RNA-seq experiment the Cellular Imaging Facility for the confocal microscopy and the Department of Electron Microscopy of the University of Lausanne for their help with the histology preparation Department of Biological and Medical Sciences GZ and APM conceived the study design; GB performed the dissections for RNA extraction; AH perform the HCR and histology experiments image analysis and signal interpretation; GZ generated the bulk RNA-seq data and wrote the manuscript; all the authors contributed to the final version of the manuscript Reprints and permissions Download citation DOI: https://doi.org/10.1038/s41598-024-65336-2 A 5-year study on men that had primary partial-gland cryoablation shows that the procedure averted cancer recurrence in most patients while preserving urinary and sexual function Led by researchers at New York University Grossman School of Medicine the study tracked the patient outcomes after primary partial-gland cryoablation a procedure during which only the malignant part of the prostate gland is treated with cryotherapy to destroy it Focal therapy such as this is increasingly used in place of the traditional whole-gland prostate cancer treatments such as prostatectomy or irradiation of the entire gland Prostatectomy offers the most thorough form of prostate cancer prevention but most men will experience urinary incontinence and sexual dysfunction in the form of erectile dysfunction Authors of the current study had previously reported that primary partial-gland cryoablation avoids incontinence and minimizes sexual dysfunction no study had yet tracked the effectiveness of the combination of partial-gland ablation with intense follow-up to watch for Published recently in the journal Urology the study showed that 91 participants (89%) achieved the main study measure of treatment success “It’s worth considering that the need to take out the whole gland was judged to be treatment failure in our study even though nearly all of these men before the advent of partial-gland removal would have had the whole-gland procedure,” said senior study author Herbert Lepor the Martin Spatz Chair of the Department of Urology at NYU Langone Health “We found that [primary partial-gland cryoablation] can avert the profound consequences that can come with gland removal while still showing excellent results in preventing recurrence.” Advances in magnetic resonance imaging (MRI) enabled the team to reliably identify the sites 91 could be evaluated for freedom-from-failure over the entire 5 years rates of freedom-from-recurrence of in-field and overall clinically significant prostate cancer were 86% The proportion with freedom-from-failure at 5 years was 89% (95% confidence interval [CI] = 83%–95%) 15 (16.5%) underwent whole-gland salvage treatment and 15 (16.5%) underwent salvage focal therapy Only intermediate-risk patients were studied because this level of disease aggressiveness would otherwise have required whole-gland treatment immediately upon diagnosis and his experience in having performed more than 5,000 radical prostatectomies in his career both argue that 80% of men with intermediate-risk disease will chose to undergo focal cryotherapy over prostatectomy if they have the choice the current study was designed with especially intense surveillance of patients over time after focal therapy Study patients underwent prostatic-specific antigen (PSA) tests “This study represents the largest comprehensive prospective study of men with intermediate-risk prostate cancer treated with partial-gland cryoablation,” said James Wysock another author of the study and Assistant Professor in the Department of Urology at NYU Langone “We ensured rigorous follow-up over 5 years Disclosure: The study was funded by private donors, many of whom had undergone the study procedure. For full disclosure of all study authors, visit www.goldjournal.net 1. Lepor H, Rapoport E, Tafa M, et al: Five-year oncologic outcomes following primary partial gland cryo-ablation prospective cohort study of men with intermediate-risk prostate cancer. Urology 196:189-195, 2025 Metrics details The European hoopoe (Upupa epops) conforms a paradigmatic example of animals cultivating bacteria in their uropygial gland that protect them against pathogenic infections We here explore the hypothesis that enterococci are the responsible bacteria of such beneficial effect We did so by comparing the antimicrobial activity against three indicator bacteria of colonies isolated from cultures of enterococci and mesophilic bacteria from the uropygial skin or secretion of nestlings and males of the subspecies longirostris in Hainan (China) enterococci isolated from nesting birds are more active than those from non-nesting birds enterococci from the uropygial secretion were more active than those isolated from the skin or than mesophilic bacteria isolates These results therefore support the hypothesis that hoopoe females and nestlings cultivate enterococci in their uropygial gland with relatively high antimicrobial activity suggests that enterococci growing in the uropygial gland of hoopoes play an important role in the reproductive success and survival prospects of this species the antimicrobial activity of isolates from those birds should be higher than that of isolates from males and non-breeding females We here explore the above-mentioned three predictions from the current knowledge of the hypothesis that hoopoes maintain a mutualistic association with the enterococci inhabiting their uropygial gland We did so in a Chinese hoopoe population of the subspecies longirostris in the tropics (Hainan) we sampled the bacterial communities of the uropygial secretion and skin of nestlings samples were cultivated in a general medium for mesophilic bacteria and in a specific medium for Enterococcus spp we quantified the antimicrobial activity of isolates from those two culture media against Bacillus licheniformis Listeria innocua and Enterococcus faecalis we explored and found support for the predicted effects of culture media and individual reproductive stage on the antimicrobial activity of isolated bacterial colonies Antimicrobial activity of colonies isolated from general (TSA for mesophilic bacteria) and selective (KF for enterococci bacteria) cultures of samples collected from the uropygial gland skin (Skin) and secretion (Secr) of nestling (N) brooding (BF) or non-brooding female (NF) and male (M) hoopoes Pooled values of nesting (nestlings and brooding females) and non-nesting (non-brooding females and males) hoopoes are also shown Showed values are means ± 95% confidence intervals the interaction between the type of hoopoe sampled (F < 0.56 P > 0.647) or the nesting stage (F < 0.40 P > 0.528) with the sampled location (uropygial gland skin or secretion) significantly explained antimicrobial activity of isolates Our main results are that (i) independently of the indicator bacteria used enterococci are more active than mesophilic bacteria isolated from hoopoes (ii) isolates from the uropygial secretion are more active than bacterial colonies from the uropygial skin and (iii) bacteria (mainly enterococci) collected from nesting individuals (nestlings and brooding females) demonstrated stronger antimicrobial activity than those from non-nesting individuals the effect of sampled location did not interact with other factors but the interaction between nesting stage and culture media of isolates explained the antimicrobial activity of symbiotic bacteria of the hoopoe subspecies longirostris Below we discuss the importance of these results supporting the main predictions of the hypothesis that hoopoe females and nestlings cultivate enterococci in their uropygial gland with relatively high antimicrobial capacity it is not surprising that the antimicrobial activity of enterococci isolates was higher than that of isolates that included some other groups of bacteria Sequencing and taxonomically identifying isolates from the skin of the uropygial gland and its secretion is necessary to test this possibility because bacteria from the secretions of males and non-breeding females did not grow in specific media for enterococci contrary to what has been detected for the Spanish hoopoes non-enterococci bacteria grew in cultures from the secretion of the subspecies longirostris Genetic identification of isolated colonies from this subspecies is necessary to confirm this possibility The non-significant interactions between culture media and sampled location on the one hand and between sample location and hoopoe breeding stage on the other might be important for Chinese hoopoes breeding in the tropics Future studies should focus on the genetic identification of bacteria that grew in cultures of different samples and individuals of the subspecies longirostris our results confirm most of the general assumptions of the mutualistic association between hoopoes and enterococci of the uropygial secretion and suggest that it may vary among subspecies of hoopoes under different selection pressures they are mainly occupied by Crested Mynas (Acridotheres cristatellus) Oriental Magpie Robins (Copsychus saularis) and White-shouldered Starlings (Sturnus sinensis) while hoopoes (Upupa epops longirostris) prefer to nest in the holes of walls of buildings in local farms The fieldwork was carried out from the beginning of May to the end of June of 2023 and consisted of an intense search for hoopoe nests in the area We also asked farmers and local people for locations where they had seen hoopoes carrying prey in the beak we looked inside and estimated the reproduction stage (eggs When the female was inside and the older nestlings were less than 8 days old we carefully removed pieces of the wall and captured the female we pasted the removed material to the wall and the female was then released into the nest through the entrance hole which we kept closed for 5 min with a cotton bag to calm the female and reduce the risk of nest abandonment Nestlings were sampled when the older one was around 18 days old None of the sampled hoopoe nests were abandoned after sampling we gently introduced a sterilized micropipette tip (1–10 µl) inside the uropygial-gland papilla and collected all the uropygial secretion that was directly included in an empty and sterilized microfuge vial The used vials were kept at approximately 4 °C in a portable icebox until their use in the lab within the next 48 h The experiments comply with the current laws of China All applicable guidelines for the care and use of animals were followed Experimental procedures for hoopoe manipulation were approved by the Animal Research Ethics Committee of Hainan Provincial Education Centre for Ecology and Environment Sample sizes were relatively small and restricted to nests with hoopoe nestlings older than 16 days and to adults caught at the right breeding stage (breeding and non-breeding females and males) nest visits and the number of sampled individuals were reduced to those allowing reliable statistical tests a broadly used general medium for growing aerobic mesophilic bacteria (Scharlau Chemie S.A. and a specific medium for enterococci (Kenner fecal agar (KFA) Scharlau Chemie S.A. The KF medium also included TTC (2,3,5-Triphenyl tetrazolium chloride solution) (1%) Petri dishes were incubated at 37 °C for 24 h and 72 h To explore the variation in antimicrobial activity of isolated bacterial strains in relation to the type of individual (nestlings sampling location (skin or secretion of the uropygial gland) and bacterial type (enterococci or mesophilic bacteria) we included these variables as fixed factors in General Linear Mixed Models (GLMM) These models also included the identity of sampled individuals (nested within the type of individual hoopoes) as the first random factor and the interactions with sampling location and bacterial type as two additional random factors These three random factors accounted for the non-independence of data from colonies isolated from the same individual as well as for the repeated-measures nature of information from samples of different locations and bacterial types from the same individual The effects of the interactions between fixed factors ((i) type of bacteria (mesophilic vs (ii) type of bacteria and type of individual and (iii) location of samples and type of individuals) on the antimicrobial activities were explored in separate models that also included main effects and the second order interaction between individual identity (nested within the type of individual hoopoes) location and bacterial types as additional random effects The effects of second order interactions between fixed factors could not be estimated because enterococci did not grow in samples from non-nesting individuals Non-significant interactions were sequentially removed with final models only including that between type of bacteria and type of individual hoopoes since the antimicrobial activity of nesting (i.e. nestlings and brooding females) and non-nesting hoopoes (i.e. males and non-brooding females) showed similar patterns (see results) we repeated all these statistical models by including information on whether sampled hoopoes were or were not in the nests (i.e. we presented the results of these last models in the Results section while models including information of the “type of hoopoes samples” were reported in the Electronic Supplementary Material (ESM) Values of antimicrobial activity were Log10 transformed before the analyses to approach a normal distribution and residuals of GLMMs were plotted against expected normal values to detect outliers (extreme and isolated points that separated from the expected values and from other points) the model was run again without considering detected outliers statistically significant factors) did not depend on considering outliers we show results that excluded those points All statistical analyses were performed in Statistica 13.034 All data generated or analysed during this study are included in this published article (and its supplementary information files) Fungus-growing ants use antibiotic-producing bacteria to control garden parasites Bacterial protection of beetle-fungus mutualism Symbiont-mediated protection in insect hosts Flórez, L. V., Biedermann, P. H. W., Engl, T. & Kaltenpoth, M. Defensive symbioses of animals with prokaryotic and eukaryotic microorganisms. Nat. Prod. Rep. 32, 904–936. https://doi.org/10.1039/C5NP00010F (2015) Symbiotic association between hoopoes and antibiotic-producing bacteria that live in their uropygial gland Characterization of antimicrobial substances produced by Enterococcus faecalis MRR 10–3 isolated from the uropygial gland of the hoopoe Upupa epops Ruiz-Rodríguez, M. et al. Antimicrobial activity and genetic profile of enteroccoci isolated from hoopoes uropygial gland. PLoS ONE 7, e41843. https://doi.org/10.1371/journal.pone.0041843 (2012) sexual and developmental differences in hoopoe Upupa epops preen gland morphology and secretions: evidence for a role of bacteria Martín-Vivaldi, M. et al. Antimicrobial chemicals in hoopoe preen secretions are produced by symbiotic bacteria. Proc. R Soc. Lond. B Biol. Sci. 277, 123–130. https://doi.org/10.1098/rspb.2009.1377 (2010) Martínez-García, A. et al. Preening as a vehicle for key bacteria in hoopoes. Microb. Ecol. 70, 1024–1033. https://doi.org/10.1007/s00248-015-0636-1 (2015) Soler, J. J. et al. Nestedness of hoopoes’ bacterial communities: symbionts from the uropygial gland to the eggshell. Biol. J. Linn. Soc. 18, 763–773. https://doi.org/10.1111/bij.12772 (2016) Martín-Vivaldi, M. et al. Special structures of hoopoe eggshells enhance the adhesion of symbiont-carrying uropygial secretion that increase hatching success. J. Anim. Ecol. 83, 1289–1301. https://doi.org/10.1111/1365-2656.12243 (2014) Ruiz-Rodríguez, M. et al. Symbiotic bacteria living in the hoopoe’s uropygial gland prevent feather degradation. J. Exp. Biol. 212, 3621–3626. https://doi.org/10.1242/jeb.031336 (2009) Bacteriocins with a broader antimicrobial spectrum prevail in enterococcal symbionts isolated from the hoopoe’s uropygial gland Martín-Vivaldi, M. et al. Acquisition of uropygial gland microbiome by hoopoe nestlings. Microb. Ecol. 76, 285–297. https://doi.org/10.1007/s00248-017-1125-5 (2018) Environmental factors shape the community of symbionts in the uropygial gland of hoopoes more than genetic factors The microbiome of the uropygial secretion in hoopoes is shaped along the nesting phase Seasonal and sexual differences in the microbiota of the hoopoe uropygial secretion Sánchez-Hidalgo, M. et al. AS-48 bacteriocin: Close to perfection. Cell. Mol. Life Sci. 68, 2845–2857. https://doi.org/10.1007/s00018-011-0724-4 (2011) Beneficial effects of cloacal bacteria on growth and fledging size in nestling pied flycatchers (Ficedula hypoleuca) in Spain Enterococci at the crossroads of food safety? Montalbán-López, M., Sánchez-Hidalgo, M., Valdivia, E., Martínez-Bueno, M. & Maqueda, M. Are bacteriocins underexploited? Novel applications for OLD antimicrobials. Curr. Pharma. Biotechnol. 12, 1205–1220. https://doi.org/10.2174/138920111796117364 (2011) The role and application of enterococci in food and health Microbial infection risk predicts antimicrobial potential of avian symbionts Ibáñez-Álamo, J. D., Rubio, E. & Soler, J. J. Evolution of nestling faeces removal in avian phylogeny. Anim. Behav. 124, 1–5. https://doi.org/10.1016/j.anbehav.2016.11.033 (2017) Martín-Vivaldi, M., Doña, J., Romero-Masegosa, J. & Soto-Cárdenas, M. In Enciclopedia Virtual de los Vertebrados Españoles (eds A. Salvador & M.B. Morales) Ch. http://www.vertebradosibericos.org/ How much variance can be explained by ecologists and evolutionary biologists? Liu, J., Zhang, F., Liu, Y. & Liang, W. Egg recognition and nestling discrimination in the Crested Myna (Acridotheres cristatellus): Size matters. Avian Res. 14, 100111–100111. https://doi.org/10.1016/j.avrs.2023.100111 (2023) Ruiz-Castellano, C., Ruiz-Rodríguez, M., Tomás, G. & Soler, J. J. Antimicrobial activity of nest-lining feathers is enhanced by breeding activity in Avian nests. FEMS Microbiol. Ecol. 95, fiz05. https://doi.org/10.1093/femsec/fiz052 (2019) The causes of the mortality of eggs and nestlings of Passer Spp An annotated checklist of pathogenic microorganisms associated with migratory birds Bacterial pathogens in wild birds: a review of the frequency and effects of infection STATISTICA (data analysis software system) Download references and their contribution contacting local people asking them for hoopoe nests was essential for collecting the samples used in the present work Cristina Soler-Zamora and Javier Martín-Vivaldi also helped us in the field and with the laboratory work Bingyu Cai and Zhufen Gao helped us in the lab plating bacterial samples The Spanish part of the research group received funds from the projects PID2020-117429GB-C21 and PID2020-117429GB-C22 funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación/10.13039/501100011033 Departamento de Ecología Funcional y Evolutiva Estación Experimental de Zonas Áridas (EEZA-CSIC) María Dolores Barón & Ester Martínez-Renau Unidad Asociada (CSIC): Coevolución: Cucos Juan José Soler & Manuel Martín-Vivaldi Ministry of Education Key Laboratory for Ecology of Tropical Islands with the help of WL designed the study and JJS performed the statistical analyses and wrote a first version of the manuscript All the authors substantially contributed to the final version of the manuscript and approved the submission Download citation DOI: https://doi.org/10.1038/s41598-024-81062-1 Metrics details A Publisher Correction to this article was published on 10 October 2024 This article has been updated Heparan sulfate (HS) regulation of FGFR function which is essential for salivary gland (SG) development is determined by the immense structural diversity of sulfated HS domains 3-O-sulfotransferases generate highly 3-O-sulfated HS domains (3-O-HS) and Hs3st3a1 and Hs3st3b1 are enriched in myoepithelial cells (MECs) that produce basement membrane (BM) and are a growth factor signaling hub Hs3st3a1;Hs3st3b1 double-knockout (DKO) mice generated to investigate 3-O-HS regulation of MEC function and growth factor signaling show loss of specific highly 3-O-HS and increased FGF/FGFR complex binding to HS Defined 3-O-HS added to FGFR pulldown assays and primary organ cultures modulates FGFR signaling to regulate MEC BM synthesis which is critical for secretory unit homeostasis and acinar function Understanding how sulfated HS regulates development will inform the use of HS mimetics in organ regeneration The functions of many FGFs are regulated by heparan sulfate (HS) either by increasing the affinity of FGF/FGFR signaling complexes or by acting as a reservoir for growth factors on the cell surface or in the basement membrane (BM) a specialized extracellular matrix (ECM) that separates epithelia from stroma recent chemoenzymatic synthesis of defined 3-O-HS structures allows us to investigate their function These advances allow more specific probing into the study of the 3-O-sulfated structure and function relationship of HS Single Hs3st3a1 or Hs3st3b1 knockout mice have subtle secretory phenotypes suggesting that the loss of only one Hs3st3 enzyme is functionally compensated for by another Hs3st3 enzyme we generated 3-O-sulfotransferase DKO mice to study the function of 3-O-sulfation in vivo We confirmed the loss of specific 3-O-sulfated domains using mass spectrometry and analyzed gene expression at multiple stages of SMG development myoepithelial cell cultures and using defined chemoenzymatically synthesized HS in explant culture to interrogate 3-O-sulfated-HS function We observe a reduction in MEC and ducts in the adult gland and a proportional increase in acinar cells which contain acinar cells surrounded by MECs and BM We discover that loss of these highly sulfated domains disrupts FGFR signaling a Data shown are the number of mice of a given genotype for each sex derived from 25 independent litters obtained from heterozygous crosses Chi-square analysis shows no significant difference from the expected ratios b Body weight and SMG weight of adult male and female mice normalized to body weight and WT d Mass spec analysis of HS disaccharides and tetrasaccharides in adult male SMGs Analysis of HS disaccharide (c) and tetrasaccharide (d) composition between WT and DKO SMGs All data shown is averaged and presented as stacked bar graphs and ΔUA-GlcNS6S-GlcA-GlcNS3S6S **p = 0.0057 ΔUA2S-GlcNAc6S not detected in WT and DKO SMGS ΔUA-GlcNS6S-IdoA2S-GlcNS3S6S not detected in DKO SMGs e Gene expression changes in HS biosynthetic enzymes in both male and female DKO SMGs normalized to the respective male or female WT SMGs and Rps29 n = 3 for WT-F (Glce and Hs2st1) and DKO-F (Glce n = 17 DKO-F (Hs3st1) and n = 18 DKO-F (Hs6st1) ***p < 0.0001 for Hs3st3a1 and Hs3st3b1 in DKO-male and DKO-female Source data are provided as a Source Data file whereas no differences were detected in Hs3st3 KO mice The DKO SMGs were more similar in their HS with only UA2S-GlcNAc6S and UA2S-GlcNS being different Further work is needed to fully understand the biological significance of these differences this analysis showed that both males and females have altered HS composition in the SMGs of DKO mice Quantitative PCR analysis in the adult DKO SMGs showed, as expected, no expression of Hs3st3a1 and Hs3st3b1, while other HS enzymes in DKO SMGs were comparable to the WT (Fig. 1e) Both WT and DKO adult SMG had no detection of Hs3st2 these analyses confirm there is a significant decrease in both ΔUA-GlcNS6S-IdoA2S-GlcNS3S6S and ΔUA-GlcNS6S-GlcA-GlcNS3S6S tetrasaccharides generated by Hs3st3 enzymes without compensatory upregulation of other biosynthetic enzymes in DKO SMG a Representative H&E images of adult male (M) and female (F) WT and DKO SMGs Ducts and acinar cells quantified per area normalized to the WT and WT-F) and n = 3 (DKO-F) SMGs with multiple areas quantified (WT-M: n = 21 and DKO-F duct ***p < 0.0001 compared to WT b RNAseq analysis of DKO SMGs identifies reduced MEC and duct genes with the increase of some acinar genes ductal and acini in DKO-M SMGs (n = 5) compared to WT-M (n = 4) The color scale represents scaled gene expression values DEGs obtained using a non-parametric Wald test with Benjamini–Hochberg adjustment (p-value < 0.05 and fold change > 2) c qPCR confirmed gene expression changes in female and male SMGs normalized to Rps29 and respective sex-matched WT (dotted line) Unpaired two-tailed t-test compared to sex-matched WT (DKO-M: Krt14 **p = 0.0045 d Confocal imaging of WT-M and DKO-M SMGs showing Acinar1 (Ac1 SMA (green) and EGF (magenta) immunostaining e Quantification of staining normalized to the WT SMGs and nuclei suggesting a cell-autonomous role for 3-O-sulfated HS in MEC differentiation and/or maintenance Validation of RNAseq by qPCR (Fig. 2c) confirmed the overall trends measured in male and female SMGs Examples of MEC genes that were reduced in expression include those for contractile proteins such as Acta2 (Smooth muscle actin) Mylk (myosin light chain kinase) and Tpm2 (tropomyosin 2) Krt14 and Postn (a secreted ECM protein involved in integrin-mediated cell migration) which are expressed in both MECs and basal ducts DEGs included growth factor genes such as Ctgf which were all confirmed by qPCR in the male and/or female DKO SMG genes associated with focal adhesions (21 genes) cell adhesion molecules (19 genes) also reflect the MEC function of producing basement membrane components and HSPGs we next investigated how changes in HS influence FGFR signaling and secretory unit function in DKO SMGs a Binding of FGF10:FGFR2b-Fc complex overlapping with COLIV Representative images of single confocal sections from WT and DKO SMGs showing FGFR2b-Fc (green) b Representative images of single confocal sections from WT and DKO male SMGs showing 3G10 c Quantification of A and B protein fluorescence intensity normalized to total nuclei and expressed as a fold change compared to WT MUC10: n = 3; 3G10: n = 3 SMGs from WT and DKO mice d Representative Western blot of 3G10 antibody shows an increase in high molecular weight HSPG cores (indicated by red arrows) in DKO male SMG lysates treated with or without heparinase III and chondroitinase ABC Bands identified by an asterisk may represent the light and heavy chains of immunoglobulins RNAseq did not identify changes in BM (Hspg2 and Col18a1) or cell surface (Sdc1 and Sdc4) HSPG transcripts in the DKO the increased LACE and 3G10 antibody staining suggested there may potentially be increased FGF/FGFR signaling in the DKO a Western blots showing pERK is increased in DKO male SMG lysate Graph shows band intensity of pERK normalized to total ERK and expressed as a fold change in ratio compared to WT The graphs are n = 10 biological samples from 3 experiments b Immunostaining showing representative images of single confocal sections from WT and DKO male SMGs of pERK (green) c Graphs show cytosolic Ca2+ change measurements stimulated by CCh with fura2 fluorescence (340/380) A representative trace from three independent experiments shows calcium reabsorption is decreased in DKO and the graph shows the average of the group responses Cells analyzed for calcium release studies are WT = 138 and DKO = 122 cells and for calcium release WT = 136 and DKO = 122 cells d Recordings of CCh-regulated volume decrease (RVD) using calcein dye Left-hand panel shows a representative trace and graph shows the average response of 125 WT and 125 DKO cells from three independent experiments Unpaired two-tailed t-test compared to WT; *p = 0.0343 e Immunostaining for a polarity marker (ZO1) Images are maximum projections of z-stacks from three biological samples f Lickometer test of drinking behavior shows adult DKO female mice drink water more frequently than WT mice g Salivary flow rates in adult male mice normalized to WT and shown as % and thus salivary function may be affected The Hs3st3a1 or Hs3st3b1 single KO mice did not show any problems with saliva flow although both these mice strains displayed increased drinking behavior our data suggest that in the adult DKO SMG the loss of 3-O-HS domains disrupts the epithelial progenitor niche increasing FGFR signaling and BM metabolism To identify the initial events causing this loss of homeostasis we analyzed earlier developmental time points predicting that increased FGFR signaling might increase BM synthesis by MECs and disrupt their development a Light micrograph of SMGs isolated from E13 embryos and cultured ex vivo for 24 h Graph is quantification of the number of endbuds (expressed as a ratio of the number at 24 h/the number at 1 h) of WT (n = 15) and DKO (n = 10) SMGs b Gene expression changes were normalized to WT and Rps29 The n values indicate the number of SMGs analyzed Unpaired two-tailed t-test compared to WT (Hs3st3a1 ***p < 0.001 d Immunostaining at E13 shows Hs3st3-dependent 3-O-sulfation (HS4C3V) in the basement membrane is reduced in DKO SMGs whereas Hs3st1-dependent 3-O-sulfation (AT488 binding) is not affected c Representative single confocal images of HS4C3V (green) Right panel shows that HS4C3V staining in the basement membrane is dependent on heparan sulfate The section was pretreated with heparinase III enzyme (HPT) which removes the 3-O-sulfated epitope recognized by the antibody d Representative single confocal images of AT488 (green) a Gene expression changes in E15 DKO SMGs were normalized to WT control and Rps29 Unpaired two-tailed t-test compared to WT (Hs3st1 **p = 0.0014 b Representative images show immunostaining at E16 with SMA (green) in MEC and perlecan (magenta) in basement membrane Graph shows quantification of protein fluorescence intensity normalized to total nuclei and expressed as a fold change compared to WT c Gene expression changes in P2 DKO SMGs were normalized to WT control and Rps29 Unpaired two-tailed t-test compared to WT (Hs3st3b1 ***p < 0.0001 d Representative images showing immunostaining at P2 show SMA (green) in MEC Graph shows protein fluorescence intensity quantification normalized to total nuclei and expressed as a fold change compared to WT a Chemical structure of chemoenzymatically synthesized 12mer HS with biotin tag Figure 7a Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license b Pulldown assays of 12mer HS and FGF:FGFR complexes Top panel show representative gels for FGFs 1,2,7 and 10 complexed with rFGFR1b bottom panel shows the same FGFs with rFGFR2b The molecular weight markers in kilodaltons (97 and 64 kDa) are indicated on each gel Full gels are provided in the source data file Graphs show quantitation of the FGFR complex separated by SDS-PAGE and silver-stained from independent samples One-way ANOVA with Dunnett’s test for multiple comparisons to non-3-O-HS 3ST3-HS ***p < 0.0001; FGF10:FGFR1b: 3ST1-HS *p = 0.0136; FGF2:FGFR2b: 3ST1-HS *p = 0.0482; FGF10:FGFR2b: 3ST1-HS ***p = 0.0002 and 3ST3-HS ***p < 0.0001 c E13 SMG epithelia cultured with FGF10 in a laminin-111 3D matrix were treated with 12mer HS and cultured for 48 h (n = 6 with 4 epithelia each) the size of the epithelia was measured as a morphogenic index d Gene expression of markers of FGFR signaling and MECs were analyzed by qPCR were normalized to non-3-O-HS and Rps29 The n values indicate the number of epithelial culture experiments analyzed One-way ANOVA with Dunnett’s test for multiple comparisons to non-3-O-HS (3ST1-HS: Hs3st3a1 **p = 0.0017 Col18a1 *p = 0.0118 and for 3ST3-HS: Acta2 **p = 0.0078 e Confocal imaging of E13 epithelia treated with 12mer HS showing SMA (magenta) and CNN1 (green) and KRT19 (yellow) we knocked out two Hs3st3 enzymes to investigate 3-O-HS regulation of FGFR signaling and MEC function during SMG development and homeostasis We confirmed the loss of specific 3ST3-HS tetrasaccharides in the SMGs suggesting overall reduced growth factor function during development but their SMGs were proportionally larger with a disrupted epithelial compartment The reduction of MEC and ducts suggested an altered progenitor microenvironment We show that increased BM gene expression was an early event that we propose led to further increases in genes related to FGFR signaling and MEC differentiation this dysregulation of the progenitor microenvironment disrupted MEC and acinar cell communication reducing secretory unit function we show that both types of 3-O-HS can form multiple FGF/FGFR signaling complexes Further study will require a generation of conditional knockouts to investigate the function of both types of 3-O-HS later in development DKO secretory units had disrupted acinar apical-basal polarity a likely consequence of improper MEC communication and/or BM metabolism Although there was reduced transcription of some BM genes enriched in MECs (Supplementary File 1) the protein staining of collagen IV and HSPGs around acinar cells was increased It is not clear if the increased BM staining is related to a lack of proteolytic turnover of BM components the mesenchyme is still the major source of FGF7 which would have to diffuse through the BM which supports the concept that 3-O-HS has different roles in different cells at discrete stages of development which was not transcriptionally affected in the DKO We speculate the loss of 3-O-HS disrupts BM metabolism allowing increased FGF diffusion and increasing acinar FGFR signaling The identity of BM or cell surface HSPGs that are responsible for this remains to be determined We also speculate that the disrupted acinar polarity and the lack of contractile MEC to expel saliva leads to the increase in gland weight due to saliva production by acinar secretory units that cannot secrete we propose a model where the genetic deletion of Hs3st3a1 and Hs3st3b1 results in the loss of the 3-O-sulfated domains in the BM and the cell surface increasing growth factor signaling to MECs This promotes gene expression associated with BM metabolism potentially generating more bioactive proteolytic BM products which increase FGFR signaling and disrupts MEC function likely altering BM architecture and MEC-acinar communication which disrupts acinar polarity and impairs calcium signaling Understanding how the 3-O-sulfated HS code regulates FGFR signaling BM metabolism and MEC biology will be essential to manipulate cellular specificity of growth factor signaling and enhance the progenitor microenvironment to drive organ regeneration No specific new materials were generated in this work, see Supplementary Table 2 for resources Mice were kept in a 14 h on/10 h off light/dark cycle at 74–78 F with 50–70% humidity timed mating was set up and a plug detected was considered day 0 due to the known sexual dimorphism in adult mouse SMGs differing outcomes between sexes was considered Sex was not considered in experiments using embryonic or neonatal glands as sexual dimorphism does not occur during development All mice were maintained and treated according to guidelines approved by the National Institute of Dental and Craniofacial Research and the National Institutes of Health Animal Care and Use Committee Mice were genotyped for Hs3st3b1 allele as previously described17 Hs3st3a1 ZFN primer Fwd (CTGGCCTTACTTCTGGACGA) and Hs3st3a1 ZFN Rev (CAAGGGAGAAGAACGGGAG) were used for the amplification of DNA PCR cycling parameters included an initial denaturing at 94 °C for 5 min; denaturing at 94 °C for 15 s To perform the mismatch-sensitive nuclease assay PCR products were denatured with T7E1 enzyme (New England BioLabs USA) at 95 °C for 2 min; followed by a 2 °C/s drop to 85 °C PCR products were separated on a 2.0% TBE agarose gel with ethidium bromide PCR products were purified using QIAquick PCR purification kit (QIAGEN Inc. The AMAC derivatization of lyophilized samples was performed as described previously51 10 μL of 0.1 M AMAC solution in DMSO/glacial acetic acid (17:3 v/v) was added to the samples and incubated at room temperature for 15 min Then 10 μL of freshly prepared 1 M aqueous sodium cyanoborohydride was added to this solution The reaction mixture was incubated at 45 °C for 2 h the reaction solution was centrifuged to obtain the supernatant which was subjected to the LC-MS/MS analysis A Vanquish Flex UHPLC System (ThermoFisher Scientific) coupled with TSQ Fortis triple-quadrupole mass spectrometry as the detector was used The ACQUITY Glycan BEH Amide column (1.7 μm UK) was used to separate di/tetra-saccharides at 60 °C Mobile phase A buffer used was 50 mM ammonium formate in water The flow rate of 0.3 mL/min and the elution gradient as follows were used: 0–15 min 83–70% B Online triple-quadrupole mass spectrometry operating in the multiple reaction monitoring (MRM) mode was used as the detector The ESI-MS analysis was operated in the negative ion mode using the following parameters: Negative ion spray voltage at 3.0 kV ion transfer tube temp at 250 °C and vaporizer temp at 400 °C TraceFinder software was applied for data processing The amount of HS was determined by comparing the peak area of native di/tetrasaccharides to each di/tetra-saccharides calibrant and the recovery yield was calculated based on the comparison of the amount of recovery calibrant disaccharide in the samples and control HS analysis of DKO and single KO males or WT and DKO male and female SMGs was performed on samples analyzed within the same analytical runs RNA-seq data are available on the Gene Expression Omnibus (GEO) website (GEO: GSE235187) DEGs were also used to identify the enriched Kyoto Encyclopedia of Genes and Genome (KEGG) pathways [16] The E16 timepoint was extracted from the integrated SEURAT objects using the SplitObject function from SEURAT and DotPlots were generated with the DotPlot function of the same package which were designed using Beacon DesgnerTM software (PREMIER Biosoft) SMG tissue from adult male and female WT and DKO mice were isolated and fixed in 4% PFA before standard paraffin embedding was performed by Histoserv Inc (Germantown Hematoxylin and Eosin (H&E) staining was performed on 5 µm thick sections (Histoserv Inc Slides were scanned using ×40 objective with either a S60 Nanozoomer Digital slide scanner (Hamamatsu) or Aperio Scanscope scanner (Leica Biosystems) and snapshots of random areas (250 µm2) were taken using similar settings in either NDP.view 2 software (Hamamatsu) or Aperio Image software Blinded quantification of ducts and acinar cells per area was performed by using the cell counter setting for manual counting in FIJI Between 13 and 21 areas from 3 to 4 glands/animals were used for quantification (Male WT: n = 21 Whole-mount staining on E16 SMGs was performed by permeabilizing the SMGs in 0.2% Triton-X-100/PBS at 4 °C for 48 h The glands were incubated with the primary and secondary antibodies at 4 °C for 48 h All slides were imaged using a Zeiss 880 confocal microscope a super-resolution airy scan was used for imaging Fluorescence was quantified in the epithelial region within the basement membrane (ROI) and normalized to that of nuclei in the ROI and then the data was normalized to control An average of five images were taken per SMG and a minimum of three glands were used for each group For whole-mount staining of isolated epithelia epithelia were fixed with ice-cold acetone-methanol for 10 min at −20 °C and antibody incubations were performed as described for the whole-mount staining of E13 SMGs adult glands fixed with 4% paraformaldehyde overnight at RT were processed for paraffin sectioning Heat-activated antigen retrieval using a 0.05% Tween-20/10 mM citric acid buffer pH 6.0 was performed on 7 μm sections The samples were blocked for 1 h at RT with 10% heat-inactivated donkey serum incubated overnight at 4 °C with 50 nM soluble recombinant rFGFR2b-Fc anti-E-cadherin (BD Transduction Labs) and anti-collagen TIV (Millipore) Cy dye-conjugated secondary antibodies were added for 1 h The Fc on the receptors was detected with Alexa Fluor® 488 AffiniPure F(ab’)2 Fragment Donkey Anti-Human IgG Fcγ fragment specific secondary antibody (Jackson ImmunoResearch Laboratories Immunofluorescence was analyzed with a Zeiss LSM880 microscope tissue sections were treated with 20 mU/mL heparinase III or 40 mU/mL chondroitinase ABC enzymes (Amsbio LLC) diluted in buffer containing 50 mM HEPES and 5 mM CaCl2 for 2 h at 37 °C after antigen retrieval The subsequent steps were performed as described earlier weighed and then sonicated in RIPA Buffer (25 mM Tris 0.1% SDS) with protease inhibitor cocktail (Pierce No and phosphatase inhibitor cocktail (Millipore Sigma) Total protein concentrations were assessed using a BCA Protein Assay Kit (Pierce NuPageTM LDS Sample Buffer and NuPageTM Sample Reducing Agent (both from ThermoFisher Scientific) were added to the homogenate and the samples were boiled at 95 °C for 5 min The sample (20 µg) was electrophoresed on NuPAGETM 4–12% Bis-Tris Gels and transferred to PVDF membranes using the Invitrogen iBlot 2 Dry Blotting System (ThermoFisher Scientific) Blots were blocked with 5% BSA in 0.1% TBS-Tween (50 mM Tris-HCl pH 7.5 0.1% Tween-20) for 1 h at room temperature following the transfer The following primary antibodies were used: phospho-p44/42 MAPK (1:1000; Cell Signaling T202/Y204 #4370) p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling #9102) All blots were incubated with a secondary antibody conjugated to horseradish peroxidase (1:10,000 The blots were developed using the SuperSignal West Dura Chemiluminescent Extended Duration Substate (ThermoFisher Scientific) Blots were imaged on GE Amersham Imager AI680 and analyzed using NIH ImageJ software Band intensities were normalized to β-actin and wildtype controls Blots were stripped using RestoreTM PLUS Western Blot Stripping Buffer (Thermo Scientific #46430) for 5 min and then blocked for 1 h with 5% BSA in 0.1% TBS-Tween 50 µg lysates were diluted and treated with or without 80 mU/mL heparinase III and 100 mU/mL chondroitinase ABC diluted in buffer containing 4 mM CaCl2 in PBS for 2 h at 37 °C Lysates were boiled with Laemmli sample buffer and reducing agent and run on SDS-PAGE gels as described early gels were transferred to nitrocellulose membranes using NUPAGE blotting buffer (ThermoFisher Scientific #NP00061) for 90 min at 95 volts Blots were blocked with 5% milk/TBST and probed with an anti-Delta-heparan sulfate 3G10 (Asmbio #370260-1) and β-actin rabbit monoclonal antibody-HRP conjugate (Cell Signaling #5125S) A training session was conducted with the mice prior to the experiment followed by a recording of the number of licks of 4 mice for 1 h Controls and double-knockout mice were tested at the same time and then retested 5 times on separate days Saliva collection was performed as previously described17 mice were weighed and intramuscularly anesthetized with ketamine (60 mg/kg) and xylazine (8 mg/kg) A subcutaneous injection of pilocarpine at 0.25 mg/kg body weight was then injected to stimulate saliva secretion Whole saliva was collected gravimetrically for 20 min using a 75-mm hematocrit tube (Drummond) into pre-weighed 1.5 mL Eppendorf tubes The amount of saliva collected was calculated as micrograms per gram of body weight and then normalized as percentage to the WT littermates Saliva samples (15 μL) collected from mice were mixed with Laemmli sample buffer and reducing agent (ThermoFisher Scientific) These samples were then separated by gel electrophoresis on 4–12% pre-cast bis-tris gels (ThermoFisher Scientific) The gels were stained overnight in Coomassie G-250 stain (SimplyBlue Safe stain followed by multiple washes with deionized water to allow visualization of protein bands Gels were imaged using GE Amersham Imager AI680 The SMGs were finely minced and digested with Liberase TL (0.5 mg/mL) twice in Eagle’s minimum essential medium for 25 min each Cells were continuously gassed with a mixture of 95% O2 and 5% CO2 isolated submandibular gland cells were loaded with Fura-2 (1 µM) for 45 min and cells were excited at 340/380 nm with an emission of 510 nm fresh dispersed SMG cells were loaded with the fluoroprobe calcein (1 µM for 10 min Emitted fluorescence was measured at 510 nm Cell volume was estimated using calcein intensity An Olympus × 51 microscope (Olympus Centre Valley) with an ORCA-ER camera (Hamamatsu) attached to a Polychrome V (Till Photonics LLC) was used USA) was used for signal acquisition and data analysis MA) was used for data analysis and display Significant difference between individual groups was tested using ANOVA Streptavidin M-280 beads (ThermoFisher Scientific) (25 µL) equilibrated with 200 µL of IP-MS cell lysis buffer (ThermoFisher Scientific) were incubated with 100 µL IP-MS buffer containing 125 nM HS for 1 h on rotor at room temperature The beads were washed three times with IP-MS cell lysis buffer Equimolar concentrations (125 nM) of recombinant FGFRs and FGFs (all from R&D Systems Inc. MN) were added to the beads and incubated for 1 h on rotor at 4 °C The supernatant was aspirated and discarded and beads were then washed once with SureQuant buffer A (ThermoFisher Scientific) followed by one wash with buffer A supplemented with 350 mM NaCl followed by one wash with buffer A supplemented with 750 mM NaCl The beads were then washed twice with SureQuant buffer B (ThermoFisher Scientific) Washed beads were boiled with SDS-loading buffer and reducing agent and run on 4–12% bis-tris gels (ThermoFisher Scientific) The gels were stained with FASTsilver Kit (G Biosciences The data was obtained from at least three independent experiments Quantification of the FGF receptor bands in the silver-stained gels was performed using the NIH ImageJ program Each experiment was repeated at least three times Explants were either lysed for RNA or fixed for immunostaining These cultures were performed as previously described28 P2 SMGs were minced and dissociated in a 15 mL gentleMACS C tube with digestion enzymes (0.575 mg/mL collagenase type II (Gibco USA) diluted in Hanks Buffer Salt Solution (HBSS) (Thermo Scientific Cell dissociation was performed in a Miltenyi gentleMACS Octo Dissociator using the manufacturer’s preset 37C_h_TDK_2 program and resuspended in smooth muscle cell growth media (Cell Applications The cells were strained through a 70-μm filter (MACS Miltenyi Biotec and 50,000 cells were plated on 24-well BioCoatTM Collagen IV multiwell plates (Corning #08–774–29) in Smooth muscle growth media (Cell Applications Inc. the media was removed and replaced with Smooth muscle differentiation media (Cell Applications Inc. #300D-250) for the duration of the experiment MECs were collected for qPCR after 7 days of culture 0.3 µm steps) of 6–12 areas were captured on a Nikon A1Rplus MP microscope using the ×20 objective (SMA WT: n = 10 Quantification of integrated density for each channel normalized to nuclear stain was done on maximum projections using the measure analysis in FIJI Data were log-transformed and analyzed with an unpaired Student’s t-test (two-tailed) or one-way ANOVA with post hoc Dunnett’s multiple comparisons test when comparing more than two groups DKO samples were compared to the WT using Prism 10 software (GraphPad Paired two-tailed t-test was used for HS analysis of the male SMGs compared to the respective WT Tables of DEGs were generated using the DESeq2 package and the non-parametric Wald test with Benjamini–Hochberg adjusted p values <0.05 and Log2 fold change The p-values for KEGG analysis tables are derived using right-tailed Fisher’s exact t-test followed by a Benjamini–Hochberg adjustment Values are expressed as mean ± standard error of the mean (SM) from three or more biological replicates (as indicated) and each staining was repeated with at least three biological replicates showing similar results Western blots and silver-stained gel images are representative and each assay was repeated with at least three biological replicates or three independent samples Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article A Correction to this paper has been published: https://doi.org/10.1038/s41467-024-53230-4 FGFR2 is essential for salivary gland duct homeostasis and MAPK-dependent seromucous acinar cell differentiation LADD syndrome is caused by FGF10 mutations and future directions for salivary gland regeneration Basement membrane dynamics and mechanics in tissue morphogenesis Basement membranes in development and disease New functional roles for non-collagenous domains of basement membrane collagens Basement membranes: cell scaffoldings and signaling platforms MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis Concurrent reduction in the sulfation of heparan sulfate and basement membrane assembly in a cell model system Cryo-EM reveals the molecular basis oflaminin polymerization and LN-lamininopathies Heparan sulfate 3-O-sulfation: a rare modification in search of a function Binding of heparin to antithrombin III: a chemical proof of the critical role played by a 3-sulfated 2-amino-2-deoxy-D-glucose residue Hs3st3-modified heparan sulfate controls KIT+ progenitor expansion by regulating 3-O-sulfotransferases Heparan sulfate biosynthesis and sulfation profiles as modulators of cancer signalling and progression Loss of Hs3st3a1 or Hs3st3b1 enzymes alters heparan sulfate to reduce epithelial morphogenesis and adult salivary gland function Analysis of 3-O-sulfated heparan sulfate using isotopically labeled oligosaccharide calibrants Increased 3-O-sulfated heparan sulfate in Alzheimer’s disease brain is associated with genetic risk gene HS3ST1 Dissecting structure-function of 3-O-sulfated heparin and engineered heparan sulfates Diverse epithelial cell populations contribute to the regeneration of secretory units in injured salivary glands Genetic and scRNA-seq analysis reveals distinct cell populations that contribute to salivary gland development and maintenance The evolving definition of salivary gland stem cells Generation of a single-cell RNAseq atlas of murine salivary gland development Context-dependent function of myoepithelial cells in breast morphogenesis and neoplasia Myoepithelial cells: their origin and function in breast morphogenesis and neoplasia Diverse progenitor cells preserve salivary gland ductal architecture after radiation-induced damage Neurotrophin signaling is a central mechanism of salivary dysfunction after irradiation that disrupts myoepithelial cells A cellular hierarchy of Notch and Kras signaling controls cell fate specification in the developing mouse salivary gland Expanding the 3-O-sulfate proteome–enhanced binding of neuropilin-1 to 3-O-sulfated heparan sulfate modulates its activity Mice deficient in heparan sulfate 3-O-sulfotransferase-1: normal hemostasis with unexpected perinatal phenotypes A comprehensive nomenclature for serine proteases with homology to tissue kallikreins and hormonal regulation of prostate-specific antigen and the extended kallikrein locus Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 signaling in brain endothelial cells Specific role for glypican-1 in glioma angiogenesis Measurement of intracellular calcium of submandibular glands using a high throughput plate reader Calcium signaling defects underlying salivary gland dysfunction Laminin alpha5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through beta1 integrin signaling Par-1b is required for morphogenesis and differentiation of myoepithelial cells during salivary gland development The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation Control of fibroblast growth factor (FGF) 7- and FGF1-induced mitogenesis and downstream signaling by distinct heparin octasaccharide motifs Structural specificity in a FGF7-affinity purified heparin octasaccharide required for formation of a complex with FGF7 and FGFR2IIIb The heparin/heparan sulfate sequence that interacts with cyclophilin B contains a 3-O-sulfated N-unsubstituted glucosamine residue The importance of heparan sulfate in herpesvirus infection HS3ST1 genotype regulates antithrombin’s inflammomodulatory tone and associates with atherosclerosis Probing structural selectivity of synthetic heparin binding to stabilin protein receptors Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer FGF2-dependent mesenchyme and laminin-111 are niche factors in salivary gland organoids Neuronal-epithelial cross-talk drives acinar specification via NRG1-ERBB3-mTORC2 signaling Myoepithelial cells: their origin and function in lacrimal gland morphogenesis Quantitative analysis of heparan sulfate using isotopically labeled calibrants Improving the sensitivity for quantifying heparan sulfate from biological samples Patel, V. N. et al. Loss of 3-O-sulfotransferase enzymes, Hs3st3a1 and Hs3st3b1, reduces kidney and glomerular size and disrupts glomerular architecture. Matrix Biol. https://doi.org/10.1016/j.matbio.2024.06.006 (2024) CERE-120 prevents irradiation-induced hypofunction and restores immune homeostasis in porcine salivary glands Integrating single-cell transcriptomic data across different conditions Comprehensive integration of single-cell data Spatial and temporal expression of heparan sulfate in mouse development regulates FGF and FGF receptor assembly Peripheral and orofacial pain sensation is unaffected by the loss of p39 Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis Specific heparan sulfate structures modulate FGF10-mediated submandibular gland epithelial morphogenesis and differentiation Download references This project was funded in part by the Intramural Program of the NIH at NIDCR laboratory was supported by R44GM142304 to Z.W Open access funding provided by the National Institutes of Health National Institute of Dental and Craniofacial Research Division of Chemical Biology and Medicinal Chemistry NIDCD/NIDCR Genomics and Computational Biology Core is a founder and chief scientific officer for Glycan Therapeutics Corp is an employee of Glycan Therapeutics Corp and has equity in the company The other authors declare no competing interests Nature Communications thanks Lena Kjellén and Elaine Emmerson for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-024-51862-0 Metrics details Diffuse large B-cell lymphoma (DLBCL) accounts for approximately 30–40% of all non-Hodgkin lymphoma cases the adrenal gland DLBCL was not been well explored We performed RNA sequencing of ten DLBCL samples from adrenal gland integrated analyzed DLBCL RNA data from multiple organs defined the new subtypes of adrenal gland DLBCL and explored their molecular signatures The special expression pattern and microenvironment immunology scores of adrenal glands DLBCL were observed when compared with other organs Natural killer T cells was predicted to significantly enrichment in adrenal gland DLBCL canonical cancer pathways such as programmed death protein 1 signaling pathways tumor necrosis factor signaling pathways and peptide antigen binding pathways were found to be correlated with adrenal gland DLBCL Further analysis defined two distant adrenal gland DLBCL sub-type by RNA expression pattern defined the molecular subtype of adrenal gland DLBCL These results expanded the organ related DLBCL data provided the new knowledge of adrenal gland DLBCL expression profile Heterozygous symptoms resulted in further complications and finally caused lots of burden and greatly impaired quality of patient’s life patients were also reported to benefit from stem cell transplantation we report a comprehensive study of DLBCL from adrenal gland and other organs discovered the specific immune signature and pathways of adrenal gland DLBCL Total RNA was extracted using TRNzol Universal Reagent (4992730 and integrity of the RNAs was investigated by Bioanalyzer 2100 system (Agilent Technologies RNA sequencing was conducted with 1 ug RNA for each sample using the ployA isolation method More than 5 Giga bases of data per sample was obtained on the Illumina NovaSeq 6000 platform for subsequent analysis immune score and cell sub type (CD4+ T cells Expression and immune pattern among organs Two clusters were observed between adrenal gland from our center and other organs from TCGA database Heterozygous immune scores pattern among among multiple organs Nonparametric Kruskal–Wallis test was utilized for comparison among multiple groups p values < 0.05 were considered significant difference (C) Microenviroment score of all DLBCL samples NKT cell score was enriched in adrenal gland samples Heterozygous NKT scores pattern among among multiple organs Differentially expressed genes between adrenal gland and lymph node (A) Dysregulated genes between adrenal gland and lymph node samples fold change > 1 were selected as thresholds (B) Heatmap of all DLBCL samples including adrenal gland and lymph node (C) Differentially expressed gene enriched in pathways by gene set enrichment analysis Expression and immune pattern among adrenal gland samples (A) Correlation plot of all adrenal gland DLBCL samples Two clusters were observed among adrenal gland samples (B) Significant different expressed genes enriched in cluster1 and cluster2 (C) NKT score between cluster 1 and cluster 2 adrenal gland DLBCL samples Nonparametric Wilcoxon test was utilized for comparison (D) CD4 memory T score between cluster 1 and cluster 2 adrenal gland DLBCL samples (E) Proliferate B cell score between cluster 1 and cluster 2 adrenal gland DLBCL samples (F) Differentially expressed gene enriched in KRAS pathways in multiple cancers by gene set enrichment analysis (G) Differentially expressed gene enriched in ATM PTEN pathways by gene set enrichment analysis DLBCL encompassed different subtypes and molecular variants that exhibited heterozygous characteristics and clinical behaviors Main subtype of DLBCL included activated B-cell-like (ABC) DLBCL primary mediastinal large B-cell lymphoma (PMBCL) multiple dysfunctions in the immune system could be observed and cell interactions in the tumor microenvironment DLBCL typically presented with enlarged lymph nodes Patients may experience symptoms such as fatigue Risk factors of DLBCL were reported such as age (older individuals) immunodeficiency (such as HIV/AIDS or post-transplant immunosuppression) The standard treatment for DLBCL was R-CHOP which involved a combination of chemotherapy drugs and immunotherapy included rituximab (an anti-CD20 antibody) This approach exhibited high response rates and improved survival outcomes CD30-targeted antibody-drug conjugate called brentuximab vedotin also shown promising results in patients with relapsed or refractory DLBCL were being investigated in clinical trials Chimeric antigen receptor (CAR) T cell therapy which modified the patient’s T cells to express a CAR that recognizes and targets CD19 demonstrated complete remissions in relapsed or failed to respond to other treatments patients type I iNKT cells produce IFN-γ and played an immunoregulatory role on the NK and CD8+ T cell populations by inducing cytokine production Given the limited effectiveness of standard therapies in certain patients researchers have explored TME-based treatments as novel strategies to establish a more immunogenic environment and enhance drug delivery ultimately improving patient response rates Emerging evidence suggests that the composition of the TME is pivotal in understanding the pathogenesis of lymphoma while also providing novel avenues for targeted therapies and tumor prognosis prediction These TME cells exhibit distinct biomarkers and fulfill various roles in the tumorigenesis and prognosis of B-cell lymphoma disrupted communication between lymphoma cells and the microenvironment contributes to the ability of lymphoma cells to evade the immune system’s surveillance Immune escape mechanisms in DLBCL include: (1) altering immune recognition by reducing or losing recognition molecules (2) suppressing the immune system’s antitumor function and (3) creating a microenvironment that supports the growth of lymphoma cells plays a significant role in DLBCL’s tumor development and progression prompting active investigation into its molecular foundations we sequenced ten DLBCL samples from adrenal gland analyzed multiple organ related DLBCL RNA data defined the new subtype of adrenal gland DLBCL and explore their molecular signatures Our study expanded the organ related DLBCL data The datasets presented in this study can be available from the corresponding author on reasonable request Genetics and pathogenesis of diffuse large B-cell lymphoma Diffuse large B-cell lymphoma (DLBCL): Early patient management and emerging treatment options featureCounts: An efficient general purpose program for assigning sequence reads to genomic features xCell: Digitally portraying the tissue cellular heterogeneity landscape Cancer prevention and therapy through the modulation of the tumor microenvironment Detachable nanoparticle-enhanced chemoimmunotherapy based on precise killing of tumor seeds and normalizing the growing soil strategy Tumor microenvironment: Sanctuary of the devil Influence of tumour micro-environment heterogeneity on therapeutic response Therapeutic targeting of the tumor microenvironment The biology of the tumor microenvironment in DLBCL: Targeting the “don’t eat me” signal Download references Yunke Li from Beijing Digitf Biotechnology Co. (Beijing) in data analysis and colleagues from Annoroad Gene Technology (Beijing Ruochen Qi and Xiaoyan Zhang contributed equally to this work The First Affiliated Hospital of Air Force Military Medical University Longlong Zhang: Clinical data collection; Xiaozheng Fan Bo Yang: Writing-original draft preparation; Guohui Wang This study was approved by the Institutional Ethics Board of The First Affiliated Hospital of Air Force Military Medical University The studies were conducted in accordance with the local legislation and institutional requirements Written informed consent was obtained from the patients in this study Download citation DOI: https://doi.org/10.1038/s41598-025-90610-2 Metrics details Canine apocrine gland anal sac adenocarcinoma (AGASACA) is a rare few cell lines are available and used to establish the current treatment protocols Organoids are three-dimensional cell cultures derived mainly from stem cells and can reproduce tissueʼs epithelial structure 10 AGASACA organoid lines were developed from surgically removed tissues of AGASACA-affected dogs and analyzed for comparison with the original tissues AGASACA organoids were successfully generated from all cases and were positive for CK7 consistent with previous reports in dogs and humans Electron microscopic imaging of AGASACA organoids showed organelles including numerous granules and fat droplets that characterize apocrine gland cells AGASACA organoids were tumorigenic in vivo in immunodeficient mice treatment of the AGASACA organoids with carboplatin and lapatinib revealed different sensitivity profiles among lineages being divided into sensitive and resistant ones mitoxantrone and toceranib showed generally high efficacy in all organoids our established AGASACA organoids have the potential to be an experimental tool for the development of novel therapies for canine and human apocrine gland adenocarcinoma Because few validated experimental models exist for human apocrine gland cancer canine AGASACA has also attracted attention as a spontaneous model for human apocrine gland cancer and have shown that these methods can reproduce histopathological or genetic characteristics of their original tumor tissues and evaluate drug sensitivity the canine AGASACA organoid culture method has not been established yet we aimed to establish an organoid culture method from canine AGASACA tissues and evaluated the histopathological characteristics and tumorigenicity of the generated organoids We also investigated the efficacy of molecularly targeted drugs The components were as follows: Advanced DMEM with 50% Wnt and R-Spondin conditioned medium; 2 mM GlutaMax; 1X B-27 supplement; 100 μg/mL Primocin (Thermo Fisher Scientific USA); 1 mM N-Acetyl-L-cysteine; 10 mM nicotinamide (Sigma-Aldrich USA); 3 μM SB202190; and 10 μM Y-27632 (Cayman Anti-cancer drugs used in the present study were as follows: carboplatin (FUJIFILM WAKO Pure Chemical Corporation mitoxantrone (Cayman Chemical); and toceranib (Sigma-Aldrich) The primary antibodies used were as follows: Cytokeratin 7 (CK7 The secondary antibodies used were as follows: Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cayman); and Dako Envision + dual Link System-HRP (Agilent Technologies Inc. Generation of canine anal sac grand carcinoma (AGASACA) organoids Schematic experimental design of generation of canine AGASACA using a piece of surgically removed tissue (A) AGASACA cells were seeded on 24-well plates with Matrigel as a 3D culture Representative AGASACA organoid images taken on days 2 Representative images of bright-field microscopy (C) and hematoxylin and eosin (H&E) staining (D) of each lineage of canine AGASACA organoids and their original tissues (AT22002 Transmission Electron Micrograph (TEM) of AGASACA organoids (E) showing the formation and maturation process of secretory granules (b) and apocrine release separation image (c) were shown Depending on the size of the tissues submitted to the experiment some were fixed in buffered formalin and routinely processed to prepare paraffin blocks for sectioning at 4 µm thickness to be used for Hematoxylin and Eosin (H&E) staining and immunohistochemistry analyses After the organoids and tissues were fixed with 4% paraformaldehyde (PFA) at room temperature (RT) for 24 h they were then dehydrated with ethanol and xylene Both organoids and tissue sections were deparaffined and stained with H&E using standard protocol Images were taken with a camera attached to a microscope (BX-52; Olympus The sections were dehydrated in xylene and graded ethanol Antigen retrieval was performed using 10 mM citrate buffer the slides were treated with 1% hydrogen peroxide for 30 min at RT to stop the endogenous peroxidase activity blocking was performed with 10% normal goat serum (NGS) for 30 min at RT they were treated with primary antibodies (CK7 1:100 and VEGF 1:100) and incubated at 4 °C overnight they were incubated with the secondary antibody (EnVision Dual Link System-HRP) and visualized by using a DAB solution (Nacalai tesque the slides were counterstained with hematoxylin for 1 min Organoids were collected into 15 ml tubes and fixed overnight with 2.5% glutaraldehyde (pH 7.4) They were then washed once with 0.1 M cacodylate (pH 7.4) and incubated at 4 °C for 2 h in a solution of 2% osmium tetroxide and 1.5% K4Fe(CN)6 in 0.1 M sodium cacodylate (pH 7.4) dehydrated in graded ethanol solutions (50% (70–110 nm) were sliced with a diamond knife using a Leica UC7 ultramicrotome and loaded onto 50-mesh copper grids coated by form bar and carbon film The sections were post-stained with uranyl acetate for 15 min at RT and lead citrate TEM images were prepared using a TEM (H-7500 Japan) with a TEM digital camera (NanoSprint500 they were treated with 5 mM ethylenediamine tetraacetic acid (EDTA)/PBS They were then trypsinized using TrypLE (Thermo Fisher Scientific Inc.) for 5 min at 37 °C the organoid cells were strained (70 µm nylon cell strainer 2 × 103 cells of organoids were mixed with 10 µl Matrigel on ice and solidified in a 37 °C CO2 incubator for 20 min The culture media were added and the plates were incubated for 24 h in a 37 °C CO2 incubator the cells were treated with dimethyl sulfoxide (DMSO) Cell viability was examined using the PrestoBlue kit (Thermo Fisher Scientific Inc.) and a microplate reader (TECAN The data of the microplate reader were analyzed and plotted using Sigma Plot software (Systat Software 17 C.B-17/IcrHsd-Prkdcscid mice were obtained from Japan SLC (Hamamatsu) Male immunodeficient mice (5 weeks old) were housed under specific pathogen-free conditions 1 × 106 cells of AGASACA organoids were subcutaneously implanted into the back of mice under isoflurane anesthesia mice were euthanized under isoflurane anesthesia and the organoid-derived tumors were isolated and used for H&E and IHC stainings These mouse experiments were carried out according to the Institutional Animal Care and Use Committee of Tokyo University of Agriculture and Technology approval (Approval number: R04-120) The studies were done in agreement with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines Data are shown as means ± standard error of the mean (SEM) Statistical evaluations were performed using a one-way analysis of variance (ANOVA) followed by Bonferroni’s test P-values < 0.05 were statistically significant These findings closely resemble apocrine gland cells indicating that canine AGASACA organoids could recapitulate the epithelial characteristics of apocrine adenocarcinoma Expression of a glandular epithelial marker CK7 (A) and HER2 (D) in each strain of canine AGASACA organoids (AT22007 Representative photomicrographs were shown (n = 4) Tumorigenesis induced by AGASACA organoids The trypsinized AGASACA organoid cells were subcutaneously implanted into the back of mice Observation of AGASACA organoid injection-induced tumor formulation at mice back at 5 weeks after injection (A Representative images of H&E staining (B n = 4) and expression of CK7 in the formed tumor tissues (C Sensitivity of canine AGASACA organoids to different anti-cancer drugs After each line of canine AGASACA organoid cells were trypsinized and seeded into Matrigel Representative phase-contrast images of canine AGASACA organoids (AT22004) treated with each anti-cancer drug are shown (A) Cell viability was assessed using a Prestoblue cell viability assay and 100% on Y axis represents cell viability for each control (B These results collectively suggest that canine AGASACA organoids may be a new experimental tool for the development of novel treatments for AGASACA diseased dogs which were consistent with the result of cell viability assay Drug sensitivity testing using organoids has the advantage of treating a wide range of drugs at once and eliminating drugs that are not effective before use thereby reducing side effects and increasing owner satisfaction and research is underway to use microfluidic flow with organoids to co-culture them with immune cells canine AGASACA organoids are expected to serve as applicable experimental models in AGASACA research and tools for determining individual treatment strategies We have successfully cultured AGASACA organoids from canine AGASACA tissues AGASACA organoids showed similar expression patterns of markers of the original tumor tissue and were tumorigenic in vivo varying sensitivities to chemotherapies and targeted drugs were evident across AGASACA organoid lines These data suggest that canine AGASACA organoids might be a new experimental platform for novel treatment approaches to AGASACA-diseased dogs Further studies on AGASACA organoids will contribute to a better understanding of canine and human AGASACA The authors declare that all the data supporting the findings of this study are available within the article Polton, G. 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J. 197, 782–787. https://doi.org/10.1016/j.tvjl.2013.05.005 (2013) Download references Tokyo University of Agriculture and Technology Issei Tsurukami and Amira Abugomaa carried out the experiments Tatsuya Usui and Kazuaki Sasaki designed the study and Kazuaki Sasaki wrote and revised the manuscript Download citation DOI: https://doi.org/10.1038/s41598-025-90623-x Metrics details Dry eye disease (DED) is a multifactorial aging disorder leading to tear film insufficiency and instability an important knowledge gap lingers in understanding senescence-associated ocular pathogenesis due to limited in vitro translational lacrimal gland (LG) models this remains a major roadblock to discover effective therapies for the restoration of tear film secretion the authors reported the magnetic bioassembly of two LG organoid platforms to recapitulate functional and aging states porcine primary LG cells were assembled into organoids via a magnetic 3D bioprinting (M3DB) platform This platform could form reproducible LG organoids with epithelial hallmarks (AQP5+) and exhibit epithelial secretory functions (lysozyme activity) DNA damage-induced senescence and cell death was induced with etoposide and LG organoid hypofunction and senescence-associated pathogenesis were observed a novel gene therapy with Box A domain of high-mobility group box-1 (HMGB1-Box A) previously established by our group was applied here to prevent LG cellular senescence for the first time HMGB1-Box A transfection prevented LG organoids from senescence-associated pathogenesis at the transcriptomic M3DB platforms could generate functional and DNA damage-induced senescence LG organoids and this latter damage could be prevented with HMGB1-Box A gene therapy we’ve shown evidence that Box A from high mobility group box 1 (HMGB1) produced DNA gaps via its molecular scissoring activity Transfer of Box A-producing plasmid increases the DNA gaps and enhances DNA durability by protecting the DNA from all types of damage including base changes and losses and single-strand and double-strand DNA breaks This Box A-induced cascade of events ultimately leads to the prevention of DNA damage and suppression of senescence-associated pathogenesis When new Youth-DNA‐GAPs by HMGB1-Box A are supplemented the complete rejuvenation is observed of in vitro senescent cells and in two aging rat models HMGB1-Box A may constitute a promising candidate for future anti-senescent gene therapies for many tissues and exocrine organs including the LG developing senescent LG organoid models mimicking DED is of ultimate importance to evaluate potential anti-senescent gene therapies the main goal of this study was to determine whether HMGB1-BoxA gene therapy can prevent cellular senescence in bioprinted LG organoids exposed to etoposide functional LG organoids were developed by magnetic 3D bioprinting and characterized using multi-omics approaches a LG organoid model with DNA damage-induced senescence was generated upon the induction with etoposide a plasmid with the HMGB1-Box A gene was transfected using a standard lipid-based nanoparticle technology to assess the prevention of cellular senescence in the LG organoid and functional preservation of fluid secretion and the tissue fragments were enzymatically digested in 1x PBS buffer with collagenase type II 1 mg/mL (Gibco A 2 mL (for 200 mg of tissue) of this enzymatic dissociation was used and place inside a 15 mL tube at 37 °C water beaker with magnetic stirring at 500 rpm and incubated for 30 min (vortex the tube every 15 min) The resulting cell suspensions were filtered through 100 μm and 40 μm cell strainers (SPL South Korea) and plated on a T75 flask coated with Cultrex basement membrane extract (Bio-Techne USA) at a density of 1\(\:\:\times\:\:\)106 cells per flask Cells were then cultured in 10 mL expansion media in a 5% CO2 incubator at 37 °C until they reached a confluency of 70–80% The expansion media was composed of DMEM/F12 (Sigma-Aldrich) the Trypan blue exclusion reagent and methodology was utilized (Thermo Fisher Scientific) and cell viability was determined with a Countess 3 FL Automated Cell Counter (Thermo Fisher Scientific) To induce differentiation of the epithelial cells the expansion medium was replaced with 10 mL of fresh epithelial enrichment media and the cells were maintained in culture for an additional 8 days The epithelial enrichment media contained Defined Keratinocyte Serum-Free basal media (DKSFM Thermo Fisher Scientific) with 20 ng/mL EGF The plasmids were transformed into competent Escherichia coli (DH5α USA) and the transformants were selected on LB agar The colonies were then cultured in LB broth with 34 µg/mL chloramphenicol at 37 °C for 16 h with shaking The plasmids were then extracted using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific USA) according to the manufacturer’s instructions The differentiated epithelial-enriched porcine LG cells at confluency of 50–60% were maintained in each well of a 6-well plate containing 2 mL of differentiated culture medium 2.0 µg of each plasmid was transfected for 24 h using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions and their size and morphology was assessed for 6 culture days using a flatbed scanner Epson Perfection V600 (Epson a monolayer of differentiated LG cells with a confluency of 70–80% was dissociated and resuspended in a culture medium at a density 1 × 106 cells/mL The cells were then magnetized with magnetic nanoparticle solution (MNP) at the ratio 10,000 cells/µL and incubated for 2 h at 37 °C with shaking (250 rpm) cells were resuspended in culture medium to a density of 3.33 × 105 cells/mL and transferred into an ultra-low attachment flat bottom 96-well plate with 150 µL of cell suspension in each well and a magnetic dot drive underneath to gain the spheroid-like shape The LG organoids were cultured in a 5% CO2 incubator at 37 °C for 24 h on top of the drive before being treated with 10 µM etoposide for another 24 h The vehicle group was treated with 0.1% DMSO Cytotoxicity or late apoptosis was evaluated with propidium iodide (PI) and nuclear Hoechst 33,342 staining (Thermo Fisher Scientific) These organoids were imaged with a fluorescence microscope (EVOS FL auto) through the z-axis to generate a z-stack (at 10 μm distance between each scan) and whole images were annotated for late apoptotic cells (PI-stained) and ImageJ software was used to determine the fluorescent intensity signal (as per measurement of “integrated density”) Cell viability in the LG organoids was evaluated by determining their ATP activity using CellTiter-Glo 3D Cell Viability Assay (Promega Corporation USA) according to the manufacturer’s protocol reagents and plates were allowed to equilibrate at RT prior to the addition of 20 µL of reagent to each well of a 96-well plate containing organoid in 100 µL of DMEM plates were vigorously mixed for 5 min to induce cell lysis followed by incubation at 37 °C for 25 min 100 µL of the resulting solution was transferred to a white opaque-walled 96-well plate and the relative luminescence units (RLU) were measured on a microplate reader (GloMax Explorer Multimode Microplate Reader Reactive oxygen species (ROS) generation was determined by ROS-Glo H2O2 assay kit (Promega Corporation) as per manufacturer’s instructions This assay was performed 48 h after cellular senescence was induced in the LG organoids while these were cultured in 5% CO2 at 37 °C the culture medium was replaced with 100 µL of medium containing H2O2 substrate and incubated for 6 h 100 µL of working ROS-Glo detection reagent was added to each well and the plate was incubated for 40 min at RT The relative luminescence units (RLU) were then measured on a microplate reader (GloMax Explorer Multimode Microplate Reader) To determine the functionality of LG organoid upon neurostimulation the intracellular Ca2+ mobilization in the organoid was determined with a Fluo-4 Calcium Imaging Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol The organoids were treated with Fluo-4AM for 25 min at 37 °C organoids were stimulated with 10 µg/mL Carbachol (Sigma USA) and Ca2+ intracellular influx was observed using a fluorescence and confocal microscopes (EVOS FL auto and Zeiss LSM 980 LG organoids were washed with 1× PBS and fixed with 4% paraformaldehyde solution for 20 min organoids were stained using the Senescence-associated β-galactosidase (SA-β-gal) staining kit (Cell Signaling Technology The SA-β-gal in organoids was visualized under a light microscope (DMi1 Inverted Microscope Germany) and quantified the results from five random fields using Image J software (NIH Thermo Fisher Scientific) was utilized to measure lysozyme activity in the conditioned media solution where LG organoids were cultured The protocol followed the manufacturer instructions a 10 mg/mL stock suspension of the DQTM lysozyme substrate (Component A) was prepared in 2 mL deionized water the Component C reagent was dissolved in 1 mL of deionized water A lysozyme standard curve was generated in a total volume of 100 µL per well whereas 50 µL of 1× of Component B was added followed by 50 µL of the 1000 U/mL stock solution of lysozyme (prepared earlier) These were mixed by pipetting in the first well and so forth until the final one is discarded lysozyme-containing experimental samples were diluted in 1× Component B to prepate a volume of 50 µL for each reaction Samples were serially diluted to determine the optimal sample concentration 50 µL of the 50 µg/mL DQ lysozyme substrate working suspension was added to each well containing the experimental samples we incubate the reaction mixtures for 30 min or longer at 37 °C and always protecting samples from light Since the assay is continuous (does not terminate) the fluorescence can be measured at multiple time points to follow the reaction kinetics We measured the fluorescence intensity of each reaction in a fluorescence microplate reader (GloMax Explorer Multimode) Digestion products from the DQ lysozyme substrate have a maximum absorption at 494 nm and maximum fluorescence emission at 518 nm these wavelengths were used to measure fluorescence intensity Three hundred sixty-six genes that have percent identity to human genes lower than 85% were excluded an analysis was started by running the analysis function section on the nSolver 4.0 software The Agglomerative Cluster analysis was run and a Z-score transformation function on genes was generated by the software This dictates how the heatmap was centered and scaled Heatmaps were generated using the R studio and GraphPad Prism version 9 (GraphPad To study proteins expressed by LG organoids after aging induction and gene therapy 20–30 organoids were harvested 48 h after etoposide treatment for each experimental group and analyzed through Western blot analysis 250 µg of protein were extracted by using 250 µL RIPA lysis buffer containing protease inhibitors (Thermo Fisher Scientific) 20 µg of denatured total proteins were resolved on 10% SDS-PAGE gels and transferred to a polyvinylidene difluoride membrane (Millipore Non-specific protein bindings were blocked in blocking solution 5% non-fat dry milk in 1× Tris-Buffered Saline with 0.1% Tween-20 (TBST) membranes were incubated with monoclonal antibodies against AQP5 (AB92320 they were incubated with horseradish peroxidase-conjugated secondary antibodies that corresponded to each primary antibody before subjecting to enhance chemiluminescence detection (Bio-Rad All experiments were performed in triplicate at minimum Data was plotted as the mean ± standard deviation (SD) The number of biological replicates is indicated in the figure captions as well as whether fold change was plotted or normalization was done D’Agostino-Pearson normality test was performed to confirm normal shape distribution of all data sets Since all data exhibited a normal distribution we used the Student’s t-test for comparison between two groups A one-way or two-way ANOVA was used for comparison of multiple groups followed by Tukey’s or Dunnett’s post-hoc tests USA) was used for all statistical analysis and significance was defined at 5% Epithelial morphology and viability of a M3DB-assembled LG organoid (A) LG organoid biofabrication workflow steps (B) Brightfield micrographs with phase contrast showing consistent spheroid formation in MNP-tagged LG primary cells (C) Fold change in total ATP of each organoid was determined by a CellTiter-Glo 3D assay (n = 4–5) and all values were normalized to day 1 (baseline) The total ATP was deemed stable through 6 days of culture (n = 5) organoid diameter was determined and consistent up to day 3 after which there was wide variation in organoid size (n = 10) *p < 0.05 when compared to baseline or day 1 with one-way ANOVA with Dunnett’s post-hoc test (E) H&E micrographs displayed a structural integrity and epithelial parenchyma with acini-like clusters (gray arrowheads top white framed inset at higher magnification) and lumen-like regions (black arrowhead bottom black framed inset at higher magnification) of the LG organoid at day 3 of culture resembling that of native LG fresh biopsy tissue Inset is a focused area arising from black frame box intracellular Ca2+ was actively mobilized in the cells within the LG-like organoid The average ratio of apoptotic cells was approximately 40% for the etoposide-treated LGO group (LGO + Eto) and 10% for the control LGO group (LGO) but one needs to account for a substantial decrease in size and number of cells in the senescent organoid (LGO + Eto) (F) Fold change in late apoptosis was calculated after normalization to control LG organoids (LGO) Both organoid groups were stained with PI and the fluorescence signal was quantified for each z-stack Student t-test was performed (n = 3–5): ***p < 0.001 Senescence markers were detected by: (G) ROS-Glo assay ****p < 0.0001 using a Student t-test (n = 3–5) and (H) Senescence-associated β-galactosidase activity ***p < 0.001 using a Student t-test (n = 3–5) the function was evaluated with a Ca2+ influx assay (I) ****p < 0.0001 using a Student t-test (n = 3–5) and lysozyme activity assay (J) ****p < 0.0001 using a Student t-test (n = 3–5) (I) and (J) graphs are plotted as fold change (FC) after normalization to the control LGO group (C,F,G) or to the LGO + Eto group (I,J) Overall, we report herein a bioprinted etoposide-exposed LG organoid that can be utilized for investigating anti-aging gene therapy for the LG. Protection against cellular senescence after Box A gene therapy in LG organoids (A) Brightfield micrographs of whole-mount staining of senescence-associated β-galactosidase (β-gal) activity and Period acid-Schiff (PAS) staining in healthy LG organoids (LGO) and senescent-associated organoids exposed to Etoposide (Eto) after scramble or Box A gene therapy Dashed black lines represent bright magenta regions with mucin deposition in vacuoles (B) The intensity of β-gal-stained cells was quantified by ImageJ ****p < 0.0001 while using Student t-test (n = 3–4) (C) Fluorescence micrographs confirmed the Ca2+ influx and secretory function before and upon cholinergic stimulation with carbachol (1 min) (D) Ca2+-labeled fluorescence signal plotted as a fold change (FC) relative Scramble + Eto group (E) Senescence-associated ROS levels were determined by ROS-Glo assay kit normalized to control naïve LG organoids (LGO) and plotted as fold change (FC) (F) Western blot displaying expression of AQP5 and NKCC1 pro-acinar proteins at 0 h and 48 h after Box A gene transfer (Box A) scrambled plasmid transfer (Scramble) in etoposide-exposed LG organoids and control naïve LG organoids (LGO) The grouping of blots was cropped from 3 different parts of the same gel for each protein and exposures are explicit by using white spaces for a clear delineation The protein content was extracted from 20–30 organoids/group and which were combined at each group level and loaded into each lane The blot was run one time for each protein of interest (G) Differential gene expression between Box A HMGB1 and Scramble plasmid gene therapy in LG organoids was quantified using a nCounter transcriptome panel and nSolver software and plotted with Rosalind software A list of significant differentially expressed genes was determined by a calculated cut-off filter as dictated by the software Default settings for the filter are at a fold change of 1.5 for upregulated and 1.5 for downregulated with a p-adjusted value of 0.05 the modeling in M3DB platforms needs further validation with LG primary cells from human biopsies epithelial acinar polarity should be assessed with large organoids in integrated fluid flow systems senescence-associated β-galactosidase activity and apoptosis (i.e indicating the development of a LG model with senescence-associated pathogenesis this is the first report showing the development of a senescence-associated LG organoid with impaired function (pathogenesis) and cellular senescence features the need for other senescence induction approaches triggering other mechanisms would be relevant to better understand the complex aging phenomena human DED cannot be fully assessed in porcine organoids because of limited homology in certain DED-related peroxidation genes (< 90%) plus our organoids have limited size (< 300 μm) these cannot produce tear film on a robust manner for proteomic studies the HMGB1-Box A gene therapy may constitute a promising anti-senescence strategy for DED prompted by a hypofunctional LG and may have potential applications in aging-associated chronic diseases affecting other exocrine glands M3DB in vitro platforms can generate functional LG organoids as well as hypofunctional LG organoids after DNA-damage induced by etoposide exposure Such senescence-associated pathogenesis phenotype can be prevented by novel gene therapies with HMGB1-Box A Future studies with human LG organoids are necessary to confirm these findings when non-pathological LG biopsies become available Datasets and figures/tables supporting the conclusions of this article are available in the OSF repository: [https://osf.io/8fdx7/?view_only=c0b1d2e859544d26af2491d7ae9d0f52] The organoid culture protocol described in this article is published on protocols.io [DOI 10.17504/protocols.io.b5ttq6nn] de Paiva, C. 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Mater.6, 402–420. https://doi.org/10.1038/s41578-021-00279-y (2021) Download references This research is funded by Thailand Science research and Innovation Fund Chulalongkorn University to JNF This project is also supported by the National Research Council of Thailand (NRCT) and Chulalongkorn University (Project number: N42A670176) to JNF (main PI) and in part from the Faculty Research Grant (Grant number: DRF66033) from Faculty of Dentistry Center of Excellence and Innovation for Oral Health and Healthy Longevity is funded by the Ratchadaphiseksomphot Endowment Fund National Science and Technology Development Agency We would like to give a special thanks to Dr Teerapat Rodboon for performing many of the 3D cell culture experiments and Mr Somchai Yodsanga from Department of Oral Pathology for the assistance to prepare and stain the organoid sections for histology Teerapat Rodboon expressed his wish not to be included in the authorship since he was not able to approve the initial final and revised version of the manuscript Center of Excellence and Innovation for Oral Health and Healthy Longevity Center of Excellence in Molecular Genetics of Cancer and Human Disease Narumol Bhummaphan & Apiwat Mutirangura Department of Microbiology and Center of Excellence on Oral Microbiology and Immunology performed preliminary transfection studies R.C.: Conception and design of histological studies analysis and interpretation of micrographs after histological staining and Y.O.: Performing porcine LG surgical dissections extraction and isolation of LG primary cells and 2D cell culture for expansion providing access and resources for LG biopsies O.M.: Providing resources for plasmid transfection experiments All authors understood the data and provided important contribution to the manuscript All authors read and approved the final manuscript Download citation DOI: https://doi.org/10.1038/s41598-024-73101-8 An official website of the United States government Español The prostate is a small gland in men that helps make semen Located just below the bladder in front of the rectum it wraps around the tube that carries urine and semen out of the body See your doctor right away if you have any of these symptoms: Here are some examples of non-cancer prostate problems: It means your prostate is enlarged but not cancerous Acute bacterial prostatitis usually starts suddenly from a bacterial infection. See your doctor right away if you have fever, chills, or pain in addition to prostate symptoms such as difficulty urinating or pain when urinating You also may need medication to help with pain or discomfort Chronic bacterial prostatitis is a recurrent infection This rare problem can be hard to treat but taking antibiotics for an extended time may work Talk with your doctor about other approaches to help you feel better Treatment may require a combination of medicines Be sure to talk with your doctor about the possible side effects of treatment Prostate cancer is common among American men Your chance of getting prostate cancer may be affected by your: To find out if prostate symptoms are caused by cancer your doctor will ask about your past medical problems and your family’s medical history Your doctor also will perform a physical exam your doctor will put a gloved finger into your rectum to check for: You may be asked to give a urine sample for testing Your doctor also may do a blood test to check the prostate-specific antigen (PSA) level PSA levels can be high in men with an enlarged prostate gland or with prostate cancer You may also need an ultrasound exam that takes computer pictures of the prostate your doctor will refer you to a specialist — a urologist — for a prostate biopsy The doctor will take small tissue samples from several areas of the prostate gland to look for cancer cells The USPSTF does not recommend routine PSA screening for men over age 70 The USPSTF is currently reviewing more recent evidence from this field to determine if changes are needed in screening recommendations PSA testing (along with digital rectal examination) can help doctors determine the nature of the problem In men who have been treated for prostate cancer the PSA test may be used along with physical exams and other tests to determine if the cancer has returned Treatment for prostate cancer depends on whether the cancer affects part or all of the prostate, or if it has spread to other parts of the body. It also depends on your age and overall health. Talk with your doctor about the best treatment choices for you and the possible side effects of treatment You may want to seek a second opinion from another doctor For more details on treatment choices for prostate cancer, call the National Cancer Institute’s Cancer Information Service at 800-422-6237 or visit their website MedlinePlusNational Library of Medicinewww.medlineplus.gov This content is provided by the NIH National Institute on Aging (NIA) NIA scientists and other experts review this content to ensure it is accurate and up to date Metrics details the specific involvement of Demodex infestation in the pathogenesis of RCES as well as the impact of MGD treatment in mitigating RCES The present study aims to investigate the prevalence of Demodex infestation and evaluate meibomian gland functionality in patients diagnosed with RCES It seeks to explore the association between Demodex infection this research endeavors to assess the therapeutic efficacy of intense pulsed light (IPL) therapy in conjunction with meibomian gland massage in managing RCES symptoms and reducing the frequency of disease recurrence All subjects were informed of the purpose and methods of the study and voluntarily signed an informed consent form All examinations were performed by the same experienced physician who outside the study and exacerbation frequency of RCES patients were kept the patient’s chin was placed on the bracket and the upper and lower lids were lifted separately to fully expose the conjunctiva for photography Evaluate the meibomian scores: 0 point for normal (no loss of the meibomian gland); 1 point for less than 1/3 loss; 2 point for 1/3 ~ 2/3 loss; Three points for more than 2/3 loss of the meibomian gland The total score for each eye ranged from 0 to 6 points combining the scores of both the upper and lower glands Blepharolipin score7: let the patient look up the investigator manually expresses the 8 Meibomian glands on the middle 1/3 area of the lower eyelid with moderate pressure evaluate the secretion characteristics of the meibomian gland 0 for crystal clear normal liquid meibum; 1 for muddy meibum; 2 for granular liquid meibum; 3 for toothpaste-like thick meibum Lid margin score7: The eyelid margin morphology was observed under a slit lamp There were four physical signs: Irregularity of eyelid margin 1 point for 1 sign; the total score was 0–4 point observe the Demodex under the photoelectric microscope CONSORT Flow diagram for the recruitment and randomization of participants Korea) intense pulse optical equipment was used for the treatment; parameters were tested in the patient’s jaw before the first treatment adjust the IPL energy according to the patient’s skin color and wore an eye mask to protect the patient’s eyes The treatment area was uniformly coated with a medical coupling agent for 5–0 mm Doctor was expected to wear goggles during the whole treatment Four therapeutic parts were selected from the left temple to the right putting the probe on the coupling agent part press the on-off button to emit the pulses of light and redo the operation until all the therapeutic parts have been done drop 0.4% bupivacaine hydrochloride (Santen Pharmaceutical co LTD Japan) into the conjunctival sac two times then massaged the upper and lowered meibomian gland with meibomian gland massage tweezers The same physician performed all procedures One week after the accomplishment of three treatments in RCES-A group and three months after treatments in RCES-B group the patients were admitted to the hospital for the meibomian gland loss score blepharolipin score and the number of Demodex evaluation the recurrence of corneal erosion between the two groups was compared Patients were interviewed about their symptoms and underwent slit lamp examinations every month for the first six months then every three months for the next six months if patients were unable to visit the clinic follow - up was conducted via telephone or internet to inquire about discomfort symptoms such as eye redness and foreign body sensation all counting data were skewed by the shapiro-wilk test and expressed by M (Q1 The Mann-Whitney U test was used to compare the meibomian gland loss score and the number of Demodex in the two groups The chi-square test was used to compare the positive detection rate of granular Demodex and the recurrence rate of the disease P < 0.05 was considered significant differences statistically RCES group including 17 males and 13 females, with an average age of 44.45 ± 7.75 (32 ~ 58) years old, while control group including 20 males and 11 females, with an average age of 44.81 ± 9.40 (33 ~ 60) years old. There was no significant difference statistically between the two groups in age and gender distribution (Table 1) The positive rate of demodex was 83.3% in RCES group and 38.7% in control group, with a significant difference between the 2 groups (χ2 = 7.60, P<0.01). In the RCES group, the maximum number of demodex in one eye was 18, while in the control group, the maximum number of demodex in one eye was 8 (Table 1) and demodex count before and after treatment in Group RCES-A During a one-year follow-up period post-treatment six patients in RCES-A group and six patients in RCES-B group with symptoms were followed up in hospital due to the absence of any ocular discomfort and the impact of the COVID-19 pandemic did not visit the clinic and were followed up via telephone or internet One patient in the RCES-A group experienced a recurrence of symptoms over five months post-treatment while five patients occasionally reported a mild sensation of a foreign body in the eye three months to a year post-treatment In those patients experiencing a foreign body sensation there are no typical symptoms of recurrent corneal erosions such as conjunctival congestion which were subject to repeated epithelial erosion six patients reported a recurrence of symptoms The difference in recurrence rates between the two groups was statistical significance This study found an association between recurrent corneal erosion (RCES) and both meibomian gland dysfunction (MGD) and Demodex infection improvements were observed not only in meibomian gland function and Demodex infection but also in the control of RCES This present research also found that all RCES patients demonstrated various extents of Meibomian Gland Dysfunction (MGD) This was characterized by a reduction in the quality of the blepharolipin along with significantly elevated blepharolipin scores when compared to the control group These findings suggest that MGD may play a contributory role in the pathogenesis of RCES aligning with conclusions drawn in prior research Although Demodex was still present after three times of IPL sessions the ultimate goal of treating MGD was to manage RCES and reduce its recurrence after three sessions of IPL with meibomian gland massage the meibomian gland function and Demodex counts were significantly improved and the corneal erosion of the patients was effectively controlled which is significantly lower than control; The patients exhibited only punctate turbidity in the corneal epithelium compared to the large corneal epithelial defects seen in previous recurrences These results suggest that treating MGD and Demodex can effectively control RCES offering a new approach to non-invasive clinical treatment of RCES Whether these drugs or physical therapy combined with IPL will have a better therapeutic effect is the direction of future research this research corroborates the association between RCES while causation has not been firmly established and further research is needed The significant reduction in RCES symptoms and recurrence rates post-IPL therapy suggests that addressing MGD and Demodex may be pivotal in managing RCES While IPL therapy’s long-term efficacy warrants further investigation this study indicate a promising non-invasive treatment modality for RCES with potential implications for improving patient quality of life The datasets generated and/or analysed during the current study are not publicly available due the overall research has not yet concluded but are available from the corresponding author on reasonable request Clinical course and risk factors of recurrent corneal erosion: observational study Rezidivierende hornhauterosion bei epithelialen hornhautdystrophien [recurrent corneal erosions in epithelial corneal dystrophies].Klin Recurrent corneal erosion: clinical features Demodex folliculorum infestation in meibomian gland dysfunction related dry eye patients CONSORT 2010 Statement: updated guidelines for reporting parallel group randomised trials The influence of congenital and developmental cataract surgery on the ocular surface in a six-month follow-up prospective clinical study Prevalence of meibomian gland dysfunction at the time of cataract surgery Sample size of 12 per group rule of thumb for a pilot study Clinical presentation and causes of recurrent corneal erosion syndrome: review of 100 patients Prevalence of Demodex folliculorum and Demodex brevis in patients with blepharitis and chalazion Demodex et pathologies de la surface oculaire [Demodex and ocular surface disease] High prevalence of demodex brevis infestation in chalazia Association of meibomian gland morphology with orifice plugging and lid margin thickening in meibomian gland dysfunction patients In StatPearls [Internet] (StatPearls Publishing Ocular Demodicosis as a potential cause of ocular surface inflammation Clinical diagnosis and management of Demodex blepharitis: the Demodex Expert Panel on Treatment and Eyelid Health (DEPTH) Occurrence of Demodex species in patients with blepharitis and in healthy individuals: a 10-year observational study The correlation between Demodex infestation and meibomian gland dysfunction at different ages Safety and efficacy of the phototherapeutic keratectomy for treatment of recurrent corneal erosions: a systematic review and meta-analysis Retsidiviruyushchaya Eroziya rogovitsy: sovremennyi vzglyad na problemu [recurrent corneal erosion: modern view of the problem] Bandage contact lenses versus deproteinized calf blood extract eye gel for recurrent corneal Erosion syndrome: a case-control study Intense pulsed light treatment for dry eye disease due to meibomian gland dysfunction; a 3-year retrospective study Efficacy of intense pulsed light and meibomian gland expression treatments in meibomian gland dysfunction: a meta-analysis of randomized controlled trials Intense pulsed light (IPL) therapy for the treatment of meibomian gland dysfunction Therapeutic effect of intense pulsed light with optimal pulse technology on meibomian gland dysfunction with and without ocular demodex infestation Therapeutic effect of intense pulsed light on ocular demodicosis Intense pulsed light treatment in meibomian gland dysfunction: a concise review Perfluorohexyloctane in dry eye disease: a systematic review of its efficacy and safety as a novel therapeutic agent Eyelid exfoliation treatment efficacy and safety in dry eye disease and contact lens discomfort patients: A systematic review Download references The funding was supported by Hubei provincial health Commission scientific research foundation (WJ2019F028) Research Fund of Aier Ophthalmology Hospital Group (AF2104D12) Wuhan Aier Ophthalmology Hanyang Eye Hospital designed the study and wrote the manuscript W.S and X.G provided and analyzed the data and commented on the manuscript X.M supervised the study and edited the manuscript This study protocol was reviewed and approved (Approval No HYEYE2018IRB01) by the clinical research ethics committee of Aier eye hospital of Wuhan university Written informed consent was obtained from the patient Download citation DOI: https://doi.org/10.1038/s41598-024-73215-z 2024 | LITTLE ROCK — A University of Arkansas for Medical Sciences (UAMS) research team has been awarded a $3 million grant from the National Institutes of Health (NIH) to test promising findings that could lead to better treatments for hormone loss caused by a malfunctioning pituitary gland The five-year R01grant from the NIH National Institute of Diabetes and Digestive and Kidney Diseases is focusing on the pituitary’s hormone-producing cells with a particular focus on manipulating a protein that the team found controls how the cells behave “Understanding how pituitary cells shift their functions to respond to changing needs could help us not only treat hormone deficiencies but also potentially control cancer cells that similarly ‘switch fates’ to resist treatments,” said Angus MacNicol co-principal investigator and a professor and vice chair for the College of Medicine Department of Neuroscience is joined on the study by two other co-principal investigators: Gwen Childs an expert in pituitary physiology and a distinguished professor an expert in cell biology and an associate professor Both are in the Department of Neuroscience an assistant professor in the Department of Neuroscience an associate professor in the College of Medicine Department of Biochemistry and Molecular Biology The three co-principal investigators joined forces more than 10 years ago and Angus MacNicol hopes the team science approach will continue to advance their work and none of us could have moved our previous projects forward to the point we are now if we had all been working independently,” he said “We have a synergy where the sum is greater than the parts.” Current hormone replacement therapies cannot fully replicate the body’s natural hormonal cycles highlighting the need for innovative approaches located at the base of the brain and no larger than a pea produces hormones that regulate critical bodily processes Cells within the gland have unique flexibility — called plasticity — to adjust which hormones are produced in response to changing conditions A protein called Musashi plays a critical role in how the cells function in hormone production Further understanding of Musashi’s regulatory mechanisms could potentially lead to the design of small molecules that would force Musashi to help cells work more effectively the hope is that we could control pituitary function to compensate for hormone loss as we age,” MacNicol said “This could lead to new ways to restore hormone balance An ability to control Musashi activity could be applicable to inhibit cancer cells from becoming resistant to treatments.” The team is using state-of-the-art mRNA (messenger RNA) and miRNA (micro RNA) sequencing alongside mass spectrometry to provide a comprehensive analysis of the gene expression and protein synthesis that govern pituitary cell function Unlike traditional research that centers on gene transcription the study is exploring how the cells behave after the genetic instructions are copied from the DNA into the mRNA “The techniques we’re applying are cutting-edge,” MacNicol said “They haven’t been applied to the pituitary before That’s why people are excited about the project.” Office: 501-686-8998 Mobile: 501-951-7260 Leslie@uams.edu Office: 501-686-8994 Mobile: 501-416-0354 Yavonda@uams.edu © 2025 University of Arkansas for Medical Sciences | Little Rock, AR Volume 14 - 2024 | https://doi.org/10.3389/fonc.2024.1419827 Adrenal gland metastases from malignant melanoma are a common but poorly characterised condition Their lack of consistent clinical features and poor response to immune checkpoint inhibitors pose a significant diagnostic and therapeutic challenge to practitioners This case report describes a 78-year-old male with no prior history of melanoma presenting with nonspecific abdominal symptoms and unintentional weight loss who was found to have undifferentiated bilateral adrenal gland metastases from malignant melanoma the primary site of the adrenal gland metastases remained unknown prompting the consideration of primary adrenal melanoma as a diagnosis The patient underwent four cycles of treatment with immune checkpoint inhibitors followed by maintenance therapy and subsequent adrenal metastasectomy the patient’s tumour was resistant to treatment and became undifferentiated The patient continued with palliative care until his death more than three years after the onset of symptoms and prognosis of this patient’s disease are discussed in detail to help inform the management of similar cases This case report describes a 78-year-old male presenting with abdominal pain who was found to have bilateral adrenal gland masses on imaging and later confirmed to be undifferentiated malignant melanoma with an unknown primary site The unique clinical presentation of this patient’s disease is explored in detail to help gain a greater understanding of AGM from metastatic melanoma and improve diagnosis and treatment of this condition A 78-year-old Caucasian male with no prior history of melanoma presented to the Thunder Bay Regional Health Sciences Center (TBRHSC) emergency room (ER) in November 2020 with left upper quadrant pain recent unintentional weight loss of 25-30 lbs and irregular bowel movements with occasional diarrhea and constipation The patient was a non-smoker and drank alcohol infrequently His medical history was significant for a one-month hospitalization in his 30s following a propane explosion This resulted in extensive burns to his arms he had a mole excised from an unknown location No pathology report is available from that procedure and it is not known whether it was a new growth undergoing dysplastic changes or if it was a long-standing mole He endorsed no significant history of sun nor artificial ultraviolent light exposure His family history was significant for his mother having died of colon cancer in her mid-80s and two of his brothers being diagnosed with cancer in their 70s; one with pancreatic cancer treated by Whipple’s resection and the other with lymphoma The patient’s left upper quadrant pain was previously investigated in August 2020 via endoscopy A patulous gastroesophageal junction was observed and biopsy of this site returned negative for any pathology Gastroesophageal reflux disease (GERD) was suspected This was ineffective in managing his symptoms prompting his visit to the ER in September wherein he was found to be hypertensive (blood pressure 142/96 mmHg) with abdominal distention and bloating that prevented deep palpation and detection of organomegaly All other physical findings and vitals were within normal limits Please refer to Figure 1 for a timeline with data from the related episodes of care Bloodwork revealed decreased hemoglobin (128 g/L) and hematocrit (38%) and elevated sodium (156 mEg/L) and bicarbonate (32 mEq/L) Venous blood gases revealed low bicarbonate (11 mEq/L) and elevated total CO2 (35 mEq/L) Urinalysis showed protein at 0.15 g/L and occult blood at 0.3 mg/L All other laboratory findings were within normal limits An initial computed tomography (CT) scan of the abdomen and pelvis revealed heterogeneously enhancing solid mass lesions measuring 4.6 x 3.4 cm in the right adrenal gland and 11 x 9.9 cm in the left adrenal gland displacing the spleen and left kidney (Figure 2) a small non-occlusive tumour thrombus was observed in the left renal vein Adrenal adenocarcinoma was suspected at this time Axial contrast enhanced CT (A) through the upper abdomen in November 2020 shows a 11 x 9.9 cm lobulated heterogeneously enhancing solid cystic mass in the left adrenal gland and a 4.6 x 3.4 cm similar appearing mass in the right adrenal gland (indicated by arrows) Coronal contrast enhanced CT (B) through the upper abdomen in November 2020 shows an intraluminal filling defect in the left renal vein consistent with thrombus associated with the left adrenal mass A few suspicious lymph nodes are present in the vicinity No metastases were found on a subsequent head and chest CT and whole-body bone scan An ultrasound (US) guided biopsy of the left adrenal mass revealed a poorly differentiated malignant spindle cell tumour positive for vimentin The mass was negative for additional immunohistochemical markers molecular testing for BRAF gene mutation was not possible The pathology report supported the diagnosis of an undifferentiated malignant neoplasm that had metastasized to the adrenal glands from an unknown primary site Consultation with medical oncology supported this diagnosis and the patient began an immunotherapy regimen in December 2020 including a skin biopsy of the upper back and right axilla in December 2022 and a diagnosis of primary adrenal melanoma was considered the tumours remained confined to the adrenal glands The patient underwent four cycles of nivolumab 90 mg IV and ipilimumab 250 mg IV he developed a rash and was administered diphenhydramine 50 mg PO and he was started on prednisone 90 mg PO daily with a taper of 10 mg every subsequent week with successful resolution of the rash the patient remained on a maintenance dose of nivolumab 240 mg IV administered on days one and 15 of a four-week cycle this was changed to 480 mg IV once per four-week cycle due to discomfort at his peripherally inserted central catheter (PICC) By his last cycle of treatment in September 2023 he had received a total of 22 cycles of maintenance nivolumab he tolerated immunotherapy well with minimal side effects In January 2023 a whole-body positron emission tomography (PET)-CT scan was compared to a previous PET-CT scan from June 2021 (Figure 3) This scan showed significant interval enlargement of the left adrenal metastasis and indicated that it was predominantly cystic Focal areas of increased uptake were seen along the periphery of the lesion [standardized uptake value (SUV) of 6.3] The overall volume of residual hypermetabolic disease was reduced and no other evidence of hypermetabolic disease was observed PET-CT scan in June 2021 (A) shows residual uptake within the periphery of the left adrenal mass (arrow) with an SUV of 14.7 There was no significant metabolic activity in the right adrenal mass PET-CT scan in January 2023 (B) shows enlargement of the left adrenal mass (arrow) which appears almost completely cystic with reduced volume and extent of metabolic uptake within the mass as compared to the previous PET-CT scan palliative radiation therapy was considered but advised against due to the large cystic volume of the tumour percutaneous drainage was recommended to help reduce the size of the cyst Subsequent cytology of the drainage revealed degenerative and atypical cells the sample was unsatisfactory for a complete evaluation owing to blood and debris the right adrenal mass was only observed and never biopsied It did not undergo specific management but showed a resolution in response to immunotherapy at the time of the metastasectomy Axial contrast enhanced CT through the upper abdomen in June 2023 shows complete replacement of the left adrenal gland by a well circumscribed fluid attenuation mass with smooth margins measuring 9.9 x 7.9 x 7.8 cm A linear area of fat attenuation is seen within the mass along the path of a previously placed pigtail catheter and the right adrenal gland appears normal In September 2023 the patient appeared alert and orientated with no signs of distress and sleep disturbances [Edmonton symptom assessment system (ESAS) score of 0-2] he was experiencing ongoing pain near the surgical site (managed by naproxen 500 mg PO twice per day) and minimal but continuous blood loss from the surgical incision due to incomplete wound healing an US scan of the abdomen revealed that the left suprarenal mass now measured 16.8 x 11.5 x 10.2 cm Due to the progressive enlargement of his tumour despite both surgical and immunotherapeutic interventions and the contraindications towards initiating radiation therapy the patient could not be recommended any further curative treatment He was admitted to a palliative unit and received hospice care until his death in early December 2023 it is suspected that his death resulted from a combination of ongoing blood loss compression of adjacent structures from tumour progression AGM from malignant melanoma are common, however, due to the variability or absence of symptoms in affected individuals, many cases are only discovered incidentally during imaging or post-mortem autopsy (8) One of the key strengths of this report lies in the multi-disciplinary approach to managing this common yet somewhat obscure diagnosis in a patient who presented with nonspecific symptoms This approach enabled the patient to receive a comprehensive diagnostic assessment and ongoing care despite the therapeutic difficulties associated with this diagnosis One of the key limitations of this approach was the uncertainty around the identification of a primary site and the absence of a pathology report from the mole excised roughly three years before the onset of symptoms this patient’s diagnosis of malignant melanoma cannot be confirmed and the possibility of this being a case of primary adrenal melanoma needs to be considered it was not possible to examine the lesion further as a new biopsy was not taken prior to the patient’s death this case report describes a 78-year-old male who presented with nonspecific abdominal symptoms and weight loss who was later confirmed to have bilateral AGM from malignant melanoma despite no prior history of melanoma He was managed with ICIs and underwent a left adrenal metastasectomy however his tumour was resistant to treatment and became undifferentiated prior his death which occurred more than three years after the onset of symptoms this patient’s case highlights the challenge of managing AGMs from malignant melanoma and reinforces the need for further advancement in the diagnosis and treatment of this condition to improve patient prognosis The original contributions presented in the study are included in the article/supplementary material Further inquiries can be directed to the corresponding author Data was collected through a retrospective chart review in accordance with the local legislation and institutional requirements Written informed consent was obtained from the individual(s) for their participation in and publication of any potentially identifiable images or data included in this article The author(s) declare that no financial support was received for the research The authors wish to thank the staff of the radiation oncology and pathology departments at the TBRHSC for their contributions to the data provided in this case report The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher Incidence of noncutaneous melanomas in the U.S Early detection of Malignant melanoma: The role of physician examination and self-examination of the skin Crossref Full Text | Google Scholar Extracapsular spread in melanoma lymphadenopathy: prognostic implications Prognostic factors analysis of 17,600 melanoma patients: validation of the American Joint Committee on Cancer melanoma staging system Improved survival with ipilimumab in patients with metastatic melanoma and long-term safety in patients with advanced melanoma receiving nivolumab 4-year survival and outcomes after cessation of pembrolizumab (pembro) after 2-years in patients (pts) with ipilimumab (ipi)-naive advanced melanoma in KEYNOTE-006 doi: 10.1200/JCO.2018.36.15_suppl.9503 Melanoma metastases to the adrenal gland are highly resistant to immune checkpoint inhibitors Melanoma adrenal metastasis: natural history and surgical 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Gastrointestinal Cancers: Gastric and Esophageal Cancers Volume 15 - 2025 | https://doi.org/10.3389/fonc.2025.1525781 Introduction: Esophageal submucosal gland duct adenoma (ESGDA) is a rare benign tumor with non-specific clinical features and imaging findings Case report: In this report we describe the clinicopathological features of a new-onset case of ESGDA and review 19 previously-reported ESGDA cases in the literature Results: The median age of the 20 patients was 70 years Lesions located in the lower esophagus accounted for 81% of the cases Chest and abdominal discomfort were the main clinical symptoms and endoscopic findings mostly included polypoid masses that were impossible to correctly diagnose prior to excision and biopsy the submucosal tumor was found to be composed of glandular cavities of various sizes bland cytology with infrequent mitotic figures and the interstitium was infiltrated by a large number of lymphocytes Immunohistochemical analysis revealed the expression of adenomyoepithelial cytokeratins (CK) CK7 the longest follow-up period was 132 months of the 20 patients Discussion: ESGDA is more common in the lower esophagus in elderly patients Taking into account its characteristic microscopic morphology and immunohistochemical markers the possibility of this rare disease should be considered to avoid misdiagnosis or missed diagnosis Complete en bloc resection with Endoscopicp may be the best strategy for both the diagnosis and treatment of this entity Esophageal submucosal gland duct adenoma (ESGDA) is a benign tumor that originates in the submucosal gland (SMG) of the esophagus and is unrelated to Barrett’s esophagus (1) only 19 such cases have been reported in the literature worldwide ESGDA has no specific clinical or endoscopic manifestations and is often misdiagnosed as one of many other types of benign and/or malignant diseases; however its pathology includes characteristic changes and its clinicopathological features are analyzed in the context of a literature review on the subject in order to improve the general understanding of this rare disease and provide a better basis for its future diagnosis and treatment Endoscopic mucosal resection (EMR) was performed and samples were sent for pathological examination shows the endoscope image and pathological images of the case (A) shows the mucosal bulge near the cardia of the esophagus; (B × 200); (D–G) shows the Immunohistochemical staining of the case (D) CK7-positive double-layered cells in ductal adenoma of esophageal submucosal gland indicating glandular epithelial differentiation; (E) CK5/6 (+) cells in the inner layer and outer layer of ductal adenoma of esophageal submucosal gland indicating myoepithelial and squamous epithelial differentiation; (F) Positive p63 in the outer basal layer of ductal adenoma of esophageal submucosal gland indicating myoepithelial differentiation; (G) proliferation index of esophageal submucosal ductal adenoma <1% Pathological diagnosis: Esophageal submucosal gland duct adenoma (ESGDA) EMR was used for complete dissection of the polyps and the patient’s postoperative recovery proceeded well No recurrence was observed after 17 months of follow-up Similar findings were observed in the present case as the lesions were located in the lower esophagus ranging from 3 to 35mm in diameter with an average of 10.4mm most are seen as raised or polypoid tumors that are difficult to distinguish from other inflammatory fibrous polyps All 20 cases of ESGDA that have been reported so far could not be correctly diagnosed prior to pathological analysis Summary of the clinicopathological features of esophageal gland duct adenoma ESGDAs typically have a polypoid appearance The cut surface is often greyish-white and solid and cystic cavity formation can be observed on larger masses the lesions are mainly composed of glandular or cystic cavities of varying sizes The inner layer of glandular epithelial cells can be multi-layered or protrude into the cavity in the form of papillary hyperplasia while the outer layer typically consists of basal-layer cells that are fusiform and arranged around the glandular epithelium Numerous inflammatory cell infiltrates are often observed around the lesions The inner-layer cells are usually positive for CK7 indicating glandular epithelial differentiation while the basal layer cells are positive for p63 and CK5/6 indicating myoepithelial and squamous epithelial differentiation suggesting that these glands have no mucus-secreting functions atrophy or hyperplasia of the normal acini in the surrounding submucosa is often observed The Ki-67 proliferation index of ESGDA is usually low There are currently no established diagnostic criteria for ESGDA we believe that the shape meets the following criteria: (1) The lobular structure is composed of many glands or cysts that are mostly double-layered with glandular inner structures The epithelium and outer layer are composed of basal or myoepithelial cells (2) The gland or cyst epithelium shows papillary hyperplasia (3) Lymphocyte infiltration and lymphoid follicle formation can be seen around the lesion along with normal acinus atrophy or hyperplasia Immunohistochemical analysis shows that the outer basal-layer cells express SMA and p63 and that the inner glandular epithelium express MUC5B and various CKs and MUC2 markers are all typically negative In terms of microscopic morphological features which are difficult to differentiate clinically and endoscopically attention should be paid to the identification of esophageal adenocarcinomas and adenoid cystic carcinomas whether these are related to the direct carcinogenesis of ESGDA remains to be explored in future studies with large sample sizes and long follow-up periods are necessary to understand the exact behavior of the disease when a polypoid mass is observed in the lower esophagus of an elderly patient under a microscope where two layers of mild cells form a cystic or papillary shape the possibility of ESGDA should be considered and the final diagnosis should be made in combination with immunohistochemical analysis This will ultimately aid in preventing misdiagnosis or missed diagnosis of ESGDAs ESGDA requires only endoscopic tumor resection The original contributions presented in the study are included in the article/Supplementary Material The studies involving humans were approved by the ethics committee of Yangpu Hospital Affiliated to Tongji University The participants provided their written informed consent to participate in this study Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article The author(s) declare financial support was received for the research This work was supported by Project of College-level Key Discipline of Yangpu Hospital Affiliated to Tongji University (No Fund of Yangpu Hospital Affiliated to Tongji University (No Project of Yangpu District Health and Wellness Committee (No YPM202414) and Science and Technology Project of Health Commission of Jiangxi Province (No.202510881) We thank Editage (www.editage.cn) for its linguistic assistance during the preparation of this manuscript The author(s) declare that no Generative AI was used in the creation of this manuscript The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2025.1525781/full#supplementary-material Refinement and reproducibility of histologic criteria for the assessment of microscopic lesions in patients with gastroesophageal reflux disease: the esohisto project doi: 10.1111/j.1440-1827.1990.tb01555.x Gi stem cells - new insights into roles in physiology and pathophysiology PubMed Abstract | Crossref Full Text | Google Scholar Hyperplasia of the submucosal glands of the columnar-lined oesophagus PubMed Abstract | Crossref Full Text | Google Scholar Esophageal submucosal gland duct adenoma: an unrecognised esophageal counterpart of minor salivary gland tumours with frequent braf V600e mutations Adenoma accompanied by superficial squamous cell carcinoma of the esophagus doi: 10.1002/1097-0142(19930415)71:8<2435::aid-cncr2820710802>3.0.co;2-a doi: 10.1097/00000478-199510000-00009 doi: 10.1111/j.1572-0241.1998.00461.x Esophageal submucosal gland duct adenoma: characteristic eus and histopathologic features Cavitary mucoepidermoid carcinoma of lung with metastases in skeletal muscles as presenting features: A case report and review of the literature Esophageal gland duct adenoma: immunohistochemical comparison with the normal esophageal gland and ultrastractural analysis doi: 10.1097/01.pas.0000213400.64945.9e PubMed Abstract | Crossref Full Text | Google Scholar Esophageal submucosal gland duct adenoma: A clinicopathological and immunohistochemical study with a review of the literature Dis esophagus: Off J Int Soc Dis Esophagus Endoscopic findings of esophageal submucosal gland duct adenoma VideoGIE: an Off video J Am Soc Gastrointestinal Endoscopy Endoscopic findings of esophageal gland duct adenoma resected by endoscopic submucosal dissection Esophageal submucosal gland duct adenoma of the esophagus-gastric junction: report of a case Zhonghua bing li xue za zhi = Chin J Pathol doi: 10.3760/cma.j.cn112151-20190913-00500 Esophageal submucosal gland duct adenoma: report of a case doi: 10.3760/cma.j.cn112151-20201109-00834 Clinicopathological characteristics of esophageal submucosal gland duct adenoma doi: 10.3760/cma.j.cn112151-20210330-00247 Porcine esophageal submucosal gland culture model shows capacity for proliferation and differentiation Intrathoracic robotic-sewn anastomosis during ivor lewis esophagectomy for cancer: back to basics Adenocarcinoma in Situ Arising from the Submucosal Oesophageal Mucous Glands doi: 10.1097/00042737-200309000-00018 Esophageal Adenocarcinoma That Probably Originated in the Esophageal Gland Duct: A Case Report doi: 10.1046/j.1440-1827.1999.00838.x Huang L and Hao H (2025) Esophageal submucosal gland duct adenoma: a case report and literature review Received: 10 November 2024; Accepted: 20 January 2025;Published: 06 February 2025 Copyright © 2025 Zhou, Zheng, Huang and Hao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Hua Hao, aGFvaHVhNDEwQHRvbmdqaS5lZHUuY24= Metrics details The “gut-brain axis” is involved in many physiological processes its role in regulating mammary gland (MG) development remains unknown we established the mice model of bilateral subdiaphragmatic vagotomy (Vago) to clarify the effects of “gut-brain axis” on MG development in pubertal mice The results showed that Vago reduced the ratio of Lactobacillus and Bifidobacterium neuronal excitability in the nucleus of solitary tract (NTS) Transplanting the gut microbiota of Vago mice to recipient mice replicated these effects and transplanting the gut microbiota of Control mice to Vago mice did not alleviate these effects which up-regulates the ratio of Lactobacillus and Bifidobacterium supplementation elevated NTS neuron excitability GOS enhances BDNF-mediated mammary gland development in pubertal mice via the “gut-brain axis” the study of the physiological mechanism of MG development during puberty has important theoretical and practical value the survival of sensory nerve fibers within the mammary fat pad (MFD) ensures the development of the ductal tree certain aspects of MG development are still unknown the specific mechanisms by which the external environment and internal physiological state interact to jointly influence MG development The in-depth study of the interaction mechanisms between the external environment and the internal physiological state not only forms an important theoretical value in the field of basic science research but also plays a key role in clinical medicine and public health management Despite the fact that the link between the gut-brain axis regarding the development and function of distant organs is now becoming more widely recognised the role of the gut-brain axis in the regulation of MG function remains unreported we are going to conduct a series of studies on the gut-brain axis (vagus nerve pathway) on MG development in pubertal mice to verify the existence of the “gut-brain-mammary gland axis” a Schematic diagram of Vago/SS mouse model protocol The grouping information is as follows: the blank control group (4-week-old pubertal mice) was not subjected to any treatment the negative control group (4/6-week-old pubertal mice) was exerted with SS and the experimental group (4/6-week-old pubertal mice) was exerted with Vago (n = 6); (b d) Changes in body weight of mice during 4 W of rearing e) Changes in food intake of mice during 4 W of rearing; (f) Representative image of whole mount staining scale bar: 5 mm; (g) Quantitative schematic representation of MG development in mice; (h i) Quantification of MG development; (j) Enclosing Radius the number of Sholl intersections (Sum inters) and Branching Density; (k) Linear Sholl plots; (l) Sholl analysis bubble map The mean ± SEM was used for all data presentations The above results suggest that Vago leads to inhibition of MG developmental processes in pubertal mice The result suggested a correlation between mammary sensory nerve fiber density and the development levels of MG The blank control group (4-week-old pubertal mice) was not subjected to any treatment and the experimental group (4/6-week-old pubertal mice) was exerted with Vago (n = 6); (a) The protein expression of Tuj1 and BDNF in MG were detected by Western blotting (n = 3); (b c) Relative protein abundance of Tuj1 and BDNF (compared to β-actin) in Fig 2a; (d) ELISA kit for measuring BDNF in serum; (e) MG frozen sections were stained with Tuj1 (red) and DAPI (blue) to visualize sensory nerve fibers (n = 5) scale bar: 100 μm; (f) Quantification of mammary sensory nerve fiber density and the experimental group (4/6-week-old pubertal mice) was exerted with Vago (n = 6) (a) NTS were stained with c-Fos (green) and DAPI (blue) to visualize the effect on NTS excitability (n = 3) scale bar: 200 μm; (b) Quantification of the activated neuron within the NTS based on c-Fos using ImageJ; (c) PVH were stained with BDNF (green) and DAPI (blue) to visualize the effect on BDNF synthesis and secretion in the PVH (n = 3) scale bar: 200 μm; (d) Quantification of the mean fluorescence intensity of BDNF within the PVH using ImageJ; (e) Hippocampus were stained with BDNF (green) and DAPI (blue) to visualize the effect on BDNF synthesis and secretion within four regions of the hippocampus (CA1 i) Quantification of the mean fluorescence intensity of BDNF within four regions of the hippocampus (CA1 the negative control group (4-week-old pubertal mice) was exerted with SS and the experimental group (4-week-old pubertal mice) was exerted with Vago (n = 6) a Schematic diagram of the Vago-FMT mouse model protocol and the experimental group (4-week-old pubertal mice) was subjected to Vago-FMT (n = 9); (b c) Changes in body weight and food intake of mice during 4 W of rearing; (d representative images of whole mount staining and quantification of MG development (n = 6) and Branching Density; (g) Linear Sholl plots; (h) Sholl analysis bubble map; (i) Schematic of cross-feeding after a female mice has given birth to her pups; (j) Weight change of pups during 4 W of rearing Vago-FMT (8 W) group compared to Control (8 W) group a Schematic diagram of the Control-FMT mouse model protocol The grouping information is as follows: the control group (4-week-old pubertal mice) was subjected to SS/Vago followed by SS-FMT/Vago-FMT and the experimental group (4-week-old pubertal mice) was subjected to SS/Vago followed by Control-FMT (n = 9); (b Vago (8 W) group compared to SS (8 W) group ****p < 0.0001; Vago (8 W)+Control-FMT group compared to SS (8 W)+Control-FMT group scale bar: 100 μm; (j) Quantification of mammary sensory nerve fiber density Vago-FMT (8 W) group compared to Control (8 W) group and Vago (8 W) group compared to SS (8 W) group The above results suggest that the GUT-VN-NTS-PVH pathway affects the expression levels of BDNF a Schematic diagram of the mouse supplemented GOS model protocol the negative control group (4-week-old pubertal mice) was subjected to SS and the experimental group (4-week-old pubertal mice) was subjected to Vago c) Representative images of whole mount staining and quantification of MG development and Branching Density; (e) Linear Sholl plots; (f) Sholl analysis bubble map; (g) NTS were stained with c-Fos (green) and DAPI (blue) to visualize the effect on NTS excitability (n = 3) scale bar: 200 μm; (h) PVH were stained with BDNF (green) and DAPI (blue) to visualize the effect on BDNF synthesis and secretion in the PVH (n = 3) scale bar: 200 μm; (i) MG frozen sections were stained with Tuj1 (red) and DAPI (blue) to visualize sensory nerve fibers (n = 5) scale bar: 100 μm; (j) Quantification of the activated neuron within the NTS based on c-Fos using ImageJ; (k) Quantification of the mean fluorescence intensity of BDNF within the PVH using ImageJ; (l) ELISA kit for measuring BDNF in serum; (m) Quantification of mammary sensory nerve fiber density Control+GOS-H group compared to the Control group ****p < 0.0001; Vago+GOS-H group compared to SS + GOS-H group A schematic model of GOS changing the ratio of gut microbiota composition and affecting BDNF synthesis and secretion via VN-mediated NTS to promote mammary gland development in pubertal mice which was shown to significantly inhibit NTS neuronal excitability and decrease synthesis and secretion of BDNF in the paraventricular nucleus of hypothalamic (PVH) and hippocampal regions a significant increase in body weight and food intake was observed in the mice while a significant increase in body weight and food intake was also observed in the Vago mice transplanted with normal microbiota This result suggests that VN has an important role in sensing gut signals and thus regulating appetite The above studies suggest that early in life gut microbiota may mediate the synthesis and secretion of BDNF via the VN This process induces neuronal maturation and differentiation in different brain regions and plays a key role in regulating the development of the host CNS It also alters the normal development of the host PNS which in turn affects the development of other organs the protein expression levels of Tuj1 and BDNF in the MG were significantly reduced whereas Tuj1 immunofluorescence staining of MG sections showed a significant reduction in mammary sensory nerve fiber density The MG development was inhibited due to the reduced levels of BDNF allowing axon terminal pruning which may be one of the effective means of activating the “gut-brain axis (vagal pathway)” by GOS Our findings indicate that Vago exerts an inhibitory effect on mammary development in pubertal mice Vago has been observed to alter the digestive capacity of mice thereby influencing the nutritional status of the organism although the VN is a principal bidirectional neural communication pathway in the gut-brain axis the findings of this study do not provide compelling evidence to support the conclusion that the brain regulates gut microbial composition through the VN The current study indicates that alterations in the composition of the gut microbiota may influence the synthesis and secretion of BDNF in the brain This suggests that further investigation into the potential use of this mechanism in the treatment of postpartum depression due to lactation in pregnant women may be of interest GOS activates the VN by up-regulating Lactobacillus and Bifidobacterium It promotes MG development in pubertal mice via the GUT-VN-NTS-PVH pathway It remains unclear how Lactobacillus and Bifidobacterium activate the VN We hypothesize that this may occur via two potential pathways: the presence of altered enteric nervous system/intestinal glands and/or altered metabolite levels The experimental animals used in this study were SPF grade ICR mice (3-4 weeks old) purchased from Liaoning Changsheng Biotechnology Co. Protection of animal welfare during husbandry and experimental operations was carried out in accordance with the Jilin University Institutional Animal Care and Use Committee (Number of permit: SY202401004) Mice were given three days to acclimatize before the start of the experiment All mice were housed in an artificially adjustable barrier environment (22-23 °C 55 ± 10% humidity) with a 12 h light-dark cycle and provided with adequate food and water A batch of 4-week-old mice were randomly divided into 5 groups: Control (4 W) group (n = 6) the SS (4/6 W) and Vago (4/6 W) groups underwent sham surgery (SS) and bilateral subdiaphragmatic vagotomy (Vago) at week 4/6 the mice were weighed at two-day intervals during the experiment serum and fecal samples (colon contents) were collected as well as bilateral MGs and intact brain tissue were also gathered Another batch of 4-week-old mice were randomly divided into 3 groups: the Control (8 W) group (n = 6) The SS (8 W) and Vago (8 W) groups underwent SS and Vago at week 8 A batch of 4-week-old mice was randomly divided into 6 groups: Control (8 W) group (n = 21) Vago (8 W) group (n = 21) and Vago (8 W)+Control-FMT group (n = 21) All groups were subjected to FMT (Vago-FMT: Vago derived Fecal Microbiota Transplant; Control-FMT: Control derived Fecal Microbiota Transplant) and reared until 8 weeks of age for subsequent experiments during which the mice were weighed at two-day intervals Fixed feeding of 9 pups per female mouse for 4 weeks and pups were weighed at two-day intervals Referring to Qiu et al. (2022)27 different concentrations of GOS (purity ≥95% China) were supplemented by gavage according to the body weight of mice A batch of 4-week-old mice was randomly divided into 4 groups: Control group (n = 6) fecal samples (colon contents) were collected as well as bilateral MGs was also gathered Another batch of 4-week-old mice were randomly divided into two parts and randomly grouped: (1) Antibiotic cocktail (ABX) group (n = 6) and vancomycin (0.5 g/L) were added to the drinking water to remove most of the gut microbiota Mice were fasted overnight before surgery and anesthetized using 10% urethane the hair near the abdominal midline was shaved and the skin was disinfected with an iodophor disinfectant The skin and muscular layer were incised along the abdominal midline to expose the liver tissue which was carefully and slowly pushed superiorly with a sterile cotton ball moistened with 0.9% saline to expose the esophagus and the required surgical field the subsequent operations were performed under an animal operating microscope: A ligature was performed at the junction of the stomach and esophagus for retraction to allow the operator to see the dorsal and ventral vagus nerve trunks extending along the esophagus which were meticulously stripped and then severed restored the stomach and liver to their normal position the muscular layer and skin were sutured layer by layer using 4-0 absorbable surgical sutures (PGA) Mice were fasted for 20 h before intraperitoneal injection of CCK-8 (8 μg/kg) and then the food intake was measured over a 2 h period The Vago mice whose food intake was reduced by more than 30% were removed during the course of the experiment and were not involved in subsequent experiments A 3-day surgical recovery period was given before proceeding to the next step of the experiment incisions of uniform size were created at the same site in the mouse using the same treatment The dorsal and ventral vagal trunks were exposed the abdominal incision was closed layer by layer using the same method fresh fecal pellets were collected from mice at 8 weeks of age and then soaked (1 fecal pellet/mL for 15 min) using sterile PBS solution with constant vibration shaking during the soaking period The suspension was obtained by centrifugation at 1000 rpm the suspension was centrifuged at 8000 rpm 4 °C for 5 min and filtered twice to obtain the final bacterial suspension mixed with an equal volume of 40% glycerol and stored at -80 °C for subsequent experiments solid media was used under anaerobic conditions and colonies were counted by the dilution coating method to determine if the bacterial concentration of 108 CFU/mL was reached in each tube of storage solution and vancomycin (100 mg/kg) were dissolved in sterile PBS solution according to the body weights of the mice and gavaged (200 μL at a time) to the mice for 5 days gavage (200 μL at a time) was carried out on day 6 using sterile PBS solution FMT (200 μL at a time) was carried out on day 7 for 1 week after which gavage was performed at 2-day intervals the limbs were immobilized and the skin was opened to expose the MGs The fourth pair of MGs of mice were removed carefully spread and placed on slides treated with poly-L-lysine solution (Sigma) and subsequently immersed in pre-prepared Carnoy’s solution (60% ethanol and 10% glacial acetic acid) for 12 h of fixation 15% ethanol and distilled water for 10 min and stained with carmine dye solution (2 g/L and the results were visualized by an optical microscope and the central lymph node was located approximately one-quarter of the way to the left of the whole MG the number of mammary ducts crossing each hypothetical line was determined to quantify MG development in mice Quantitative method 2: Referring to the method of Stanko et al. (2017)55 Sholl analysis was carried out on the microscopic photograph of the whole MG using the Sholl analysis plugin in ImageJ and the radius step size was set to 0.2 mm while performing the analysis for enclosing radius: the length of the MG was measured from the starting point as the primary mammary duct to the end point as the farthest terminal duct; for the mammary epithelial area (MEA): measurement of the area surrounded by the mammary ducts; for the number of Sholl intersections (Sum inters) and Sholl decay coefficients (Sholl decay): statistics by the Sholl analysis plugin; for branching density: Sum inters / (MEA - LNA) The skin is incised above the 1st-2nd lumbar vertebrae along the dorsal midline and the skin is carefully and slowly separated from the abdominal wall using forceps making sure not to damage the surrounding tissues part of the sensory nerve extending from the dorsal root ganglia can be observed and severed After no significant bleeding was observed the skin was finally sutured using 4-0 absorbable surgical sutures (PGA) The skin and abdominal wall are carefully and slowly separated using forceps making sure not to damage the surrounding tissue and not to sever the sensory nerves the back incision was closed in the same way the collected intact brain tissues were immersed in 4% paraformaldehyde (w/v) for 3 days then embedded in paraffin and cut the tissue blocks into 5 μm sections The paraffin sections underwent deparaffinization with xylene and ethanol the collected MG tissues were embedded in OCT (Sakura Finetek USA and the tissue blocks were cut into 8 μm sections the frozen sections were air-dried to dry out the water and fixed in cold acetone for 10 min and then washed with PBS three times after the acetone was completely dry antigen repair was performed in 20 mM EDTA (PH = 8.0) for 9 min at medium heat The sections were blocked with 3% BSA (w/v) for 30 min primary antibody was incubated overnight at 4 °C secondary antibody was incubated at room temperature for 1 h protected from light DAPI was incubated at room temperature for 10 min protected from light and the sections were sealed with fluoromount-G (Wuhan Servicebio Technology Co. images were acquired using an upright fluorescent microscope (Nikon Eclipse C1) Secondary antibody: Alexa Fluor 488-labeled goat anti-rabbit IgG (1:400 C-Fos+ cells were calculated using the ImageJ Mean fluorescence intensity=Integrated Density (IntDen) / Area Referring to the method of Liu et al. (2012)8 MG sensory nerve fiber density was quantified using ImageJ Fiber density (%) = Area of Tuj1 staining within the band / Total area of 15 µm band surrounding the mammary duct × 100% EPH4EV Cell) were purchased from American Type Culture Collection (ATCC CRL-3063TM) and the bovine mammary epithelial cells (BMECs MAC-T) were purchased from Inner Mongolia Wanrui Biotechnology Co. The cells were cultured in a humidified incubator at 37 °C with 5% CO2 and the medium was DMEM (Gibco) containing 10% FBS (Inner Mongolia Opcel Biotechnology Co. Cell culture consumables (CellSafeTM) were purchased from Guangzhou Jet BioFiltration Co. they were treated with varying concentrations of GOS (0 Subsequently used for protein extraction or the effect of varying concentrations of GOS on the cell proliferative viability was assayed using the Cell Counting Kit-8 (Invigentech Inc IV08-500) according to the manufacturer’s instructions P0013F) containing Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific 78443) was added to the sample tubes containing mouse mammary gland tissues and fully grinded using a fully automated sample rapid grinder (Shanghai Jingxin Industrial Co. The protein concentration was determined and partitioned using the BCA Protein Concentration Assay Kit (Proteintech extracted proteins (30 μg/lane) were electrophoresed by 4% and 12% SDS-PAGE and then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes which were blocked for 2 h with 5% skimmed milk (w/v) The PVDF membranes were incubated with primary antibody at 4 °C overnight and then washed five times with Tris-buffered saline with Tween 20 (TBST) for 10 min each time The membrane was incubated for 1 h at room temperature with secondary antibody (1:5000 BA1056/BA1050) corresponding to the primary antibody and then washed five times with TBST for 10 min each time the secondary antibody was made to fluoresce with a hypersensitive luminescent solution (Applicygen and then the protein content was visualized and photographed using a Tanon 4600 image analysis system The Mouse BDNF ELISA Kit (Thermo Fisher Scientific Inc EEL088) was used to detect the changes in BDNF levels in mouse serum according to the manufacturer’s instructions The 16S rRNA sequencing of fecal bacteria obtained from the experiment was done on the Illumina NovaSeq platform (Nanjing Personal Biotechnology Co. Nonrepetitive sequences were clustered into different optional taxon units (OTUs) according to a 97% similarity A minimum of 6 mice were present in each group of animal experiments and all data are expressed as mean ± standard error of the mean (SEM) and bubble maps were performed using GraphPad Prism 9.0 software and differences between groups were determined by t-test or one/two-way ANOVA All relevant data about this research can be requested from the corresponding author The data for 16S rRNA gene sequencing have been deposited with links to BioProject accession number PRJNA1145029 in the NCBI BioProject database and will be accessed upon published Mammary gland stem cells: more puzzles than explanations Mammary development in the embryo and adult: a journey of morphogenesis and commitment Critical review on physiological and molecular features during bovine mammary gland development: recent advances Overexpression of igf-1 during early development expands the number of mammary stem cells and primes them for transformation Niacin stimulates eph4ev mammary epithelial cell proliferation and mammary gland development in pubertal mice through activation of akt/mtor and erk1/2 signaling pathways High-fat diet alters circadian rhythms in mammary glands of pubertal mice Sexually dimorphic bdnf signaling directs sensory innervation of the mammary gland Balance between bdnf and semaphorins gates the innervation of the mammary gland The central nervous system and the gut microbiome The gut-brain axis: how microbiota and host inflammasome influence brain physiology and pathology Achyranthes bidentata polysaccharide ameliorates type 2 diabetes mellitus by gut microbiota-derived short-chain fatty acids-induced activation of the glp-1/glp-1r/camp/pka/creb/ins pathway The gut microbiota modulate locomotion via vagus-dependent glucagon-like peptide-1 signaling The microbiota-gut-brain axis: from motility to mood Gut microbiota changes require vagus nerve integrity to promote depressive-like behaviors in mice Alleviating effect of vagus nerve cutting in salmonella-induced gut infections and anxiety-like behavior via enhancing microbiota-derived gaba Neurotrophins: roles in neuronal development and function The pattern of c-fos expression and its refractory period in the brain of rats and monkeys Neurotrophins and their receptors: a convergence point for many signalling pathways Gut-brain communication and obesity: understanding functions of the vagus nerve The role of the vagus nerve in appetite control: implications for the pathogenesis of obesity The vagus nerve modulates bdnf expression and neurogenesis in the hippocampus Silibinin decreases hepatic glucose production through the activation of gut-brain-liver axis in diabetic rats The gut microbiota and the brain-gut-kidney axis in hypertension and chronic kidney disease Strain specificity of lactobacilli with promoted colonization by galactooligosaccharides administration in protecting intestinal barriers during salmonella infection Gos ameliorates nonalcoholic fatty liver disease induced by high fat and high sugar diet through lipid metabolism and intestinal microbes Gut microbe to brain signaling: what happens in vagus… Ly6c(hi) monocytes provide a link between antibiotic-induced changes in gut microbiota and adult hippocampal neurogenesis Pleiotropic effects of bdnf on the cerebellum and hippocampus: implications for neurodevelopmental disorders Pleiotropic functions of neurotrophins in development Neurotrophin regulation of neural circuit development and function Normal gut microbiota modulates brain development and behavior A next-generation probiotic: akkermansia muciniphila ameliorates chronic stress-induced depressive-like behavior in mice by regulating gut microbiota and metabolites Adult hippocampal neurogenesis is regulated by the microbiome bdnf and their high-affinity receptors in ovine mammary glands during development and lactation Cell death and sexual differentiation of the nervous system Stress-sensitive neural circuits change the gut microbiome via duodenal glands Involvement of intestinal microbiota in adult neurogenesis and the expression of brain-derived neurotrophic factor and other mental disorders as well as the protective effects of dietary components Crosstalk between dietary pomegranate and gut microbiota: evidence of health benefits Nutrition and supplementation in ulcerative colitis Pectin alleviates high fat (lard) diet-induced nonalcoholic fatty liver disease in mice: possible role of short-chain fatty acids and gut microbiota regulated by pectin Effects of different galacto-oligosaccharide supplementation on growth performance and appetite-related hormones in holstein calves Allobaculum involves in the modulation of intestinal angptlt4 expression in mice treated by high-fat diet Gut commensal bacteroides acidifaciens prevents obesity and improves insulin sensitivity in mice Bacteroides thetaiotaomicron ameliorates mouse hepatic steatosis through regulating gut microbial composition gut-liver folate and unsaturated fatty acids metabolism Short-chain fatty acids (scfas) alone or in combination regulate select immune functions of microglia-like cells Communication of gut microbiota and brain via immune and neuroendocrine signaling Loss of vagal anti-inflammatory effect: in vivo visualization and adoptive transfer Neuroprotective effects of fecal microbiota transplantation on mptp-induced parkinson’s disease mice: gut microbiota glial reaction and tlr4/tnf-alpha signaling pathway Neuropilin-2 promotes branching morphogenesis in the mouse mammary gland Quantifying branching density in rat mammary gland whole-mounts using the sholl analysis method Download references This study was supported by the National Natural Science Foundation of China (Project No Jilin Scientific and Technological Development Program (Project No These authors contributed equally: Yusong Ge State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases Key Laboratory for Zoonosis Research of the Ministry of Education participated in the experimental operation participated in the compilation of experimental data All authors were informed and agreed to publish the manuscript Download citation DOI: https://doi.org/10.1038/s41522-024-00607-4 Metrics details The research team began their study by exploring age-related changes in mammary glands during physiological aging in rats because based on our prior studies they better reproduce the heterogeneity and immune escape of human breast cancer and also have ER+ tumors — the subtype that is most common in older women,” explains Kornelia Polyak Prices may be subject to local taxes which are calculated during checkout Nature Aging https://www.nature.com/nataging/ Reprints and permissions Download citation DOI: https://doi.org/10.1038/s43587-024-00783-0 Mastitis: What to do when your breasts are painfully inflamed How — and why — to fit more fiber and fermented food into your meals UTI in older women: Why postmenopausal women are susceptible to urinary tract infection Some adults may need a measles booster shot Counting steps is good — is combining steps and heart rate better Can saw palmetto treat an enlarged prostate half of all men will have an enlarged prostate a condition also known as benign prostatic hyperplasia While BPH does not increase your risk of getting prostate cancer or having sexual problems specifically by causing annoying and embarrassing urination problems "Since prostate enlargement happens gradually men often think more frequent trips to the bathroom are a natural part of aging," says Dr chief medical editor at Harvard Health Publishing and an assistant professor of medicine at Harvard-affiliated Brigham and Women's Hospital "But a little medication can help relieve symptoms meaning less urinary urgency and fewer nighttime awakenings to use the bathroom." An enlarged prostate places pressure on the urethra your prostate can grow from the size of a walnut to about the size of a lemon It's not clear why the prostate grows like this but it's believed certain male hormones such as dihydrotestosterone tend to act more strongly on the prostate gland later in life Because the prostate is located just below the bladder when it becomes larger it can place pressure on the urethra the tube that carries urine from the bladder through the penis and out of the body This may lead to a variety of urination problems and feel like you have not fully emptied your bladder Urine that doesn't get expelled and collects in the bladder can increase the risk of infection which in turn makes it painful to urinate and causes even more bathroom trips and potentially loss of bladder control Urinary tract (or bladder) infections can also lead to a kidney infection Early research suggested that 5-alpha-reductase inhibitors (5-ARIs) a class of drugs used to treat prostate enlargement might increase the risk of developing more aggressive prostate cancer newer studies have found that not only do the drugs appear to pose no extra risk they may even protect against prostate cancer research from the Prostate Cancer Prevention Trial study in 2013 showed that taking the 5-ARI finasteride (Proscar) for seven years could lower the chance of getting low-grade prostate cancer by 25% among men ages 55 and older issue of the Journal of the National Cancer Institute also showed that finasteride lowered the risk by a similar amount (21%) and found the protective effect lasted for at least 16 years See your doctor if you have any of these problems A digital rectal exam can often confirm an enlarged prostate and your doctor may take a urine sample to check for a bladder infection that can be treated with antibiotics If your enlarged prostate is causing symptoms your doctor will likely offer you medication to improve and manage them Two main classes of drugs are used: alpha blockers and 5-alpha-reductase inhibitors (5-ARIs) Your doctor may prescribe one or both types depending on your symptoms and the size of your prostate gland These drugs relax the muscles around the prostate and the opening of the bladder Common BPH symptoms often improve within two days They are most effective for men with minimally to moderately enlarged prostates Commonly prescribed drugs in this class include alfuzosin (Uroxatral) Drugs in this class slowly shrink the prostate so it stops pressing on the urethra Treatment often reduces the prostate's size by one-quarter after six months to a year The two common drugs are finasteride (Proscar) and dutasteride (Avodart) Men might opt for surgery to remove excess tissue from the prostate if medications do not relieve symptoms sufficiently or cause undesirable side effects or if there are complications like urinary retention or recurring urinary tract infections For men who have both erectile dysfunction and symptoms of BPH once-daily low-dose tadalafil (Cialis) is another option Get the latest in health news delivered to your inbox Do not sell my personal information | Privacy Policy and Terms of Use is yours absolutely FREE when you sign up to receive Health Alerts from Harvard Medical School Sign up to get tips for living a healthy lifestyle with ways to fight inflammation and improve cognitive health plus the latest advances in preventative medicine Get helpful tips and guidance for everything from fighting inflammation to finding the best diets for weight loss...from exercises to build a stronger core to advice on treating cataracts the latest news on medical advances and breakthroughs from Harvard Medical School experts Sign up now and get a FREE copy of theBest Diets for Cognitive Fitness Stay on top of latest health news from Harvard Medical School get a FREE copy of the Best Diets for Cognitive Fitness Metrics details Although immune checkpoint inhibitors (ICIs) are effective in some patients with salivary gland carcinoma (SGC) biomarkers which predict the efficacy and prognosis of SGC patients treated with pembrolizumab have not been identified We conducted a multi-institutional retrospective cohort study to evaluate the efficacy and safety of pembrolizumab monotherapy in patients with recurrent and/or metastatic SGC and to determine optimal cut-off values of the combined positive score (CPS) and tumor proportion score (TPS) as numerical expression levels of programmed death-ligand 1 (PD-L1) which predict the efficacy of pembrolizumab we investigated the association of patient characteristics and hematological markers with clinical outcomes Optimal cut-off values of CPS and TPS were 15 and 25% ORRs of CPS-high and TPS-high were 55.6 and 75.0% and significantly higher than those of CPS-low and TPS-low patients with a low platelet-lymphocyte ratio (PLR) had a significantly longer PFS No grade 4 or greater adverse events were observed This study demonstrated the efficacy and safety of pembrolizumab monotherapy and identified optimal cut-off values of CPS and TPS the majority of patients with R/M SGC are not candidates for these targeted therapies the impact of PD-L1 expression on the efficacy of pembrolizumab in SGC patients remains controversial This study aimed to clarify the efficacy and safety of pembrolizumab monotherapy in R/M SGC in a real-world setting and to measure CPS and tumor proportion score (TPS) as real values for PD-L1 expression to identify their optimal cut-off values for this treatment we investigated the association of patient background factors and hematological markers with clinical outcomes of pembrolizumab Twenty-seven patients with R/M SGC were treated with pembrolizumab monotherapy during the study period. No patients received pembrolizumab with chemotherapy concurrently. Patient characteristics are summarized in Table 1 and median follow-up period for all patients was 6.9 months (range The most common histopathological type was salivary duct carcinoma (SDC) (n = 15; 56%) Sixteen patients (59%) had received chemotherapy before pembrolizumab all of whom had disease progression within 6 months prior to pembrolizumab initiation Median follow-up period of patients with SDC was 6.9 months (range While 4 patients received pembrolizumab as the first-line treatment 11 of 15 (73%) patients with SDC had received chemotherapy and then had disease progression prior to pembrolizumab monotherapy Trastuzumab was administered to 6 patients with HER2-positive SGC 10 patients (37.0%) continued to receive pembrolizumab whereas 17 patients (63.0%) discontinued treatment due to progressive disease (PD) (n = 16; 94.1%) or immune-related adverse event (irAE) (n = 1; 5.9%) The median number of cycles of pembrolizumab administered was 4 (range received 1 or more sequential chemotherapy regimens after pembrolizumab treatment Kaplan–Meier survival curves of progression-free survival (a) and overall survival (b) of all patients Representative images of the tumor before and during pembrolizumab monotherapy in a patient with pancreatic metastasis of salivary duct carcinoma (a) Pre-treatment of the patient with pancreatic metastasis (b) Tumor shrinkage observed 63 days after the initiation of pembrolizumab monotherapy (c) Tumor disappeared 154 days after the initiation of pembrolizumab monotherapy All AEs reported are listed in Table 3 Nine patients (33.3%) experienced irAE during treatment One patient (3.7%) showed grade 3 irAE diabetes One patient discontinued treatment due to the irAE of grade 2 uveitis Kaplan–Meier survival curves of SGC patients according to CPS (a) and TPS (b) on PFS In 6 patients with AdCC, median CPS and TPS was low at 2.5 (range 0–5) and < 1% (range, 0–1%), respectively. SDC patients had significantly higher CPS than AdCC patients (Supplementary Figure S3) In one patient each with lymphoepithelial carcinoma and acinic cell carcinoma One patient with adenocarcinoma was negative for CPS and TPS In all 27 patients, median neutrophil–lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR) and lymphocyte-monocyte ratio (LMR) was 3.3 (range 1.1–12.7), 194.1 (range 68.3–1331.3) and 3.1 (range 1.1–12.9), respectively (Table 1) PLR and LMR against objective response was 3.0 We divided the patients accordingly into high and low groups to clarify the impact of NLR and 9 (33%) patients were categorized in the NLR- and 18 (67%) patients were categorized in the NLR- Regarding the modified Glasgow Prognostic Score (mGPS) 24 (89%) and three (11%) patients were assigned scores of 0 and 1 Regarding hematological biomarkers, patients with higher PLR had a significantly worse PFS than those with lower PLR among all patients [Table 4 we demonstrated the efficacy of immunotherapy with pembrolizumab in patients with R/M SGC Compared to patients with CPS-L and TPS-L SGC patients with CPS-H and TPS-H SGC had significantly higher ORRs CPS and TPS may be useful biomarkers for the selection of pembrolizumab for SGC suggesting promising efficacy in this cohort Results from ongoing trials of pembrolizumab plus docetaxel (NCT03360890) and pembrolizumab plus pemetrexed (NCT04895735) in R/M SGCs are awaited the ORR of pembrolizumab in patients with R/M SGC with low PLR was 38.9% which was higher than the 0% in those with high PLR Patients with low PLR also had a significantly longer PFS These results suggest that PLR might be a predictive marker of clinical outcome in patients with R/M SGC treated with pembrolizumab The frequency of irAE in patients with SGC in our cohort was consistent with previous reports and no unexpected toxicity was observed Almost all patients were able to continue treatment without discontinuation due to irAEs except for one patient with grade 2 uveitis this study demonstrated the efficacy and safety of pembrolizumab monotherapy in patients with R/M SGC We identified optimal cut-off values of CPS and TPS in predicting the clinical outcomes of this treatment where CPS-H and TPS-H SGC had significantly better ORR than CPS-L and TPS-L SGC We also showed that PLR was significantly associated with PFS in patients with SGC This study was conducted under a retrospective cohort design in 27 patients with R/M SGC treated with pembrolizumab from March 2016 to April 2021 at seven facilities in Japan following approval from the ethics committee of the participating institutions (Approval number of each institution: International University of Health and Welfare 70–00-0145; Tokyo Medical University Hospital 2021–0086; Yokohama City University Hospital This study was also performed in accordance with the Declaration of Helsinki informed consent was waived by the Institutional Review Boards of all participating institutions We provided information regarding the research plan via a web-based public release and patients and their families were provided an opportunity to opt-out of the study under the policy of the Japanese government The charts of all 27 patients who received pembrolizumab monotherapy were retrospectively reviewed and information on patient characteristics treatment modality and clinical outcomes was collected Twenty-five tumor samples were pathologically reviewed and evaluated by a pathologist with expertise in SGCs (T.N.) Carcinoma ex pleomorphic adenoma was classified according to individual carcinoma components We excluded patients for whom therapeutic effect could not be evaluated Pembrolizumab was administered at 200 mg/body every 3 weeks imaging examination by CT or MRI was performed every 6–9 weeks The efficacy of pembrolizumab was evaluated with regard to ORR CBR (defined as the percentage of patients who achieved CR DCR (defined as the percentage of patients who achieved CR Treatment efficacy was evaluated according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) PFS was defined as the time from the start of pembrolizumab to the diagnosis of PD OS was defined as the period from the day of initiation of pembrolizumab to the day of death regardless of cause or the day of last follow-up AEs were assessed according to CTCAE version 5.0 up to 30 days after the last administration of pembrolizumab Administration of pembrolizumab was terminated when PD was confirmed the patient experienced unacceptable adverse events additional treatment with other agents was permitted To clarify the impact of PD-L1 expression on survival and response each categorized into two groups by optimum cut-off value PD-L1 expression in samples was assessed using the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies USA) and characterized by CPS and TPS through central pathological review (H.H.) CPS and TPS were defined as the number of PD-L1-positive cells (tumor cells and macrophages) divided by the total number of tumor cells × 100 and as the number of PD-L1-positive tumor cells divided by the total number of tumor cells × 100 Optimum cut-off values of CPS and TPS were evaluated using area under the receiver operating characteristic curve (AUROC) against objective response we also examined associations between survival or response and age Eastern Cooperative Oncology Group (ECOG) performance status (PS) LMR and mGPS as examined in peripheral blood just before the start of pembrolizumab treatment These markers were categorized into two groups by optimum cut-off value evaluated using AUROC against objective response patients with both elevated C-reactive protein (CRP; > 1.0 mg/dL) and decreased albumin (Alb; < 3.5 g/dL) were assigned a score of 2; those with an elevated CRP (> 1.0 mg/dl) and non-decreased Alb (≥ 3.5 g/dl) were assigned a score of 1; and those with a non-elevated CRP (≤ 1.0 mg/dl) were assigned a score of 0 OS and PFS were estimated by the Kaplan–Meier product-limit method and tested by the log-rank test in both the SGC and SDC groups To evaluate the impact of clinical factors on PFS we estimated HRs and 95% CIs using univariate Cox proportional hazards models to evaluate the impact of these factors on objective response All statistical analyses were performed using STATA version 16 (Stata Corp. and p-values < 0.05 were considered statistically significant The datasets generated in the current study are available from the corresponding author on request Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries Management of salivary gland malignancy: ASCO guideline Salivary gland cancer: ESMO-European reference network on rare adult solid cancers (EURACAN) clinical practice guideline for diagnosis Phase II trial of trastuzumab and docetaxel in patients with human epidermal growth factor receptor 2-positive salivary duct carcinoma A prospective phase II study of combined androgen blockade in patients with androgen receptor-positive metastatic or locally advanced unresectable salivary gland carcinoma Le, X. et al. Larotrectinib treatment for patients with TRK fusion-positive salivary gland cancers. Oncol. https://doi.org/10.1093/oncolo/oyac080 (2022) Association of tumour mutational burden with outcomes in patients with advanced solid tumours treated with pembrolizumab: prospective biomarker analysis of the multicohort Evaluation of pembrolizumab monotherapy in patients with previously treated advanced salivary gland carcinoma in the phase 2 KEYNOTE-158 study A phase II trial of nivolumab for patients with platinum-refractory recurrent or metastatic salivary gland cancer retrospective study of the efficacy and safety of nivolumab for recurrent and metastatic salivary gland carcinoma Fayette, J., Even, C., Digue, L., Geoffrois, L. & Rolland, F. NISCAHN: A phase II trial of nivolumab in patients with salivary gland carcinoma (Unicancer ORL-08). BMJ Oncol. https://doi.org/10.1136/bmjonc-2023-000065 (2023) Pembrolizumab for the treatment of advanced salivary gland carcinoma: findings of the phase 1b KEYNOTE-028 study Vos, J. L. et al. Nivolumab plus ipilimumab in advanced salivary gland cancer: A phase 2 trial. Nat. Med. https://doi.org/10.1038/s41591-023-02518-x (2023) . NCCN Clinical Practice Guidelines in Oncology, Head and Neck Cancers Version 2.2024 https://www.nccn.org/professionals/physician_gls/pdf/head-and-neck.pdf Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): A randomised Pembrolizumab plus chemotherapy in advanced triple-negative breast cancer Pembrolizumab as first-line therapy for patients with PD-L1-positive advanced non-small cell lung cancer: A phase 1 trial The immune microenvironment and neoantigen landscape of aggressive salivary gland carcinomas differ by subtype Programmed death ligand-1 expression is associated with poor disease free survival in salivary gland carcinomas Prognostic value and clinicopathological roles of the tumor immune microenvironment in salivary duct carcinoma Prognostic value of nutritional and inflammatory markers in patients with hepatocellular carcinoma who receive immune checkpoint inhibitors Prognostic values of systemic inflammation and nutrition-based prognostic indices in oropharyngeal carcinoma Inflammation and nutrition-based biomarkers in the prognosis of oesophageal cancer: A systematic review and meta-analysis Prognostic role of platelet-to-lymphocyte ratio and lymphocyte-to-monocyte ratio in operated rectal cancer patients: Systematic review and meta-analysis Hematological predictive markers for recurrent or metastatic squamous cell carcinomas of the head and neck treated with nivolumab: A multicenter study of 88 patients Biomarkers of systemic inflammation predict survival with first-line immune checkpoint inhibitors in non-small-cell lung cancer Prognostic value of hematologic parameters in advanced non-small cell lung cancer patients receiving anti-PD-1 inhibitors Blood cell count biomarkers predicting efficacy of pembrolizumab as second-line therapy for advanced urothelial carcinoma Neutrophil-to-lymphocyte ratio to predict the efficacy of immune checkpoint inhibitor in upper gastrointestinal cancer Contribution of platelets to tumour metastasis Pembrolizumab with or without chemotherapy in recurrent or metastatic head and neck squamous cell carcinoma: Updated results of the phase III KEYNOTE-048 study Download references This work was supported by a JSPS Grant-in-Aid for Scientific Research (C) to Dr. Yuichiro Tada [Grant number 21K09616], Dr. Daisuke Kawakita [Grant number 20K10508], Dr. Toshitaka Nagao [Grant number 23K06432], Dr. Hideaki Hirai [Grant number 22K06969], Dr. Hideaki Takahashi [Grant number 19K09873], and Dr. Takashi Matsuki [Grant number 20K07597]. The authors thank Guy Harris of Dmed (https://dmed.co.jp) for the English language review of this paper Nagoya City University Graduate School of Medical Sciences Tokyo Medical University School of Medicine Department of Otolaryngology-Head and Neck Surgery Department of Otolaryngology Head and Neck Surgery Niigata University Graduate School of Medical and Dental Sciences Department of Head and Neck Oncology and Surgery International University of Health and Welfare Mita Hospital and Y.A.; analysis and interpretation of data: Y.T. and Y.T.; drafting of the manuscript: T.M. and H.T.; review and approval of manuscript: all authors Download citation DOI: https://doi.org/10.1038/s41598-024-70779-8