Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5+ basal epithelial cells, we identify a new marker, Tetraspanin8 (Tspan8). Fractionation based on Tspan8 and Lgr5 expression uncovered three distinct mammary stem cell (MaSC) subsets in the adult mammary gland. These exist in a largely quiescent state but differ in their reconstituting ability, spatial localization, and their molecular and epigenetic signatures. Interestingly, the deeply quiescent MaSC subset (Lgr5+Tspan8hi) resides within the proximal region throughout life, and has a transcriptome strikingly similar to that of claudin-low tumours. Lgr5+Tspan8hi cells appear to originate from the embryonic mammary primordia before switching to a quiescent state postnatally but can be activated by ovarian hormones. Our findings reveal an unexpected degree of complexity within the adult MaSC compartment and identify a dormant subset poised for activation in response to physiological stimuli.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Visvader, J. E. & Clevers, H. Tissue-specific designs of stem cell hierarchies. Nat. Cell Biol. 18, 349–355 (2016).
Stingl, J. et al. Purification and unique properties of mammary epithelial stem cells. Nature 439, 993–997 (2006).
Barker, N., Tan, S. & Clevers, H. Lgr proteins in epithelial stem cell biology. Development 140, 2484–2494 (2013).
Plaks, V. et al. Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis. Cell Rep. 3, 70–78 (2013).
Wang, D. et al. Identification of multipotent mammary stem cells by protein C receptor expression. Nature 517, 81–84 (2015).
de Visser, K. E. et al. Developmental stage-specific contribution of LGR5(+) cells to basal and luminal epithelial lineages in the postnatal mammary gland. J. Pathol. 228, 300–309 (2012).
Rios, A. C., Fu, N. Y., Lindeman, G. J. & Visvader, J. E. In situ identification of bipotent stem cells in the mammary gland. Nature 506, 322–327 (2014).
Van Keymeulen, A. et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature 479, 189–193 (2011).
Cicalese, A. et al. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell 138, 1083–1095 (2009).
Dos Santos, C. O. et al. Molecular hierarchy of mammary differentiation yields refined markers of mammary stem cells. Proc. Natl Acad. Sci. USA 110, 7123–7130 (2013).
Smith, G. H. Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands. Development 132, 681–687 (2005).
Boras-Granic, K., Dann, P. & Wysolmerski, J. J. Embryonic cells contribute directly to the quiescent stem cell population in the adult mouse mammary gland. Breast Cancer Res. 16, 487 (2014).
Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).
Zhang, L. et al. Establishing estrogen-responsive mouse mammary organoids from single Lgr5+ cells. Cell. Signal. 29, 41–51 (2016).
Greco, C. et al. E-cadherin/p120-catenin and tetraspanin Co-029 cooperate for cell motility control in human colon carcinoma. Cancer Res. 70, 7674–7683 (2010).
Hemler, M. E. Tetraspanin functions and associated microdomains. Nat. Rev. Mol. Cell Biol. 6, 801–811 (2005).
Zoller, M. Tetraspanins: push and pull in suppressing and promoting metastasis. Nat. Rev. Cancer 9, 40–55 (2009).
Giraddi, R. R. et al. Stem and progenitor cell division kinetics during postnatal mouse mammary gland development. Nat. Commun. 6, 8487 (2015).
Arai, F. & Suda, T. StemBook (Cambridge, 2008).
Cheung, T. H. & Rando, T. A. Molecular regulation of stem cell quiescence. Nat. Rev. Mol. Cell Biol. 14, 329–340 (2013).
Zeng, Y. A. & Nusse, R. Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture. Cell Stem Cell 6, 568–577 (2010).
Fukada, S. et al. Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25, 2448–2459 (2007).
Liu, L. et al. Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging. Cell Rep. 4, 189–204 (2013).
Chambers, S. M. et al. Hematopoietic fingerprints: an expression database of stem cells and their progeny. Cell Stem Cell 1, 578–591 (2007).
Lien, W. H. et al. Genome-wide maps of histone modifications unwind in vivo chromatin states of the hair follicle lineage. Cell Stem Cell 9, 219–232 (2011).
Genander, M. et al. BMP signaling and its pSMAD1/5 target genes differentially regulate hair follicle stem cell lineages. Cell Stem Cell 15, 619–633 (2014).
He, X. C. et al. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-β-catenin signaling. Nat. Genet. 36, 1117–1121 (2004).
Prat, A. et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res. 12, R68 (2010).
Lim, E. et al. Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat. Med. 15, 907–913 (2009).
Pal, B. et al. Global changes in the mammary epigenome are induced by hormonal cues and coordinated by Ezh2. Cell Rep. 3, 411–426 (2013).
Wu, D. et al. ROAST: rotation gene set tests for complex microarray experiments. Bioinformatics 26, 2176–2182 (2010).
Westphalen, C. B. et al. Dclk1 defines quiescent pancreatic progenitors that promote injury-induced regeneration and tumorigenesis. Cell Stem Cell 18, 441–455 (2016).
Makarem, M. et al. Developmental changes in the in vitro activated regenerative activity of primitive mammary epithelial cells. PLoS Biol. 11, e1001630 (2013).
Spike, B. T. et al. A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer. Cell Stem Cell 10, 183–197 (2012).
Asselin-Labat, M. L. et al. Control of mammary stem cell function by steroid hormone signalling. Nature 465, 798–802 (2010).
Orford, K. W. & Scadden, D. T. Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation. Nat. Rev. Genet. 9, 115–128 (2008).
Wilson, A. et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 135, 1118–1129 (2008).
Rodgers, J. T. et al. mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature 510, 393–396 (2014).
Barker, N. et al. Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010).
Jaks, V. et al. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat. Genet. 40, 1291–1299 (2008).
Buczacki, S. J. et al. Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature 495, 65–69 (2013).
Fuentealba, L. C. et al. Embryonic origin of postnatal neural stem cells. Cell 161, 1644–1655 (2015).
Furutachi, S. et al. Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells. Nat. Neurosci. 18, 657–665 (2015).
Bowie, M. B. et al. Identification of a new intrinsically timed developmental checkpoint that reprograms key hematopoietic stem cell properties. Proc. Natl Acad. Sci. USA 104, 5878–5882 (2007).
Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84–88 (2006).
R Development Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2012); http://www.R-project.org
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
Shi, W., Oshlack, A. & Smyth, G. K. Optimizing the noise versus bias trade-off for Illumina whole genome expression BeadChips. Nucleic Acids Res. 38, e204 (2010).
Barbosa-Morais, N. L. et al. A re-annotation pipeline for Illumina BeadArrays: improving the interpretation of gene expression data. Nucleic Acids Res. 38, e17 (2010).
Smyth, G. K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. https://doi.org/10.2202/1544-6115.1027 (2004).
Ritchie, M. E. et al. Empirical array quality weights in the analysis of microarray data. BMC Bioinf. 7, 261 (2006).
Smyth, G. K., Michaud, J. & Scott, H. S. Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 21, 2067–2075 (2005).
Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Robinson, M. D. & Oshlack, A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 11, R25 (2010).
Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014).
Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 11, R14 (2010).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Yu, G., Wang, L. G. & He, Q. Y. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 31, 2382–2383 (2015).
We are grateful to the Animal, FACS, Monoclonal Antibody (Bundoora), Imaging and Histology facilities at WEHI and to M. Milevskiy for discussions. This work was supported by the Australian National Health and Medical Research Council (NHMRC) grants no. 1016701, no. 1024852, no. 1054618, no. 1059622, no. 1085191, no. 1086727, no. 1100807; NHMRC IRIISS; the Victorian State Government through Victorian Cancer Agency (VCA) funding and Operational Infrastructure Support; and the Australian Cancer Research Foundation. N.Y.F. and A.C.R. were supported by a National Breast Cancer Foundation (NBCF)/Cure Cancer Australia Fellowship; B.P. by a NHMRC Fellowship no. 1016571 and a VCA Fellowship; G.K.S., G.J.L., M.E.R. and J.E.V. by NHMRC Fellowships no. 1058892, no. 1078730, no. 1104924, no. 1102742.
The authors declare no competing financial interests.
Integrated supplementary information
(a) Whole-mount 3D confocal image and optical section of a ductal portion located in the distal part of the mammary gland from an adult (9 week-old) Lgr5-GFP-IRES-creERT2 female (representative of 4 mice, 3 independent experiments) immunostained for K5 (blue). Lgr5+ cells only appear in the basal population of the adult mammary glands. Scale bars, 50 μm (whole-mount); 10 μm (optical sections). (b) Heat map showing all the DE genes in any pairwise comparison amongst the luminal, Lgr5-GFP+ (Lgr5+) and Lgr5-GFP− (Lgr5−) basal populations. Expression values are on a log2 scale and are mean-corrected for each gene. Three biological replicates were sorted from 9 week-old Lgr5-GFP-IRES-CreERT2 females and their transcriptomes analysed by microarray. The cutoff for DE was FDR < 0.05 and absolute fold-change ≥2 (70 mice were analysed in a total of n = 3 independent experiments). (c) Heat map for the top 100 upregulated genes for Lgr5+ versus Lgr5− basal cells in (a). (d) GO enrichment analysis of DE genes between Lgr5+ versus Lgr5− basal cells in (b).
Supplementary Figure 2 FACS analysis of Tspan8-expressing cells in the adult mammary gland and representative outgrowth generated by different basal subpopulations.
(a) Representative FACS plots showing Tspan8-expressing cells in the mammary glands of 9 week-old C57BL/6 females (15 mice, 3 independent experiments). (b) Representative FACS plots showing Tspan8-expressing cells in the mammary glands of 9 week-old FVB/N females (10 mice, 3 independent experiments). (c) Representative FACS plots showing overlap of Elf-GFP+ and Tspan8+ cells in the luminal population from the mammary glands of 9 week-old Elf5-rtTA-GFP females (2 mice, 2 independent experiments). (d) Transplantation of the different subpopulations (50 cells) into the cleared fat pads of 3-week old recipient female mice. Glands were collected 10 weeks post-transplantation. Representative whole-mount images are shown (6 mice per population). Scale bar, 2 mm. (e) Transplantation of the different subpopulations (400 cells) into the cleared fat pads of recipient females. Females were mated 9–10 weeks after transplantation and mammary glands were collected at 18.5 days of pregnancy. Representative whole-mounts of outgrowths are shown (6 mice per population). Scale bar, 2 mm. (f) Representative whole-mount images of secondary outgrowths. Basal cells were sorted from primary outgrowths generated from the indicated subsets defined by Lgr5 and Tspan8 expression, and transplanted into the cleared fat pads of secondary recipient females. The secondary outgrowths were collected 10 weeks post-transplantation. Scale bar, 2 mm (5–6 mice for each population per experiment, 3 independent experiments). (g) Representative FACS plots showing the DNA and RNA content of the luminal compartment isolated on the basis of Tspan8 expression (all Lin−CD29loCD24+). Sorted cells from the mammary glands of 9 week-old Lgr5-GFP-IRES-creERT2 females were fixed and stained with 7-AAD and pyroninY (pY). G0 is defined as G1 cells with low RNA content (30 mice, 3 independent experiments).
Supplementary Figure 3 Gene expression signatures of quiescent mammary stem cells and comparison with the major subtypes of human breast cancer.
(a) Relative expression (log2 cpm values) of known basal markers across the four different myoepithelial/basal subpopulations as determined by RNA-seq analysis. (b,c) Heat maps showing expression of the top 100 DE genes on comparison of Lgr5+Tspan8hi cells versus the average of the other three subsets, either upregulated (b) or downregulated (c). Two biological replicates were sorted by flow cytometry from the mammary glands of 9 week-old Lgr5-GFP-IRES-creERT2 females and their transcriptomes were determined by RNA-seq (90 mice were analysed in a total of n = 2 independent experiments). (d) GO enrichment analysis of DE genes for Lgr5+Tspan8hi versus Lgr5+Tspan8− cells (90 mice were analysed in a total of n = 2 independent experiments). (e) Box plots of quiescent MaSC signature scores by tumour subtype. The signature scores for the Lgr5+Tspan8hi sub-population compared to any other population are strongly correlated with the claudin-low subtype of breast cancer. Boxes show 25%, 50% and 75% percentiles. Whiskers extend to minimum and maximum values. Values more than 1.5 interquartile ranges from the box are shown as individual points. Two RNA samples per mammary subpopulation were used to interrogate the breast cancer database: Claudin-low: n = 19; LumA: n = 70; LumB: n = 37; HER2: n = 22; Basal: n = 32 tumours. (f) Barcode plot depicting the strongly associated gene expression signatures of quiescent MaSCs and claudin-low tumours compared to the basal-like subtype. Genes are ordered from right to left as most upregulated to most downregulated in claudin-low cancer. The red lines designate upregulated genes in quiescent MaSCs (Lgr5+Tspan8hi versus All Others), whereas blue lines designate downregulated genes. The cutoff for DE is FDR < 0.05 and absolute fold-change ≥2 (90 mice were analysed in a total of n = 2 independent experiments). P values measuring the overall correlation were derived from the ‘roast’ function of the limma software package.
(a) Representative FACS plots showing Tspan8+ luminal cells in the proximal and distal areas of mammary glands isolated from Lgr5-GFP-IRES-creERT2 females at 5 weeks, 9 weeks or 6 months of age (15 mice were analysed for each age in a total of 3 independent experiments). (b) Representative FACS plots showing Tspan8hi basal cells in the proximal and distal areas of mammary glands from C57BL/6 females after completion of three pregnancy cycles (3 mice, 3 independent experiments). (c) Representative FACS plots showing the basal subpopulations defined by expression of Lgr5 and Tspan8 in the nipple, middle and distal areas of mammary glands from the Lgr5-GFP-IRES-creERT2 females at the age of 9 months (3 mice, 3 independent experiments).
(a) Whole-mount 3D confocal image of an entire mammary rudiment from a Lgr5-GFP-IRES-creERT2 embryo at E18.5 immunostained for GFP (green), Keratin 5 (red) and K8/K18 (blue) (representative of 3 embryos). (b) Colocalisation channel (white) built in the Imaris software showing the colocalisation pattern for K5 (basal) and K8/K18 (luminal). Note that colocalisation mainly occurs in the growing ducts. (c) Enlargement from (a) showing the trunk portion of the embryonic mammary gland, where the epithelial layers are more defined and there are rare double-positive cells (for K5 and K8/K18). (d,e) Enlargement from (a) showing developing branches. (e) shows the colocalisation channel (white). Most cells are double-positive for K5 and K8/K18 (3 embryos, 3 independent experiments). Scale bars, 100 μm (whole-mounts); 20 μm (optical sections). (f) Bar chart showing the percentage of Lgr5-GFP+ K5/K8+, K5+ and K8+ cells in the growing ducts (buds) and the trunk (n = 3 embryos). K8 in a–f designates the combined K8/K18 Troma antibody (n = 3 embryos, 3 independent experiments). Error bars represent mean ± s.e.m.; *P < 0.05; ****P < 0.0001, Student’s t-test. (g) Representative image of a mammary rudiment from a Lgr5-GFP-IRES-creERT2 female embryo at E18.5 (representative of 40 embryos). Scale bars, 500 μm. (h) Representative FACS plot showing Lgr5 and Tspan8 expression in fetal mammary glands and skin. Embryos were harvested at E18.5 from pregnant Lgr5-GFP-IRES-creERT2 females. Mammary glands from female embryos were dissected under a fluorescence microscope and GFP+ mammary rudiments from 8–10 embryos were pooled for the preparation of single cell suspensions for FACS analysis (8–10 female embryos for each experiment, 2 independent experiments). (i) Comparison of the expression levels of Lgr5-GFP in the fetal and adult MaSC-enriched populations (3 mice or 16 female embryos, 3 independent experiments).
Supplementary Figure 6 Contribution of fetal Lgr5+ cells and embryonic origin of Lgr5+ Tspan8hi cells in the adult mammary gland
(a) Representative confocal images of an E18.5 female mammary ductal tree stained with Tspan8 (red), p63 (green) and DAPI (blue) (3 embryos, 3 independent experiments). (b) Embryonic Lgr5-expressing cells contribute to the luminal and basal lineages in the adult mammary gland. b1, Whole-mount image of an entire mammary gland from Lgr5-GFP-IRES-creERT2/R26R-tdTomato mice 11 weeks after tamoxifen injection at 17.5 days of pregnancy (representative of 5 mice, 3 independent experiments). Scale bars: 1 cm. a2, Whole-mount 3D image of a ductal portion labelled for E-cadherin (blue). Inset, optical section from the enlargement showing Tomato+ luminal and myoepithelial cells labelled in the duct. Scale bars, 100 μm (whole-mount); 50 μm (optical section). (c) Representative FACS plots demonstrating the mammary repopulating activity of embryonic Lgr5-expressing cells. Lgr5-GFP-IRES-creERT2/R26R-tdTomato mice were analysed at 11 weeks after tamoxifen injection at 17.5 days of pregnancy (4 mice, 4 independent experiments). (d) Whole-mount 3D confocal image of the proximal portion of a ductal tree from a Lgr5-GFP-IRES-creERT2 mouse. EdU was IP injected twice per day from E14.5 to E18.5 and then chased for 11 weeks. The whole-mount was labelled for GFP (green), EdU (red) and E-cadherin (blue). I, Optical section from the whole-mount image showing a ductal portion emanating from the nipple area, with no EdU retention. II, Optical section from the whole-mount image showing a ductal portion in the nipple area displaying EdU retention (representative of 2 mice, 2 independent experiments). Scale bars, 300 μm (whole-mount); 50 μm (optical sections).
(a–b) Whole-mount 3D confocal images and optical sections of the proximal (a) and distal (b) parts of a mammary gland from a Lgr5-GFP-IRES-creERT2/R26R-tdTomato mouse two weeks after tamoxifen injection at 9 weeks and immunolabelled for E-cadherin (blue). Mice were treated with vehicle. (I, II) Enlarged areas showing sparsely distributed dtTomato+ myoepithelial and luminal cells in vehicle-treated mice (a). Representative images are shown (4 mice, 2 independent experiments). (c–f) Whole-mount 3D confocal images and optical sections of the proximal (c,d) and distal (e,f) parts of a mammary gland from a Lgr5-GFP-IRES-creERT2/R26R-tdTomato mouse two weeks after tamoxifen injection at 9 weeks, during adulthood and immunolabelled for E-cadherin (blue). Mice were treated with the synthetic progestin medroxyprogesterone acetate (MPA) plus estrogen (E + MPA). (d) Enlargement from (c) depicting a branching bud with luminal and basal Tomato+ cells derived from Lgr5+ cells in mice treated with E + MPA. Right hand panel, optical section of the alveolar bud. (f) Enlargement from (e) showing a ductal branch in the distal area comprising only Tomato+ basal cells. Representative of 4 mice, 2 independent experiments. Scale bars: 300 μm (whole-mounts), 20 μm (optical sections). (g) FACS plots showing EdU+ cells in the luminal and basal populations isolated from Lgr5-GFP-IRES-creERT2 mice treated with vehicle or E + MPA. Representative plots are shown (10 mice, 2 independent experiments).
(a–d) Whole-mount 3D confocal images and optical sections from enlargements of the proximal (a,b) and distal (c,d) parts of a mammary gland from a Lgr5-GFP-IRES-creERT2/R26R-tdTomato mouse two weeks during involution after tamoxifen injection at 9 weeks of age. Glands were immunolabelled with E-cadherin (blue). Expansion can be visualised in both regions of the gland. Scale bars: 200 μm (whole-mounts), 20 μm (optical sections). Representative images are shown (3 mice, 3 independent experiments).
Supplementary Information (PDF 6961 kb)
This movie shows Lgr5+ (GFP, green) and K5+ (blue) cells. Representative video of 3 embryos, 3 independent experiments. (AVI 22722 kb)
About this article
Cite this article
Fu, N., Rios, A., Pal, B. et al. Identification of quiescent and spatially restricted mammary stem cells that are hormone responsive. Nat Cell Biol 19, 164–176 (2017). https://doi.org/10.1038/ncb3471
Physiological Reviews (2020)
Characterization of radioresistant epithelial stem cell heterogeneity in the damaged mouse intestine
Scientific Reports (2020)
LGR5 in breast cancer and ductal carcinoma in situ: a diagnostic and prognostic biomarker and a therapeutic target
BMC Cancer (2020)
Mammary development in the embryo and adult: new insights into the journey of morphogenesis and commitment