SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24+ CD49fhi Mammary Stem Cell-Enriched Compartment

doi:10.1016/j.stemcr.2016.12.022

. 2017 Feb 14;8(2):417-431.

doi: 10.1016/j.stemcr.2016.12.022. Epub 2017 Jan 26.

SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24⁺ CD49f^hi Mammary Stem Cell-Enriched Compartment

Affiliations

¹ Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia.
² Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia.
³ Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences/NIH, Research Triangle Park, NC 27709, USA.
⁴ MRC Centre for Inflammation Research, University of Edinburgh, Queen's Medical Research Institute, Edinburgh EH16 4TJ, UK.
⁵ The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC 3010, Australia.
⁶ Hudson Institute of Medical Research, Clayton, VIC 3168, Australia.
⁷ Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia.
⁸ Institute of Cancer Sciences, College of MVLS, University of Glasgow, Glasgow G12 8QQ, UK.
⁹ School of Veterinary Medicine, College of MVLS, University of Glasgow, Glasgow G61 1QH, UK.
¹⁰ The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia; La Trobe University, Bundoora, VIC 3083, Australia.
¹¹ Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia.
¹² Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia; The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address: kara.britt@petermac.org.

PMID: 28132885
PMCID: PMC5312257
DOI: 10.1016/j.stemcr.2016.12.022

SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24⁺ CD49f^hi Mammary Stem Cell-Enriched Compartment

Genevieve V Dall et al. Stem Cell Reports. 2017.

. 2017 Feb 14;8(2):417-431.

doi: 10.1016/j.stemcr.2016.12.022. Epub 2017 Jan 26.

Authors

Affiliations

¹ Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia.
² Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia.
³ Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences/NIH, Research Triangle Park, NC 27709, USA.
⁴ MRC Centre for Inflammation Research, University of Edinburgh, Queen's Medical Research Institute, Edinburgh EH16 4TJ, UK.
⁵ The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC 3010, Australia.
⁶ Hudson Institute of Medical Research, Clayton, VIC 3168, Australia.
⁷ Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia.
⁸ Institute of Cancer Sciences, College of MVLS, University of Glasgow, Glasgow G12 8QQ, UK.
⁹ School of Veterinary Medicine, College of MVLS, University of Glasgow, Glasgow G61 1QH, UK.
¹⁰ The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia; La Trobe University, Bundoora, VIC 3083, Australia.
¹¹ Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia.
¹² Cancer Genetics Laboratory, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC 3800, Australia; The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address: kara.britt@petermac.org.

PMID: 28132885
PMCID: PMC5312257
DOI: 10.1016/j.stemcr.2016.12.022

Abstract

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα⁺ luminal cells and not the mammary stem cells (MaSCs) that are ERα^neg. Since ERα⁺ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα⁺ subset of EpCAM⁺/CD24⁺/CD49f^hi MaSCs. We show that the MaSC population has a distinct SCA-1⁺ population that is abundant in pre-pubertal mammary glands. The SCA-1⁺ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1^neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1⁺ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα⁺, estrogen-sensitive subpopulation within the CD24⁺/CD49f^hi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.

Keywords: Sca-1; estrogen; mammary stem cells.

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Figures

**Figure 1**
The CD24⁺ CD49f^hi Population Contains Both SCA-1⁺ and SCA-1^neg Cells with the Proportions Changing with the Age of the Mice (A and B) FACS profiles from 3-week-old (A) and 6-week-old (B) mammary glands showing the CD24⁺ CD49f^hi population that contains a distinct subpopulation of SCA-1⁺ and negative cells. (C–E) Changes in the percentage of CD24⁺ CD49f^hi (C) cells present within the lineage negative population with age. Changes in the CD24⁺ CD49f^hi SCA-1⁺ (D) and CD24⁺ CD49f^hi SCA-1^neg (E) cells with age. Graphs show means ± SEM. Statistical analysis completed by nonparametric one-way ANOVA with the Dunn multiple comparisons post hoc test in GraphPad Prism. n = 4–11 pools of 20–30 animals. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

**Figure 2**
The SCA-1⁺ Subset Expresses MaSC and Luminal Markers TaqMan qPCR analysis of MaSC and luminal markers in CD24⁺ CD49f^hi SCA-1^neg (pink bars), CD24⁺ CD49f^hi SCA-1⁺ (green bars), CD24+ CD49f^lo SCA-1^neg (dark gray bars), and CD24⁺ CD49f^lo SCA-1⁺ (light gray bars). Mean fold change ± SEM is shown relative to 6 week CD24⁺ CD49f^hi SCA-1^neg sample normalized to 1. (A) Reported MaSC markers *Lgr5*, *α-SMA*, *Delta 1*, *Id4*, *Fzd7*, *p63*, and *Aldh1a1*. (B) Mixed lineage and luminal markers *Jag1*, *Mef2C*, *Hes1*, *Hey1*, *Hey2*, *Notch1*, and *Keratin 18* as well as *Cyclin D1* and *Cylcin D2*. (C) Luminal progenitor markers *Elf5* and *C-kit*. For all graphs n = 3 pools of 20–30 animals. ^∗p < 0.05, ^∗∗p < 0.01.

**Figure 3**
SCA-1 Does Not Enrich for In Vivo MaSC Activity (A) Limiting dilution mammary fat pad transplant data from CD24⁺ CD49f^hi SCA-1⁺ (SCA-1⁺) and CD24⁺ CD49f^hi SCA-1^neg (SCA-1⁻) cells. The fractions denote the number of positive outgrowths as a percentage of the total number of transplants. The percentage of positive outgrowths is given in brackets. Circles show the filled proportion of the fat pad. Stem cell enrichment was determined using the ELDA program. (B–I) Mammary outgrowths were assessed using carmine-stained wholemount analysis (B and F), H&E (C and G), ERα (D and H), and CK14/CK18 immunostaining (E and I) to confirm the architecture and presence of cell lineages. Blue, DAPI; green, CK14; red, CK18. Arrowheads in (D) and (H) show ERα-positive cells; (B–E) show results from an SCA-1⁺ outgrowth and (F–I) from an SCA-1^neg outgrowth.

**Figure 4**
The CD24⁺ CD49f^hi SCA-1⁺ Subset Is Highly Proliferative Compared with the SCA-1^neg Subpopulation Hoechst 33342 and Ki67 flow analysis of various MaSC populations at different ages. (A–E) Representative analysis of total epithelial cells at 3 weeks of age (A). Representative analysis of CD24⁺ CD49f^hi SCA-1^neg population (B) and of the CD24⁺ CD49f^hi SCA-1⁺ population (C) at 6 weeks of age. Graphical representation of cell-cycle proportions of SCA-1^neg and SCA-1⁺ CD49f^hi cells at 3 weeks of age (D) and 6 weeks of age (E). The SCA-1 and Ki67 gates were determined using fluorescence minus one staining controls (Figure S5).

**Figure 5**
CD24⁺ CD49f^hi SCA-1⁺ Are Enriched in Terminal End Buds (A–D) Schematic of pubertal mammary glands with TEBs and a representative image of a TEB captured on a dissection microscope (A). Representative FACS profiles of lineage depleted epithelial populations in TEB samples (B), TEB-depleted DUCTs samples (C), and TEB- and DUCT-depleted samples (D). (E–G) Representative flow cytometry profiles of SCA-1 populations within the CD24⁺CD49f^hi populations of TEBs (E), TEB-depleted DUCTs (F), and TEB- and DUCT-depleted samples (G).

**Figure 6**
The SCA-1⁺ Subset Is ERα Positive (A) ERα mRNA levels in mammary epithelial cell subpopulations at 3 and 6 weeks of age. ^∗p < 0.05, ^∗∗p < 0.01. (B–E) ERα protein levels in FACS-sorted populations from 4-week-old mice. ERα protein (red), DAPI nuclear stain (blue). (B) CD24⁺ CD49f^hi SCA-1⁺, (C) CD24⁺ CD49f^hi SCA-1^neg, (D) CD24⁺ CD49f^lo SCA-1⁺, (E) CD24⁺ CD49f^lo SCA-1^neg. (F) Mean fold change ± SEM of mRNA levels of PR, *Rank*, *Rankl*, and *Egfr* relative to 6 week CD24⁺ CD49f^hi SCA-1^neg sample normalized to 1, n = 3 pools of 20–30 animals. CD24⁺ CD49f^hi SCA-1^neg (pink bars), CD24+ CD49f^hi SCA-1⁺ (green bars), CD24+ CD49f^lo SCA-1⁻ (dark gray bars), and CD24+ CD49f^lo SCA-1⁺ (light gray bars). (G) Data for all high-affinity EREs in the mouse genome (Bourdeau et al., 2004) was mined. Stem cell markers containing EREs are shown. The ERE needed to be only 2 base deviations from consensus as shown and within −10 to +5 kb of the transcriptional start site.

**Figure 7**
SCA-1⁺ Cells Are Estrogen Responsive (A–H) The percentage of SCA-1⁺ and SCA-1^neg cells within the CD24+ CD49f^hi MaSC subset is shown in contour FACS plots from wild-type C57BL/6 (A–C), Ex3αERKO (D–F), AF2ERKI (G), and ArKO (H) mammary glands. (I) The mean percentage of SCA-1⁺ CD24⁺ CD49f^hi cells in each mouse strain at 6 weeks of age ± SEM, n = 3–5. (J) Mammary gland wholemounts from wild-type, ArKO, and ArKO+ estradiol (E2) treatment. Boxed area highlights ductal growth in the ArKO mice. (K) The mean percentage of SCA-1⁺ CD24⁺ CD49f^hi cells within ArKO mice treated with either placebo or estradiol (ArKO + E2) ± SEM, n = 3–7. ^∗p < 0.05, ^∗∗p < 0.01.

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References

1. Alvi A.J., Clayton H., Joshi C., Enver T., Ashworth A., Vivanco M.M., Dale T.C., Smalley M.J. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res. 2003;5:R1–R8. - PMC - PubMed
1. Arao Y., Hamilton K.J., Ray M.K., Scott G., Mishina Y., Korach K.S. Estrogen receptor alpha AF-2 mutation results in antagonist reversal and reveals tissue selective function of estrogen receptor modulators. Proc. Natl. Acad. Sci. USA. 2011;108:14986–14991. - PMC - PubMed
1. Asselin-Labat M.-L., Shackleton M., Stingl J., Vaillant F., Forrest N.C., Eaves C.J., Visvader J.E., Lindeman G.J. Steroid hormone receptor status of mouse mammary stem cells. J. Natl. Cancer Inst. 2006;98:1011–1014. - PubMed
1. Asselin-Labat M.L., Vaillant F., Sheridan J.M., Pal B., Wu D., Simpson E.R., Yasuda H., Smyth G.K., Martin T.J., Lindeman G.J., Visvader J.E. Control of mammary stem cell function by steroid hormone signalling. Nature. 2010;465:798–802. - PubMed
1. Bai L., Rohrschneider L.R. s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue. Genes Dev. 2010;24:1882–1892. - PMC - PubMed

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[1] Alvi A.J., Clayton H., Joshi C., Enver T., Ashworth A., Vivanco M.M., Dale T.C., Smalley M.J. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res. 2003;5:R1–R8. - PMC - PubMed

[2] Alvi A.J., Clayton H., Joshi C., Enver T., Ashworth A., Vivanco M.M., Dale T.C., Smalley M.J. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res. 2003;5:R1–R8. - PMC - PubMed

[3] Arao Y., Hamilton K.J., Ray M.K., Scott G., Mishina Y., Korach K.S. Estrogen receptor alpha AF-2 mutation results in antagonist reversal and reveals tissue selective function of estrogen receptor modulators. Proc. Natl. Acad. Sci. USA. 2011;108:14986–14991. - PMC - PubMed

[4] Arao Y., Hamilton K.J., Ray M.K., Scott G., Mishina Y., Korach K.S. Estrogen receptor alpha AF-2 mutation results in antagonist reversal and reveals tissue selective function of estrogen receptor modulators. Proc. Natl. Acad. Sci. USA. 2011;108:14986–14991. - PMC - PubMed

[5] Asselin-Labat M.-L., Shackleton M., Stingl J., Vaillant F., Forrest N.C., Eaves C.J., Visvader J.E., Lindeman G.J. Steroid hormone receptor status of mouse mammary stem cells. J. Natl. Cancer Inst. 2006;98:1011–1014. - PubMed

[6] Asselin-Labat M.-L., Shackleton M., Stingl J., Vaillant F., Forrest N.C., Eaves C.J., Visvader J.E., Lindeman G.J. Steroid hormone receptor status of mouse mammary stem cells. J. Natl. Cancer Inst. 2006;98:1011–1014. - PubMed

[7] Asselin-Labat M.L., Vaillant F., Sheridan J.M., Pal B., Wu D., Simpson E.R., Yasuda H., Smyth G.K., Martin T.J., Lindeman G.J., Visvader J.E. Control of mammary stem cell function by steroid hormone signalling. Nature. 2010;465:798–802. - PubMed

[8] Asselin-Labat M.L., Vaillant F., Sheridan J.M., Pal B., Wu D., Simpson E.R., Yasuda H., Smyth G.K., Martin T.J., Lindeman G.J., Visvader J.E. Control of mammary stem cell function by steroid hormone signalling. Nature. 2010;465:798–802. - PubMed

[9] Bai L., Rohrschneider L.R. s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue. Genes Dev. 2010;24:1882–1892. - PMC - PubMed

[10] Bai L., Rohrschneider L.R. s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue. Genes Dev. 2010;24:1882–1892. - PMC - PubMed

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SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24⁺ CD49f^hi Mammary Stem Cell-Enriched Compartment

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SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24⁺ CD49f^hi Mammary Stem Cell-Enriched Compartment

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