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Molecular hierarchy of mammary differentiation yields refined markers of mammary stem cells.

ABSTRACT: The partial purification of mouse mammary gland stem cells (MaSCs) using combinatorial cell surface markers (Lin(-)CD24(+)CD29(h)CD49f(h)) has improved our understanding of their role in normal development and breast tumorigenesis. Despite the significant improvement in MaSC enrichment, there is presently no methodology that adequately isolates pure MaSCs. Seeking new markers of MaSCs, we characterized the stem-like properties and expression signature of label-retaining cells from the mammary gland of mice expressing a controllable H2b-GFP transgene. In this system, the transgene expression can be repressed in a doxycycline-dependent fashion, allowing isolation of slowly dividing cells with retained nuclear GFP signal. Here, we show that H2b-GFP(h) cells reside within the predicted MaSC compartment and display greater mammary reconstitution unit frequency compared with H2b-GFP(neg) MaSCs. According to their transcriptome profile, H2b-GFP(h) MaSCs are enriched for pathways thought to play important roles in adult stem cells. We found Cd1d, a glycoprotein expressed on the surface of antigen-presenting cells, to be highly expressed by H2b-GFP(h) MaSCs, and isolation of Cd1d(+) MaSCs further improved the mammary reconstitution unit enrichment frequency to nearly a single-cell level. Additionally, we functionally characterized a set of MaSC-enriched genes, discovering factors controlling MaSC survival. Collectively, our data provide tools for isolating a more precisely defined population of MaSCs and point to potentially critical factors for MaSC maintenance.

SUBMITTER: dos Santos CO

PROVIDER: S-EPMC3645536 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Molecular hierarchy of mammary differentiation yields refined markers of mammary stem cells.

dos Santos Camila O CO Rebbeck Clare C Rozhkova Elena E Valentine Amy A Samuels Abigail A Kadiri Lolahon R LR Osten Pavel P Harris Elena Y EY Uren Philip J PJ Smith Andrew D AD Hannon Gregory J GJ

Proceedings of the National Academy of Sciences of the United States of America 20130411 18

The partial purification of mouse mammary gland stem cells (MaSCs) using combinatorial cell surface markers (Lin(-)CD24(+)CD29(h)CD49f(h)) has improved our understanding of their role in normal development and breast tumorigenesis. Despite the significant improvement in MaSC enrichment, there is presently no methodology that adequately isolates pure MaSCs. Seeking new markers of MaSCs, we characterized the stem-like properties and expression signature of label-retaining cells from the mammary gl ...[more]

PMID: 23580620

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Project description:Triple-negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis.Mammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low-dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power.A set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. A total of 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis.Our findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC.

Project description:BackgroundMammary physiology is distinguished in containing adult stem/progenitor cells that are actively amending the breast tissue throughout the reproductive lifespan of women. Despite their importance in both mammary gland development, physiological maintenance, and reproduction, the exact role of mammary stem/progenitor cells in mammary tumorigenesis has not been fully elucidated in humans or animal models. The implications of modulating adult stem/progenitor cells in women could lead to a better understanding of not only their function, but also toward possible breast cancer prevention led us to evaluate the efficacy of rapamycin in reducing mammary stem/progenitor cell activity and malignant progression markers.MethodsWe analyzed a large number of human breast tissues for their basal and luminal cell composition with flow cytometry and their stem and progenitor cell function with sphere formation assay with respect to age and menopausal status in connection with a clinical study (NCT02642094) involving a low-dose (2 mg/day) and short-term (5-7 days) treatment of the mTOR inhibitor sirolimus. The expression of biomarkers in biopsies and surgical breast samples were measured with quantitative analysis of immunohistochemistry.ResultsSirolimus treatment significantly abrogated mammary stem cell activity, particularly in postmenopausal patients. It did not affect the frequency of luminal progenitors but decreased their self-renewal capacity. While sirolimus had no effect on basal cell population, it decreased luminal cell population, particularly in postmenopausal patients. It also significantly diminished prognostic biomarkers associated with breast cancer progression from ductal carcinoma in situ to invasive breast cancer including p16INK4A, COX-2, and Ki67, as well as markers of the senescence-associated secretary phenotype, thereby possibly functioning in preventing early breast cancer progression.ConclusionOverall, these findings indicate a link from mTOR signaling to mammary stem and progenitor cell activity and cancer progression. Trial registration This study involves a clinical trial registered under the ClinicalTrials.gov identifier NCT02642094 registered December 30, 2015.

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Molecular hierarchy of mammary differentiation yields refined markers of mammary stem cells.

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Molecular hierarchy of mammary differentiation yields refined markers of mammary stem cells.

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