Investigation of human mammary stem and progenitor subpopulations from BRCA1 mutation carriers and noncarriers

Abstract

504 Background: We have previously isolated discrete populations of mouse mammary epithelial cells (MECs) and defined a population that expresses ‘basal’ markers and is highly enriched for mammary stem cells. This subset was ‘triple negative’ for ER, PR and ErbB2 expression and is reminiscent of the basal subtype of breast cancer, suggesting that the mammary stem cell may be the ‘cell of origin’ for this poor prognosis group. Since tumors arising in BRCA1 mutation carriers often exhibit a ‘basal’ phenotype, we hypothesized that the mammary stem cell pool is aberrant in these women. Our objective was to define the human MEC hierarchy and evaluate the functional characteristics of MECs from BRCA1± and normal breast tissue. Methods: Single cell suspensions prepared from fresh human BRCA1± and normal breast tissue were sorted by flow cytometry. Hematopoietic (CD45+, CD235a+) and endothelial (CD31+) cells were removed, and the remaining viable ‘lineage minus’ (Lin-) MECs fractionated based on cell surface markers. These subpopulations were evaluated by immunostaining, RNA profiling, in vitro culture in Matrigel and in vivo xenotransplantation into ‘humanised’ mammary fat pads of immunocompromised mice. Results: Lin- cells fractionated with α6-integrin (CD49f) and epithelial cell adhesion molecule (EpCAM) revealed three discrete MEC subpopulations. A stem/progenitor population that preferentially yields breast outgrowths in vivo has been identified. These subpopulations gave rise in vitro to colonies that were either large and heterogenous or small and homogenous. Only the latter differentiated into milk-producing cells when exposed to lactogenic stimuli. Immunostaining confirmed their origins from myoepithelial and luminal progenitor cells respectively. Intriguingly, BRCA1± MECs exhibited perturbed in vitro growth properties when compared to normal controls. Conclusions: Our studies provide evidence for a human MEC hierarchy, a model to interrogate the perturbation noted in pre-neoplastic tissue derived from BRCA1 mutation carriers and the identification of the cell of transformation in BRCA1 associated breast cancer. These findings may have implications on developing prevention strategies for hereditary breast cancer. No significant financial relationships to disclose.