Abstract
Abstract Objectives: The majority of our understanding of mammary epithelial cell hierarchy is from mouse models. In addition, due to limited replication potential, primary cells of either mouse or human origin have rarely been used for functional studies. In this study, we took advantage of the recently developed epithelial cell-reprogramming assay that utilizes irradiated fibroblasts and ROCK inhibitor to characterize breast epithelial cells from individuals with varying degree of breast cancer risk. Methods: Primary breast tissues were collected at the time of surgery and cultured either immediately or at convenient time after freezing in liquid nitrogen with ROCK inhibitor. Flow cytometry sorting was employed to fractionate and propagate stem-like/basal (CD49f+/EpCAM-), luminal progenitor (CD49f+/EpCAM+), and mature (CD49f-/EpCAM+) cell enriched fractions. Similar fractionation/characterization was performed using CD133, CD10, CD271, EGFR and ALDEFLUOR. Results: Inter-individual variability was observed in proportion of CD49f+/EpCAM+, CD49-/EpCAM+ and CD133+ cells. Epithelial cells from a patient considered high risk based on the history of hyperplasia as well as cells from BRCA1 and BRCA2 patients displayed unique CD49f and EpCAM staining pattern compared to cells isolated from adjoining “normal” tissue of two cancer patients. In addition, CD133+ population was higher in BRCA1 and BRCA2 mutant patients. Morphologically, cells isolated from BRCA1 patient displayed higher levels of cells with mesenchymal phenotype. Upon short-term culturing of cells under regular 2D culture without reprogramming conditions, cell surface marker profile remained unchanged suggesting that most of the cell surface marker profile is cell intrinsic. CD49f+/EpCAM-, CD49f+/EpCAMhigh, CD49f+/EpCAMmedium, CD49f-/EpCAM+, CD133+/EpCAM+, and CD10+/EpCAM+ cells were amicable for further propagation in culture. CD49f+/EpCAMmedium cells cultured for two weeks remained CD49f+/EpCAMmedium. Additional marker profiling including cancer stem cell markers is underway to further refine stem, progenitor, and mature cells. Conclusion: Ability to propagate distinct subpopulation of normal cells from patients with different breast cancer risk would allow investigation of the impact of cell type origin of cancer on the course of the disease including response to therapy and the development of in vitro assay for breast cancer risk assessment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-05-05.
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