Characterization of Breast Cancer Stem Cell (BCSC) populations in MCF7 and MDA-MB-231 cell lines.
(A) Fluorescence activated cell sorting (FACS) analysis of cell surface proteins showing proportions of Cancer stem cells (CD44+/CD24low/−) in breast cancer cell lines. (B) FACS analysis of CD44 and ESA showing proportions of CSCs in the MDA-MB-231 (triple negative) cell line. Three biological experiments were done for each cell line. (C) BCSCs (CD44+/CD24low/−) and non-BCSCs (CD44−/CD24+) populations isolated from MCF7 were tested for their clonogenic potential in soft agar assays (4,000 seeded cells). BCSCs showed increased clonogenic ability (n = 3, error bars are +/− s.e.m, *p < 0.05). (D) To confirm the clonogenic potential of MDA-MB-231 BCSCs (ESA+), limited dilution assays were performed. CD44+/ESA+ or CD44−/ESA− cells colony numbers grown at low cell density. (n = 3, error bars are +/− s.e.m, *p < 0.05). (E) Cancer stem cell markers (NANOG, OCT4, SOX2, ALDH1A1, ALDH8A1, and ALDH1A3) expression was analyzed by Real time PCR (RT-qPCR) in MCF7 BCSC and non-BCSC populations. MCF7 BCSCs showed higher expression levels of CSC markers. (n = 3, error bars are +/− s.e.m, *p < 0.05). (F) Real Time-PCR analysis of CSC markers showed that CD44+/ESA+ cells expressed higher levels of SOX2, OCT4, NANOG and ALDH181. (n = 2, error bars are +/− s.e.m).