Isolation and Culture of Breast Cancer Stem Cells

The sorting of breast CSCs and NCSCs from the MCF-7 and ZR-75-1 breast cancer cell lines was conducted using an ALDEFLUOR^TM kit (Cyagen Biosciences Inc., USA) according to our previous report 31 (link), 32 (link). Breast CSCs were cultured in DMEM/F-12 (Invitrogen, USA) supplemented with 20 ng/mL epidermal growth factor (Beyotime Biotechnology, Jiangsu, China), 10 ng/mL basic fibroblast growth factor (Beyotime, China), 5 μg/mL insulin (Beyotime, China) and 2% B-27 (Sigma, USA) at 37 °C in a humidified atmosphere with 5% CO₂. MCF-7 breast cancer cells and NCSCs were cultured in MEM supplemented with 10% fetal bovine serum (FBS) (GIBCO, USA), 2 nM glutamine (Thermo Fisher Scientific, USA) and 5 μg/mL insulin (Beyotime, China) at 37 °C in a humidified atmosphere with 5% CO₂. ZR-75-1 breast cancer cells and NCSCs were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (GIBCO, USA) at 37 °C in a humidified atmosphere with 5% CO₂.