Isolation and Culture of Breast Cancer Cells

Breast tumor differentiated cells from two patients (#3,#4) and BTSCs (Breast tumor stem cells) were obtained as previously described [26 (link),45 (link)], and were used for the miR array. T47D (ER-positive) and MDA-MB-MB-231 (TNBC) differentiated cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/mL penicillin/streptomycin [46 (link)]. For mammosphere cultures, single cells were plated at a density of 1,000 cells/mL for T47D and 50,000/mL for MDA-MB-231. T47D and patients’ cells were grown in serum-free DMEM-F12 (Sigma, Milan, Italy), supplemented with B27 (Life technologies, Milan, Italy), 10 ng/mL EGF (Sigma), 20 ng/mL FGF (BD Biosciences, Milan, Italy) and 1X antibiotic-antimycotics (Life technologies). MDA-MB-231 stem cells were grown in Mammary Epithelial Cell Growth Medium (Lonza, Milan, Italy) supplemented with BPE, hEGF, insulin, hydrocortisone, GA-1000 (Lonza), B27 (Life technologies), and 20 ng/mL FGF (BD Biosciences, Milan, Italy). After 4–7 days, mammospheres, appearing as spheres of floating viable cells, were collected by gentle centrifugation (800 rpm) and dissociated with 0.25% trypsin for 5 min.