Breast Cancer Cell Culture and Characterization

MDA-MB-231, MCF-7, and SkBr3 breast cancer cells were obtained from the ATCC (Manassas, USA). MDA-MB-231 and MCF-7 cells were maintained in DMEM/F12 (Life Technologies, Milan, Italy), 10% fetal bovine serum (FBS) and 1% of penicillin/streptomycin, while SkBr3 cells were maintained in RPMI-1640 (Life Technologies, Milan, Italy), 10% fetal bovine serum (FBS), and 1% of penicillin/streptomycin (Life Technologies, Milan, Italy). Cells were used less than six months after resuscitation and mycoplasma negativity was tested monthly. CAFs were extracted from invasive mammary ductal carcinomas obtained from mastectomies, while normal fibroblasts (NFs) were isolated from a non-cancerous breast tissue at least 2 cm from the outer tumor margin, as previously described [25 (link),28 (link),34 (link)]. Primary cells cultures of breast fibroblasts were characterized by immunofluorescence. Cells were incubated with anti-vimentin (V9, sc-6260), anti-cytokeratin 14 (LL001 sc-53,253) and anti-fibroblast activated protein α (FAPα) (H-56) antibodies that were obtained from Santa Cruz Biotechnology (DBA, Milan, Italy) (Supplementary Figure S1). All cells were grown in a 37 °C incubator with 5% CO₂ and switched to medium without serum and phenol red the day before treatments to be processed for immunoblot and quantitative PCR (qPCR) assays.