Mda mb 231 231

Human mesenchymal-like breast cancer cell lines MDA-MB-231 (231) and MDA-MB-436 (436) were obtained from the American Type Culture Collection (ATCC). Cells were cultured in low glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS in a humidified 5% CO2 incubator. Human breast CAF2 was obtained from Robert Weinberg [31 (link)]. The CAFs T53, T73 and T68 isolated from lesions of triple-negative breast cancer patients were characterized with antibodies as vimentin-positive and pan-cytokeratin-negative [32 (link)]. The Ast CAF (specimen 87322A1) – also isolated from triple-negative breast cancer lesions – were obtained from Asterand Inc. Normal human skin fibroblasts CRL-2575 were obtained from ATCC. All fibroblasts were cultured in DMEM supplemented with 10% FCS in a humidified 5% CO2 incubator. HUVECs were acquired from the Center for Excellence in Vascular Biology, Brigham & Women's Hospital, Harvard Medical School, Boston, MA and maintained in EGM medium (2% FBS, brain bovine extract, heparin, hEGF, and hydrocortisone) (Lonza). Cancer cell spheroids were generated by seeding 2 × 10⁵ cells/ml on low-adhesion surface (Corning) for 72 h. For RNA and protein extraction, indirect co-culture of cancer cell spheroids and CAFs was performed in 0.4 μm Transwell inserts (Corning).

MCF-10A (10A), MDA-MB-231 (231), and MCF-7 cell lines were from American Type Culture Collection. HEK293T (293 T) cell lines were from the Cell Bank of the Chinese Academy of Sciences. All cell lines were characterized by DNA fingerprinting and isozyme detection. The 231 and 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA). MCF-7 cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 10 μg/ml human recombinant insulin (Sigma–Aldrich, Germany). The 10 A cells were cultured in DMEM/F12 with 5% horse serum (Gibco, USA), 0.02 μg/ml human EGF (Peprotech, USA), 0.5 μg/ml hydrocortisone (TCI, Japan), 0.1 μg/ml cholera toxin (Sigma–Aldrich) and 10 μg/ml insulin. All cell lines were grown at 37 °C under 5% CO₂ in a humidified chamber.

Human breast cancer cell lines MCF-7, ZR-7530, MDA-MB-231(231) (expressing low STC2) and lentiviral packaging cell line (293T cell) were purchased from American Type Culture Collection (Manassas, VA). MDA-MB-231 HM (231 HM) cells (expressing high STC2) were established by Breast Cancer Institute of Fudan University Shanghai Cancer Center [27 (link)]. All cell lines were maintained in DMEM medium, supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 μg/mL). All cell cultures were incubated at 37°C in 5% CO2 atmosphere.