Isolation and Culture of Mammary Tumor Cells

Single cell suspensions were established from spontaneous mammary tumors arising in FVB MMTV-PyVmT females as previously described (43 (link)). Tumors were minced and incubated at 37^oC for 2 hours in 5 ml of Ham’s F12K medium containing 1 mg/ml collagenase (Roche), 2 mg/ml soybean trypsin inhibitor (Sigma) and 2% bovine serum albumin (BSA). After addition of fetal bovine serum (FCS)-containing medium, the suspension was passed through a 70 mm nylon filter (Fisher Scientific). Single cells were pelleted by centrifugation and resuspended in PBS/2% BSA for immediate flow cytometric analysis. Stained cells were analyzed by a FACS Canto cytometer (BD Biosciences). Antibodies for flow cytometric analysis are listed in the Table S3. For establishment of MEC cell lines, cells were cultured in DMEM: F12 (1:1) medium mix containing 5% FCS, 2.5 mg/ml amphotericin B, 10 mg/ml penicillin–streptomycin and MITO+ (BD Biosciences). Non-epithelial cells were removed by differential passaging and epithelial origin of established cell lines were confirmed by CD326 (Biolegend) staining after five passages (CD326⁺ > 98%). The cell lines have been tested as mycoplasma-free using mycosensor PCR assay kit (Agilent Technologies) with the latest test in 2018. The cell line authentication was not routinely performed. The cells were used within two month after thawing.