Human breast cancer cell lines (MDA-MB-468 and HCC1954 from ATCC), the human HER293 cell line (ATCC), and murine mammary tumor cell lines EO771 (CH3 Biosystems) and 4T1 (ATCC) were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. For primary breast cancer cell culture, tumor tissues were collected and digested with MACS tumor dissociation kit (mouse, 130-096-730; human, 130-095-929; Miltenyi Biotec). Tumor tissues were sliced into small pieces in diameter around 2–4 mm, then incubated in 37°C water bath for 40 minutes and dissociated using the gentleMACS Octo Dissociator (Miltenyi Biotec). After centrifugation at 300 g for 7 minutes, single cells were cultured in DMEM/F-12 medium supplemented with 5% FBS, 1% penicillin/streptomycin, 20 ng/mL EGF, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 μg/mL insulin. After 1–2 days of culture, cells were trypsinized, washed, and ready for the next procedures.

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