Murine 4T1 cells were cultivated in Dulbecco’s modified Eagle medium (DMEM; Biochrom GmbH, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS; Biochrom AG, Heidelberg, Germany) and 1% penicillin/streptomycin (Biochrom GmbH, Berlin, Germany). 4T1 sublines derived from primary tumors, CTCs, DTCs, and metastases were cultured in selection medium containing 60 μM 6-TG (Sigma-Aldrich, Saint Louis, USA) in DMEM with 10% FBS and 1% penicillin/streptomycin (Gibco, Planegg, Germany). DTC1-derived CTC sublines, which grew in a less adhesive manner, were propagated for both adherent and semi-adherent cells. All cell lines were grown in a 5% CO2 atmosphere at 37°C.

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