Experimental design
Ethical statements
Mouse experiments
Metaphase preparation and spectral karyotyping analysis
Flow cytometry
Immunocytochemistry, IHC, and EMT scoring
Proliferation assay, MTT assay, and 2D and 3D colony formation assays
Adhesion, invasion, and scratch assay
Generation of single cell–derived clones
Quantitative real-time polymerase chain reaction
Patients
SE of CTCs and DTCs
Identification of CTCs and DTCs by iFISH
Collection of single CTC and amplification
Quality control of WGA products and library preparation
Whole-genome sequencing, CNV, and GO analysis
Statistical analysis
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.