Isolation and Culture of Tumor Cells

Tumor tissues were dissected from patients during surgery and collected in sterile Hibernation media (Supplementary Table 1) and transported on ice to the laboratory within two hours. Each tumor sample was transferred into one 100-mm culture dish and washed several times with ice-cold Hibernation media. Blood clots and gross necrosis were removed while washing the sample. The tissue was dissociated mechanically by chopping into small pieces with scissors. After primary mincing, they were collected into a 15-ml conical tube and washed twice with ice-cold Hibernation media. Next, the tissue was digested in DMEM with DNase I (250 U/ml) and collagenase type IV (1 mg/ml) (Invitrogen) for 30–60minutes at 37°C. The ratio of enzyme mixture solution and mechanically dissociated sample volume was 5:1. The primary tissue was triturated with intermittent pipetting every 10 minutes until there was no visible mass in the solution, to obtain single cell suspension. Cell suspension was centrifuged at 450g for 10 min at 4°C. Cells were washed twice with cold DMEM and were resuspended in Hibernation medium. Cells were then plated in PLO/Laminin coated 6-well plate. Serum-free medium composition was as following: DMEM (GIBCO, Invitrogen), L-glutamine, sodium pyruvate, B27, N2, insulin (20ug/ml), bFGF (20ng/ml), EGF (20ng/ml), PDGF-AB (20 ng/ml) and heparin (10 ng/ml)(Invitrogen). Cells were refed every three days and passaged every 1–2 weeks.