Establishment of EGFR-mutant NSCLC Cell Lines

The human NSCLC cell lines HCC827 and HCC4006, which harbor EGFR activating mutations (16 (link),17 (link)), were purchased from the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in RPMI-1640 medium (BioWhittaker^®; Lonza Group, Ltd., Basel, Switzerland) supplemented with 10 mM HEPES, 1 mM L-glutamine, 100 U/ml penicillin/streptomycin (BioWhittaker^®; Lonza Group) and heat inactivated 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and grown at 37°C in a 5% CO₂ atmosphere. Gefitinib (Iressa; AstraZeneca, Macclesfield, UK) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a concentration of 10 mM and stored at −20°C. Gefitinib-resistant HCC827R and HCC4006R cells were established by initially culturing with 1 µM Gefitinib in DMEM plus 10% FBS for 6 weeks. Subsequently, a 2-µM concentration of Gefitinib was used to treat the surviving cells for 8 weeks and 5 µM for another 8 weeks. Eventually, the Gefitinib-resistant NSCLC cell lines were successfully established by culturing the cells in 10 µM Gefitinib.