Characterizing NSCLC Cell Lines and Gefitinib Resistance

Human NSCLC cells, PC-9 (EGFRdelE746-A750), HCC827 (EGFRdelE746-A750), H1975 (EGFRL858R/T790M) and H1703 (EGFR wild-type), and immortalized non-transformed NL20 lung cells were purchased from the American Type Culture Collection. These cells tested negative for mycoplasma (Figure S8; MycoAlert assay from Lonza LT07-218) and were authenticated via DNA fingerprinting (Figure S9). These cells were maintained in either RPMI or MEM (Gibco) supplemented with 10% FBS (Hyclone), 2g/L glucose and penicillin/streptomycin (P/S). All cell lines were grown at 37°C under 5% CO₂. Cells were grown to ~80% confluence, harvested with trypsin, and suspended to the cell density required for each assay. PC-9 Gefitinib resistance (PC-9GR) was generated through chronic exposure of drug at gradual dose increments (from 50 nM up to 5 μM) for 6 months 67 (link). The IC50 of PC-9GR was determined to be about 3 μM (versus 50 nM for parental PC-9) and cells were then maintained in 1 μM of Gefitinib prior to all experiments. Further, DNA from PC-9GR were analysed to rule out the relation between the development of acquired resistance to Gefitinib of PC-9GR cells and secondary EGFR T790M mutation or MET gene amplification.