Establishment and Characterization of Erlotinib-Resistant Lung Cancer Cells

Human lung cancer cell lines used in this study were from the established collections in our labs or as reported in our previous studies [8 (link)–10 (link)]. PC-9 erlotinib resistant cells were established from PC-9 cells by stepwise exposure to erlotinib from 0.005 μM to 5 μM for about 4 months, and the clone named PC-9 ER clone 5 was isolated with PicoPipet (Nepa Gene, Chiba, Japan). Cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1x penicillin/streptomycin solution (Mediatech, Inc., Manassas, VA) at 37°C in a humidified tissue culture incubator with 5% CO₂. All experiments using acquired resistance cells were performed following the removal of drug exposure to avoid the direct effects of drugs on PD-L1 expression. IFN-gamma (Cell Signaling Technology, Dancers, MA) stimulation for 24 hours was performed to mimic an immune cell interaction.