Cell viability assay

Cell viability was measured by cell counting kit‐8 (CCK8: Selleck) assay. PC9 and PC9/GR cells were plated in 96‐well plates at a density of 3 × 10³/well and incubated overnight. The cells were subsequently exposed to increasing concentrations of gefitinib (0.01, 0.1, 1.0, 2.0, 5.0, and 10.0 μM), anlotinib (1.0, 2.0, 4.0, 8.0, 16.0, and 32.0 μM), or a gefitinib–anlotinib combination (gefitinib: 0.01, 0.1, 1.0, 2.0, 5.0, and 10.0 μM; with anlotinib 2.0 μM) for 72 h. Then, 10 μl of CCK8 was added into each well and incubated for 1 h. The absorbance (optical density, OD) at 450 nm was detected using a microplate reader (Tecan). Cell viability was calculated as: cell viability = (OD_sample − OD_blank)/(OD_control − OD_blank) × 100%. The values of the half maximal inhibitory concentration (IC50) were calculated by GraphPad Prism 8.3.0 (GraphPad Software). Experiments were repeated in triplicate, and an average was obtained. The combination index for the combination of gefitinib and anlotinib was calculated using CompuSyn software.