Combination of EGFR TKIs and an AXL inhibitor overcomes primary resistance caused by AXL signal activation. (A) KPP-03, PC-9, and H1975 cells were treated with EGFR TKIs alone (erlotinib 100 nmol/liter or osimertinib 100 nmol/liter), ONO-7475 (100 nmol/L) alone, ramucirumab (150 ng/μL) alone, EGFR TKIs with ONO-7475, or EGFR TKIs with ramucirumab for 72 hours. Cell viability was assessed using the MTT assay. (B) KPP-03 cells were treated with EGFR TKIs alone (erlotinib 100 nmol/L or osimertinib 100 nmol/L), ONO-7475 (100 nmol/L) alone, ramucirumab (150 ng/μL) alone, EGFR TKIs with ONO-7475, or EGFR TKIs with ramucirumab for 9 days. Cells were stained with crystal violet. (C) KPP-03 cells were incubated with EGFR TKIs alone (erlotinib 100 nmol/L or osimertinib 100 nmol/L), ONO-7475 (100 nmol/L) alone, EGFR TKIs with ONO-7475, or EGFR TKIs with ramucirumab (150 ng/μL) for 4 hours, and the indicated proteins were then detected by Western blotting assay. (D) KPP-03 cells were treated with EGFR TKIs alone (erlotinib 100 nmol/L or osimertinib 100 nmol/L), ONO-7475 (100 nmol/L) alone, EGFR TKIs with ONO-7475, or EGFR TKIs with ramucirumab (150 ng/μL) for 48 hours, and apoptotic cells were then detected by flow cytometry. p-, phosphorylated-; TKI, tyrosine kinase inhibitor.