Establ Ishment Of An Osimertinib-resi Stant Cell Line Of Human Non-small Cell Lung Cancer And The Subsequent Sensitivity Of Themotherapeutic Drugs

ObjectiveEpidermal growth factor receptor-tyrosine kinase inhibitor（EGFR-TKI）is the first line treatment of for the NSCLC patients with EGFR mutation.However,acquired resistance develops after a median of 9-14 months.The most common mechanism of TKI resistance is a second-site mutation（T790M）in the EGFR kinase domain.Osimertinib,an irreversible selective EGFR TKI,has been used in clinic to reverse gefitinib-induced resistance.Unfortunately,acquired resistance still occurs after approximately 10 months.Machanisms of osimertinib-induced resistance remain unknown.Chemotherapy is one of the strategies for EGFR-TKI resistant patients.The most effective regiment for the patients of osimertinib-induced resistance is needed to be investigated.In this study,we chose gefitinib acquired resistance cell line,PC-9/ZD（EGFR 19 exon deletion/T790M mutation）to develop an osimertinib-resistance cell line,to explore possible mechanisms of osimertinib-induced resistance and the subsequent sensitivity of themotherapeutic drugs.Methods1)The osimertinib-resisitant cell line was induced by the increasi ng dose of osimertinib to PC-9/ZD;2)CCK-8 assay was used to detect the sensitivity of drugs of cell lines;3)Western blot was used to explore the related protein expresstion of EGFR,the signal transduction protein and the epithelial-mesenchymal transition（EMT）.4)NGS was used to detect the gene mutation of cells;5)Growth curve and cell cycle distribution were used to test the proliferation of cells.Results1.NGS data showed that the PC-9/ZD cell line was EGFR exon 19 deletion and exon 20 T790M mutations with EGFR amplification.The cytotoxicity test results showed that PC-9/ZD cells were significantly insensitive to gefitinib,compared to their parental cells.The IC50 value was 5.12±0.364 and 0.437±0.078 μ mol/L,respectively（P<0.05）.The resistance index（RI）was 11.71.The results demonstrated that PC-9/ZD was gefitinib-resistant cells.2.The acquired osimertinib-resistant cell line（PC-9/ZDOR）was established by adding osimertinib to parental PC-9/ZD cells from 0.08 to 3.2μ mol/L for 7 months.The IC50 of osimertinib for PC-9/ZD and PC-9/ZDOR was 0.08±0.16 and 3.522±0.038 μ mol/L,respectively（P<0.05）.The RI was 44,indicating that the osimertinib-resistant cell line was successfully established.3.NGS data showed that no EGFR mutations and amplification were founded in PC-9/ZDOR cells.31 mutation genes were detected,such as MET c.2584-13₂584-9ddel、FGFR p.A343V、KRAS p.G13D、ERBB3 p.Q261 et al.4.Western blot showed that the expression of EGFR,P-EGFR was significantly reduced in PC-9/ZDOR cells as compared with those in PC-9/ZD cells.The expression of E-Cadherin and N-Cadherin was significantly reduced in PC-9/ZDOR cells with no increase of Vimentin.5.According to the results of NGS in PC-9/ZDOR,we chose four mutation sites with abundances over 45%.Combination of osimertinib and their antagonists including crizotinib（MET inhibitor）、PD173074（FGFRlinhibitor）、sapitinib（Erbb3 inhibitor）and selumetinib（MEK 1/2 inhibitor）in PC-9/ZDOR,respectively,could not reverse the osimertinib-induced resistance.6.PC-9/ZDOR cell line was still showing a resistance to first generation TKI,gefitinib.PC-9/ZDOR showed a higher sensitivity to docetaxel,gemcitabine and paclitaxel than cisplatin and pemetrexed（P<0.05）.Conclusion1.Acquired osimertinib-resistant cell PC-9/ZDOR was successfully established,showing the loss of EGFR gene mutation maybe one of the mechanisms of acquired resistance to osimertinib.2.Combination of osimertinib and crizotinib、PD173074、sapitinib and selumetinib,respectively,failed to increase the sensitivity to osimertinib in PC-9/ZDOR.3.The osimertinib-resistant cell line,PC-9/ZDOR,showed a higher sensitivity to taxanes and gemcitabine.