Evolution Of Immune Microenvironment And Mechanism Of Drug Resistance In EGFR L858R/T790M Co-mutant Non-small Cell Lung Cancer With Osimertinib Treatment

Background:Lung cancer is the most common cancer and a leading cause of death from cancer in men and women in the world.Epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)are considered as the first-line treatment of EGFR mutated Non-small cell lung cancer(NSCLC).However,almost all patients eventually develop acquired resistance to EGFR-TKIs.Previous studies focused too much on drug resistance caused by tumor cells,and few on tumor immune microenvironment.Therefore,it is imperative to study the mechanism of tumor immune microenvironment in tumor drug resistance.Methods:The RNA-sequence data of EGFR L858R/T790M co-mutant NSCLC cell line H1975 treated with Osimertinib were download from Gene Expression Omnibus(GEO)database.Data analysis was performed using R package.Gene Ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of the differential genes was performed using Database for Annotation,Visualization and Integrated Discovery(DAVID).The correlation among signal pathways,cytokines and immune cells was analyzed by Tumor IMmune Estimation Resource(TIMER2.0).Survival analysis of signaling pathways,cytokines and immune cells was performed using Kaplan-Meier Plotter.The resistance of Osimertinib treatment was studied by subcutaneous transplanted tumor animal model.The human EGFR L858R/T790M mutation overexpressing KLN-205 cell lines were established retrovirally using a H102-puro vector.The tumor samples were harvested for flow cytometry,ELISA,WB,IHC at three stages:Osimertinib naive,Osimertinib sensitivity and Osimertinib resistance.The changes of tumor immune cell infiltration ratio were analyzed by Flow cytometry and immunohistochemistry(IHC).The changes of cytokines secreted by tumor and immune cells were analyzed by Enzyme linked immunosorbent assay(ELISA).The changes of signal pathway in tumor cells were analyzed by Western blotting(WB).Results:Based on GEO database,the MAPK,NF-?B,TGF-? and VEGF signal pathways were activated after Osimertinib resistance.The four signal pathways were negatively correlated with CD8+T and CD4+T cells,positively correlated with M2 type tumor associated macrophages(M2),myeloid-derived suppressor cells(MDSCs)and regulatory T cells(Tregs),and positively correlated with IL-1?,IL-6,IL-10,IL-13,IL-34,M-CSF.These cytokines were negatively correlated with CD8+T and CD4+T cells,and positively correlated with M2 and Tregs.In vivo experiments,the number of CD4+T,CD8+T cells,M1 type tumor associated macrophages(M1)was increased at stage of Osimertinib sensitivity,and decreased after Osimertinib resistance.The number of MDSCs,Tregs and M2 was decreased at stage of Osimertinib sensitivity,and increased after Osimertinib resistance.The NF-?B and MAPK signal pathways activated after Osimertinib resistance.The expression of arg1,IL-4,IL-10,IL-34 was increased and the expression of IFN-y was decreased after Osimertinib resistance.Inhibiting both NF-?B and MAPK signal pathways after Osimertinib resistance could reduce the tumor size.The number of MDSCs,Tregs,M2 and the expression of argl,IL-4,IL-10,IL34 was decreased,the number of CD4+T cells,CD8+T cells,M1 and the expression of IFN-y was increased after blocking both NF-?B and MAPK signal pathways.Conclusion:The tumor immune microenvironment of EGFR L858R/T790M mutant NSCLC was activated in the stage of Osimertinib sensitivity and suppressed in the stage of Osimertinib resistance.The activation of NF-?B and MAPK signal pathways induced the immune suppressive tumor microenvironment through recruiting immune suppressive cells like MDSCs,Tregs,M2 and reducing immune activated cells like CD4+T cells,CD8+T cells,M1 by secreting immune suppressive cytokines like IL-4,IL-34.Immune suppressive cells inhibited the function of CD8+T cells by secreting argl and IL10,which caused the reduction of IFN-y secreted by CD8+T cells,These factors shaped the immune suppressive tumor microenvironment.Blocking both NF-?B and MAPK signal pathways could reverse Osimertinib resistance by decreasing the number of immune suppressive cells through reducing the secretion of immune suppressive cytokines.