The Characteristics Of Immune Microenvironment Of EGFR Mutant Lung Adenocarcinoma And Its Correlation With The Efficacy Of Immunotherapy

BackgroundsImmune checkpoint inhibitor(ICIs)has now become one of the standard treatments for non-small cell lung cancer(NSCLC).However,immunotherapy is less effective in patients with epidermal growth factor receptor(EGFR)-mutant NSCLC,the resistance mechanism is not clear.Lung adenocarcinoma(LUAD)is the major histological type of NSCLC and with the highest EGFR mutation incidence.To further understand the impact of EGFR mutations on LUAD tumor microenvironment(TME)and the underlying resistant mechanism of this type of patients treated with immune checkpoint inhibitors,we initiated this study to explore the specific tumor microenvironment characteristics of lung adenocarcinoma with different EGFR gene status.MethodsPart 1:1.The immune microenvironment characteristics of EGFR-mutant and EGFR wild-type LUAD were analyzed using single-cell transcriptome sequencing(sc-RNA seq).Nine surgically resected lung adenocarcinoma samples without treatment were collected for sc-RNA seq,of which 5 were EGFR positive and 4 were EGFR negative.The cellular components such as epithelial cells,T/natural killer cells,macrophages,fibroblasts,B cells,and dendritic cells in the tumor microenvironment of samples with different EGFR mutation status and the interactions between each cell subcluster were analyzed in order to compare the characteristics of LUAD tumor microenvironment with different EGFR mutation status.2.Bioinformatics methods were used to integrate and compare the single-cell sequencing data of 9 treatment naive samples with other 3 samples with different response to immunotherapy published in previous literature to explore the factors that may affect the immune response in the EGFR mutant LUAD tumor microenvironment.Part 2:Paraffin slides of tumor tissues paired with single-cell sequencing samples were collected,and CD3,CD4,CD8,CD103,CXCL13,CD11C,CD1C and CD68 antibodies and several immune checkpoint protein antibodies were selected to perform multiplex immunohistochemical staining(mIHC)based on the results from previous single-cell sequencing,to validate and further analyze the sc-RNA seq results in terms of cell composition,protein expression and spatial location.ResultsPart 1:1.Comparative analysis of single-cell sequencing data revealed that EGFR-mutant malignant epithelial cells had similar characteristics to the epithelial cells in non-responders.EGFR-mutant LUAD lacked CD8+tissue-resident memory(TRM)cells,which could promote tertiary lymphoid structure(TLS)generation by secreting CXCL13.2.Other cell types,including tumor-associated macrophages and cancer-associated fibroblasts,which are capable of recruiting,retaining,and expanding CD8+TRMs in the TME,were also deficient in EGFR-mutant LUAD.3.EGFR-mutant LUAD had significantly less crosstalk between T cells and other cell types via programmed cell death 1/programmed death-ligand 1(PD-1/PD-L1)or other immune checkpoints compared with EGFR wild-type LUAD,which explained the poor response to PD-1/PD-L1 monotherapy.Part 2:1.Multiplex fluorescence immunohistochemical analysis verified the presence of a higher proportion of CD8+TRMs in EGFR wild-type LUAD,along with high expression of CXCL13 by CD8+ T cells and massive TLS formation in tumor stromal areas.2.Except for TIGIT,other immune checkpoints including PD-1,PD-L1,CTLA4,LAG-3,TIM-3 and CD47 were with lower expression in EGFR-mutant LUAD tumor tissues,which may be the direct reason for the poor response to PD-1/PD-L1 treatment for this type of tumor.ConclusionsIn this study,the cellular components,cellular function and cellular interaction of the tumor immune microenvironment of EGFR mutant lung adenocarcinoma were comprehensively described at the single-cell level.Based on the results of the study,EGFR gene mutation may affect a series of gene expression from the transcriptome and protein level,so that LUAD with EGFR mutation forms an immunosuppressive tumor microenvironment and leads to poor response to immunotherapy.In addition to PD-1/PD-L1,other immune checkpoint inhibitors may also be ineffective in this type of tumor.Subsequent treatment of this type of patients should focus on improving the suppressive immune microenvironment and consider a combination therapy approach to develop treatment strategies for patients with EGFR-mutant lung adenocarcinoma.