Integrated Application Of Single-Cell Sequencing Technologies To Study The Immune Microenvironment Of Lung Adenocarcinoma

Objective:Lung adenocarcinoma is an aggressive malignancy,and a growing number of studies have demonstrated the effectiveness of targeting the tumor microenvironment for the treatment of advanced lung adenocarcinoma.Fewer studies have been performed to resolve the efficacy of immunotherapy in lung adenocarcinoma by single cell.In this study,we combined single-cell and bulk sequencing to characterize the immune microenvironment and metabolic profile of lung adenocarcinoma to predict the efficacy of immunotherapy.Methods:In this study,we identified immune subtypes by NMF clustering based on TCGA-LUAD data,and explored the Scissor tumor cells most associated with immune subtypes by Scissor algorithm.Scissor cells interacting with immune subtypes were analyzed by cellphonedb and cellchat with immune cells and stromal cells.Subsequently metabolic reprogramming of tumor cells playing different roles was explored based on ss GSEA and MEBOCOST.We then analyzed key transcription factors that drive tumor cells to perform different biological functions based on the python tool SCENIC and calculated marker genes for different tumor cell subtypes.The key transcription factors and marker genes were then incorporated into Cytoscape based on PPI protein interaction network to construct a gene regulatory network.Finally,we collected 42 samples of lung adenocarcinoma patients from China Medical University Hospital I to verify the expression of key genes using immunohistochemistry.Results:Two immune subtypes,C1 subtype with "cold" tumor and C2 subtype with "hot" tumor,were identified based on the Scissor algorithm,and two groups of epithelial cells associated with the immune subtypes were identified,Scissor＿C1 cells were associated with significant epithelial mesenchymalization and immunosuppressive features,and the inverse convolution of Scissor＿C1 may be associated with tumor invasion and metastasis as well as immunotherapy tolerance.cellchat revealed that the hypoxic microenvironment induced high expression of ANGPTL4 in Scissor＿C1 and endothelial cells,which could increase vascular permeability and thus achieve distant metastasis across the endothelium.The low metabolic activity of Scissor＿C1 may be related to its hypoxic microenvironment,and the uptake of Scissor＿C1 from TAM via SLC38A2 transporter protein may cause lung cancer.Scissor＿C1 may cause glutamine addiction in lung adenocarcinoma.EPHB2,which is significantly associated with immune checkpoint immunodepletion markers,may be a marker of immunosuppression in the tumor microenvironment.The combination of bulk sequencing and single-cell sequencing can explain the efficacy of immunotherapy from clinical information.