Single Cell Transcriptome Based Study Of Epithelial Cell Heterogeneity And Microenvironment Immune Signature In Gastric Cancer

Objective: Gastric cancer is a common tumor worldwide,which is difficult to detect at an early stage and less effective in late stage clinical treatment.Developing new therapeutic strategies for gastric cancer requires a deep understanding of the tumor microenvironment of gastric cancer.Here,this study bioinformatically parses gastric cancer single-cell sequencing data to characterize gastric cancer epithelial cell heterogeneity and microenvironmental immune signatures.Methods: Based on the GEO database GSE183904,single-cell sequencing data of a total of 50061 cells from 9 gastric cancer samples and 9 adjacent noncancerous tissue samples were collected in this study.Seurat was used in this study to quality the data with the following filtering criteria: number of genetic tests 500-10000,UMI count 1000-100000,mitochondrial gene read fraction < 30%,hemoglobin gene read fraction< 5%.SCTransform was used for data normalization.After principal component analysis,the first 15 principal components were selected for construction of the SNN map and UMAP embedding.The Clustree package was used to select the appropriate number of clustered subgroups.The Find All Markers function was used to determine the signature genes of cell clusters.In this study,single-cell literature associated cell markers and the CELLMarker website were used as references to manually annotate them by the signature genes of each cell cluster.Cluster subgroups were annotated for the first time as epithelial,immune and stromal cells.After that,cluster subgroups were rerun in subsets.After the end of cell annotation.This study calculates cell proportions,measures the intergroup differences of individual cell taxa,and enrichment analysis and GSEA are used to explore the functions of individual cell clusters.The Add Module Score function scores the feature gene set.Infer CNV computes the chromosomal copy number variation landscape of tumor epithelial cells.PROGENy evaluates epithelial and stromal cell oncogenic signaling pathway activity.SCORPIUS analysis of differentiation trajectories of epithelial and stromal cells.Cellchat was used to infer and analyze intercellular communication networks between the annotated cell subpopulations.Based on the annotated cell types,principal component analysis was used to search for immune signatures of the gastric cancer microenvironment and applied to transcriptome datasets of gastric cancer.Results: 1.Epithelial cell heterogeneity in gastric cancer: three malignant epithelia(EPL,EPM,EPH)have been defined based on tumor pathway activity scores and functional heterogeneity of three malignant epithelia has been found.In which EPL retained the characteristics of part of normal gastric epithelium,while Eph possessed higher epithelial mesenchymal transition(EMT)properties.Pseudotime analysis identified a differentiation trajectory of gastric cancer epithelial cells with EPL as the starting point and EPH as the end point,and the gene expression profile of gastric cancer epithelial cells has a continuity with the differentiation trajectory,ie,as gastric cancer evolves,gastric cancer epithelial cells gradually evolve from normal gastric mucosa characteristics of EPL(GKN1,GKN2),to tumor cell metabolism of Eph with enhanced cancer cell stemness and EMT features(S100A10,TNFRSF12 A,IFITM3).Genes with altered expression profiles during this process are defined as key molecules in gastric cancer evolution.Among them,ERGIC2,IFITM3,S100A10,RPL22L1,TNFRSF12 A,TP53I11,MET,AC004922.1,C4orf3,KRAS,RALA were associated with shorter survival time in gastric cancer patients.2.Immune features of the gastric cancer microenvironment: immune cells are initially divided into five subsets,T cells,B cells and plasma cells,NK cells,monocytes/ macrophages and mast cells.T cells account for the predominant proportion in all patients.T cells clustered into 7 subsets CD8 Trm,CD8Tem,CD8 T na?ve,CD8 Tex,CD4 T na?ve,CD4 Th,Treg.Even though T cell subsets varied in proportion between patients,all types were almost stably present.This distribution state of T cells provides strong support for the functional importance of T cells in the gastric cancer microenvironment.There are few naive CD8 T cells that emerge among normal tissues in the tumor microenvironment.Higher exhaustion scores and immunosuppression scores were present for CD8 Tex with increased proportions in the tumor microenvironment.Tregs in the gastric cancer microenvironment expressed the highest immune checkpoint score.Macrophages cluster into five subsets,TAM＿Angio,TAM＿Inflam,TAM＿Inflam＿LA,TAM＿Prolif,TAM＿C1QC.Tumor associated macrophages in the gastric cancer microenvironment are not strictly distinguished by M1 / M2 status,in which TAM＿C1QC and Tam＿Inflam＿La exhibited a higher M2 signature.TAM＿Inflam＿LA,TAM＿Inflam and Tam＿Angio has a higher proangiogenic score.TAM＿Inflam＿LA,TAM＿Angio represents a worse prognosis.RGS5 + fibroblasts involved in angiogenesis dominate the stromal microenvironment of gastric cancer.Cell communication revealed that EPH associates with TAM＿Angio,TAM＿Inflam,TAM＿C1QC have a interaction of MIF signaling pathways.Whereas MIF is thought to protect cancer cells from immunogenic cell death and impair antitumor immune responses.MIF related receptor ligand pairs exhibit promising value in tumor immunotherapy and antitumor strategies for gastric cancer treatment.Finally,based on the cellular components of the gastric cancer microenvironment,RGS5 +CAFs,Tregs,TAM＿Angio as an immune signature the gastric cancer microenvironment exhibits a worse prognostic status.Conclusions: This study describes heterogeneity among malignant epithelial cells of gastric cancer based on single-cell transcriptome and finds differentiation trajectories among epithelial cells based on functional analysis and pseudotime analysis.These results,at a certain level,annotate the transcriptional changes of gene expression profiles during tumor progression and contribute to our better understanding of epithelial cell heterogeneity in gastric cancer.On the other hand,our delineation of the immune and stromal cell landscape characterizes the gastric cancer microenvironment as highly complex and heterogeneous,revealing various classes of cell proportion changes and functional characteristics.We identified a subtype of cells that promote the gastric cancer microenvironment into an immunosuppressive state.Finally,we found that RGS5 + CAFs,Tregs,TAM＿Angio as an immune signature of the tumor microenvironment can respond to patient survival,which may help predict treatment response and reveal possible target cell populations for future therapeutic approaches.