ScATAC-seq Reveals The Microenvironment Of Gallbladder Cancer And Its Heterogeneity

Background&Aims:Gallbladder cancer(GBC)is a tumor with a high degree of cellular heterogeneity and has a very poor treatment and prognosis.With the development of immunotherapy,the treatment of cancer patients has changed dramatically and brought hope to the treatment of cancer patients and researchers.There are a number of clinical trials targeting immunotherapy for gallbladder cancer,allowing patients to achieve clinical remission,but only a small percentage of patients can benefit.Cell types and cell-cell interactions in the microenvironment of gallbladder cancer tumors affect the effectiveness of immunotherapy.Therefore,analyzing the tumor microenvironment of gallbladder cancer is expected to explore potential therapeutic directions and therapeutic targets for gallbladder cancer while gaining a deep understanding of the tumor.Each cell subpopulation in the tumor microenvironment has different functions.CD8+T cells can directly recognize and kill cancer cells after recognizing neoantigens,while CD4+T cells can also coordinate various immune responses to enhance or suppress immunity against cancer cells.Dendritic cells(DCs)initiate and regulate innate and adaptive immune responses against tumors by presenting tumor antigens and secreting pro-and anti-inflammatory cytokines and chemokines.Macrophages can promote cancer development,progression,and even metastasis by stimulating angiogenesis,promoting non-productive inflammation,and suppressing antitumor immunity.Endothelial cells can promote angiogenesis and lymphangiogenesis,thereby promoting distant tumor metastasis.In addition,there are frequent,such as ligand-receptor,interactions between these immune cells to achieve immunosuppressive or immune enhancing effects.Current research progresses reveal that CD8+T cells in the tumor microenvironment are generally depleted,and effector CD8+T cells can also be transformed into depleted CD8+T cells upon antigen stimulation;most subtypes of CD4+T cells have immunosuppressive effects;and macrophages have a major role in immunosuppression and promotion of tumor growth.In recent years,several studies have identified new and potential targets for tumor therapy by resolving the role of different immune cells in the tumor microenvironment.In recent years,single-cell sequencing technology has provided a powerful tool in resolving the tumor microenvironment,allowing us to obtain multi-layer information on the epigenomic,genomic,transcriptomic and proteomic features of individual cells and their combinations,so as to fully understand the tumor cell types,various subtypes of immune cells,differential genes of different cells,intercellular correlations and transformations in the tumor microenvironment.In turn,new gene targets,as well as biomarkers based on the genetic level,are continuously identified.With the development of single-cell sequencing technology,single-cell transcription sequencing(scRNA-seq)and single-cell Assay for Transposase-Accessible Chromatin with highthroughput sequencing(sc ATAC-seq)have received a great deal of attention.scRNA-seq can provide information on single-cell transcriptomics to understand the gene expression status of individual cells,i.e.,the transcriptional state of cells.And sc ATAC-seq can reveal the gene landscape of cellular transcription-related regulation.Combined analysis of scRNA-seq and sc ATAC-seq can link gene expression to the accessibility of regulatory elements and can more accurately reconstruct the molecular processes underlying cellular physiology.Our group has previously studied gallbladder cancer by scRNA-seq and resolved the cellular heterogeneity and interactions in the tumor microenvironment,and found that immune cells in the microenvironment are generally immunosuppressive,but there is a gap in the study on the transcription-related regulatory gene landscape of gallbladder cancer cells based on sc ATAC-seq,so the use of sc ATAC-seq and scRNA-seq The combined sc ATAC-seq and scRNA-seq analysis protocol will provide an adequate scientific basis to fully reveal the microenvironment and heterogeneity of gallbladder cancer and to identify novel precision therapeutic targets.Methods:In this study,primary gallbladder tumors and paraneoplastic tissues were obtained from enrolled patients with gallbladder adenocarcinoma for analysis.Sequencing methods were selected from 10X Genomics for Tn5 transposase sequencing and single cell transcriptome sequencing.Then we used Harmony,Signac,Seurat,Monocle,Cicero and Cellchat algorithms to cluster and functionally enrich the gallbladder cancer microenvironment and explore the intercellular transformation relationships and the interactions between different cell subpopulations.Results:1.40129 cells were obtained by scRNA-seq study and 13844 cells were obtained by sc ATAC-seq,from which 8 cell populations were identified,including epithelial cells,T&NK cells,B cells,myeloid cells,endothelial cells,fibroblasts,pericytes and mast cells.2.12 subpopulations of T&NK cells were identified,among which CXCL13~+CD4~+T cells,FOXP3~+CD4~+T cells and GZMB~+CD8~+T cells have strong immunosuppressive effects in gallbladder cancer tissues,and their highly expressed immunosuppressive genes(HAVR2,LAG3,PDCD1,TIGIT)may be potential therapeutic targets for gallbladder cancer;in addition,cytotoxic GZMK~+CD8~+T cells could be transformed into GZMB~+CD8~+T cells in gallbladder cancer,reflecting the prevalence of immunosuppressive expression of T cells in gallbladder cancer.Eleven subpopulations of myeloid cells were identified,and macrophages mainly underwent M2 polarization in gallbladder cancer and M2-type macrophages were in the majority,suggesting immunosuppressive and pro-cancer roles of tumor-associated macrophages in gallbladder cancer;LAMP3~+dendritic cells have multiple functions in the tumor microenvironment.Two subpopulations of endothelial cells were identified,mainly involved in angiogenesis and lymphangiogenesis,which may play an important role in promoting distant metastasis of gallbladder cancer.Four subpopulations of epithelial cells were identified,and Epi＿C4 was found to have an important role in multiple functions as a tumor-specific cell in the tumor.3.Extensive cellular communication existed between fibroblasts,endothelial cells and numerous cells,with particularly active communication with tumor epithelial cells;in addition,close interactions were formed between fibroblasts and tumor epithelial cells,macrophages and T cells,and tumor epithelial cells and T cells through ligand-receptor binding.Conclusion:In this study,eight characteristic cell populations and their subpopulations were identified in gallbladder cancer,and the interactions between various cell types in the microenvironment were elucidated,and the related findings provide potential intervention targets for targeted or immunotherapy of gallbladder cancer.