Analysis Of Transcriptional Heterogeneity Of Tumor Microenvironment In Bladder Cancer Tissues Based On Single-cell RNA Sequencing

Background:Bladder cancer(BC)is a highly heterogeneous tumor and the most common malignant tumor of the genitourinary system in men in China.Although patients with bladder cancer generally have a long survival period,no matter how the treatment methods are improved,the recurrence rate and metastasis rate are still high,mainly due to the high heterogeneity of epithelial cells,T lymphocytes,and fibroblasts in the tumor microenvironment of bladder cancer tissue,resulting in the lack of effective targeted therapy or specific markers of immunotherapy,and ultimately further progression of BC patients.However,there are few studies on the heterogeneity of epithelial cells,T lymphocytes,and fibroblasts in the tumor microenvironment of BC tissue,and most of the related studies have not been explored in depth at the single-cell level.Methods:R and R package Seurat mainly analyzed the single-cell RNA sequencing data of2 bladder cancer tissue specimens and 2 corresponding non-malignant tissue specimens in the GEO database.The single cells with similar information were re-clustered and labeled with Gene annotations associated with cell types.Combining copy number variation(CNV)and a series of algorithms to distinguish malignant epithelial cells from non-malignant epithelial cells,using the Find Markers function to analyze differentially expressed genes between malignant cells and non-malignant cells,showing malignant epithelial cells and non-malignant epithelial cells HALLMARK-specific pathways enriched in upregulated genes.Furthermore,the T cells and fibroblasts in the tumor microenvironment of bladder cancer were re-clustered and grouped,and the corresponding subtypes were annotated with marker genes.Monocle2 was used for trajectory analysis and Metascape was used for gene GO and Hallmark pathway enrichment analysis.In addition,402 bladder cancer samples with prognostic information in the TCGA database were used for survival analysis.The Kaplan-Meier method and log-rank test were used to construct and compare survival curves.Results:Select suitable specimens from the GEO database,obtain 15,821 cells after filtering and quality control,and conduct subsequent analysis on them.Cell annotation found that the tumor microenvironment of bladder cancer mainly includes epithelial cells,fibroblasts,B lymphocytes,T lymphocytes,monocytes/macrophages,dendritic cells,RAG,and HSPs gene highly expressed cell types.The analysis of differentially expressed genes(DEGs)(p<0.05,|Log2FC| ≥0.5)between malignant epithelial cells and non-malignant epithelial cells showed that CSTB,DRG1,IGFBP3,HILPDA,and SLC2A1 and other genes were significantly expressed in bladder malignant epithelial cells.The expression of the RPL34 gene and other genes was down-regulated.HALLMARK-specific pathways enriched for upregulated genes in malignant and non-malignant epithelial cells were also shown to differ.A total of 402 bladder cancer samples with prognostic information in the TCGA database were divided into a high group and a low group according to the median abundance of CD8+ T cells.Survival analysis showed that there was a statistically significant difference between the two groups(p=0.006).When GO enrichment analysis and pseudo-chronological analysis were used to analyze the differences in cell signaling pathways of CD8+ T cell up-regulated genes enriched in cancer tissue and paracancerous tissue,the results obtained were basically consistent.The bladder cancer samples with TCGA prognostic information were divided into a high group and a low group according to the median of CAFs cell abundance.Survival analysis showed that there was a statistical difference between the two groups(p=0.05).The enrichment of signaling pathways and heat maps of gene expression levels related to invasion,cell migration,and extracellular matrix remodeling were different for each subpopulation of fibroblasts.In different subpopulations of fibroblasts,the DCN gene was independently highly expressed in the m CAF subpopulation of normal tissues.Stratified according to the expression level of DCN gene m RNA,the overall survival curve of bladder cancer patients in the Tumor Genome Atlas(TCGA)was drawn,showing that the survival of patients with high DCN gene expression group and lower expression group was significantly different(p=0.029).Conclusion:1.There is significant transcriptional heterogeneity of epithelial cells,T cells,and fibroblasts in the tumor microenvironment of bladder cancer tissue,which promotes carcinogenesis of bladder tissue at the cellular and molecular levels.2.CSTB,NDRG1,IGFBP3,HILPDA,SLC2A1,RPL34,and DCN genes may be used as diagnostic marker genes and potential therapeutic targets for bladder cancer in the future.At the same time,hypoxia and enhanced iron death function of tumor cells mediated by CD8+ T cells are also therapeutic targets that cannot be ignored in the treatment of bladder cancer.3.The carcinogenic effect of fibroblasts may be related to m CAF with high expression of the DCN gene.