The Role Of Structural Cells In Shaping The Microenvironment Of Bladder Cancer

Part 1:Comparison of the Biological Characteristics of Urothelial Cells and Tumor Cells from NMIBC and MIBCBackground and objective:Bladder cancer can be classified as non-muscle-invasive bladder cancer(NMIBC)and muscle-invasive bladder cancer(MIBC)based on depth of tumor invasion.There are significant differences in clinical features,treatment as well as molecular mechanisms between NMIBC and MIBC.So far,there has been no consensus on the pathological grading nor the molecular subtypes based on bulk RNA-seq of bladder cancer.Single cell transcriptome sequencing(scRNA-Seq)is RNA sequencing performed at a single cell resolution.This technique has great potential for advance understanding the types and heterogeneity of tumor cells at the single-cell level and the further screening of tumor drug targets.In this part,we performed scRNA-Seq for bladder cancer,focusing on the analysis of urothelial cells and tumor cells,so as to have a comprehensive and systematic understanding of the different biological characteristics of tumor cells in NMIBC and MIBC.Methods:Bladder cancer specimens from the Urology Department of the First Affiliated Hospital of Guangxi Medical University and the Urology Department of the Affiliated Tumor Hospital of Guangxi Medical University were collected according to the inclusion and exclusion criteria and sampling standards established by the research group.After the samples were prepared into single cell suspension,the Chromium single-cell V3 kit was used to generate the single-cell libraries,and the Illumina Hiseqxten was used for sequencing.Cell Ranger software was used to compare and quantify the FASTQ data generated by sequencing output with the human Reference genome.The Seurat R package was applied for further analysis.Analysis steps included filtering low quality cells,normalizing the data,identifying the top 2000 variable genes,eliminating the batch effect of merged data,performing principal component analysis,identifying the major groups of cells using "FindNeighbors" and "FindClusters"function,clustering and differential gene expression analysis of the urothelial cells,calculating copy number variation of urothelial cells using "InferCNV"function,RNA velocity analysis by performing the velocyto.R program,survival prognosis analysis and finding potential of targeted drugs by searching GEPIA and DrugBank database.Results:Tumor cells and normal urothelial cells were identified by combining the results of CNV analysis and transcriptional counting.Normal urothelial cells can be transformed into different tumor cells,such as basal cell-c2-DUOXA2 can be transformed into cancer cell-c4-IDO1 and cancer cell-c5-KRT6A.Cancer cell-c4-IDO1 can also be transformed into Cancer cell-c5-KRT6A.We found patient-specific cell populations in tumor cells as well as heterogeneity within tumor cells.Among them,marker genes CLIC3 and DAPL1 in tumor cells were associated with overall survival.These genes have potential predictive prognostic value and are also potential drug therapeutic targets for bladder cancer.The enrichment analysis of oncogenes showed that the up-regulated oncogenes in all tumor cell subtypes were mainly enriched in KRAS pathway,supporting the malignant status of these tumor cell subtypes.In addition,MIBC tumor cells are enriched in the pathway related to interferon response,while NMIBC tumor cells are just the opposite.We also found that NMIBC tumor cells are closely related to angiogenesis and androgen action,and MIBC tumor cells are closely related to hypoxia.Metalloproteinase inhibitor related genes were highly expressed in NMIBC.Compared with MIBC tumor cells,the expression of MHC-I related genes in NMIBC tumor cells was significantly decreased.Conclusions:Urothelial cells can be transformed into different tumor cells,and there is a certain transformation relationship between tumor cells.Tumor cells of NMIBC and MIBC have different biological characteristics.In addition to differences in gene expression,tumor cells of NMIBC are closely related to angiogenesis and androgen.The tumor cells of MIBC are closely related to hypoxia.Metalloproteinase-inhibitor-related genes are highly expressed in NMIBC,which may be related to the fact that tumor cells in NMIBC are not easy to infiltrate into stroma and muscle.The expression of MHC-I related genes in NMIBC tumor cells was significantly decreased,which may be related to the immune escape mechanism of the tumor cells.Part 2:The Different Roles of Structural Cells in Shaping NMIBC and MIBC Tumor MicroenvironmentBackground and objective:Bladder cancer is not only composed of malignant cells,but also a variety of immune cells,structural cells(epithelial cells,cancer-associated fibroblasts(CAF),vascular endothelial cells,lymphatic endothelial cells and pericytes)and extracellular components.All these cells and extracelluar components create the the tumor microenvironment(TME)and affect a series of biological behaviors of tumor cells such as proliferation,progression,invasiveness as well as drug resistance.The TME is constantly changing and remodeling dynamically.The composition of the TME also differs in different stages of the disease and among different tumor types.Therefore,in this part,we will systematically study the TME of NMIBC and MIBC and compare the differences between them by performing scRNA-Seq technology,so as to comprehensively understand the role of various structural cellular and extracellular components in creating different TME between NMIBC and MIBC.There might be differences in TME between NMIBC and MIBC,howerver,so far few studies have been carried out on this field,especially on structural cells in TME.Methods:Clustering and differential gene expression analysis of the fibroblasts,endothelial cells and pericytes were performed by using Seurat R package"FindAllMarkers" function.RNA velocity analysis was conducted by using the velocyto.R program.Gene regulatory networks analysis was carried out with SCENIC package.Survival prognosis analysis and finding potential of targeted drugs was performed by searching GEPIA and DrugBank database.Immunofluorescence(IF)technique was used to analyze the distribution of iCAF,myCAF and myofibroblasts in bladder cancer tissues.Results:There are five functional fibroblast subtypes present in bladder cancer:normal fibroblasts,iCAF,myofibroblasts,myCAF and antigen-presenting CAF(apCAF).Different CAF subtypes have different functions,such as different types of collagen secreted by different CAF subtypes.The COL4A1 and COL14A1 were highly expressed in iCAF,while the COL5A1,COL5A2,COL8A1 and COL6A2 were highly in myCAF.In addition,iCAF cells highly expressed CXCL14,CXCL12,and CXCL2.Hyaluronic acid synthase was highly expressed in iCAF derived from MIBC.The myCAF-C1-MMP11 from MIBC highly expressed MMP2,MMP11 and MMP14.Our data also showed increased expression of VEGFA and VEGFB genes in MIBC-derived CAFs,and high expression of HIF1A in MIBC-derived CAFs.The myofibroblast can be converted into iCAF,and myCAF can be converted into myofibroblast and iCAF,and apCAF can be converted into iCAF.Myofibroblast can also be converted into vascular endothelial cells.Pericytes can transform into endothelial cells.Endothelial cells can be divided into 5 cell subtypes.Bec-c3-CXCL12 and Bec-c4-CXCL2,which are mainly distributed in normal tissues,might have the function of recruiting immune cells.Bec-c2-ESM1,which is mainly distributed in tumor tissues,is closely related to the formation of tumor angiogenesis.The transcription factor HOXD10 regulates the process of tumor angiogenesis of Bec-c2-ESM1 by acting on target genes(FAM84A,HOXD9,HOXD10,SMOX,SLC39A8,HOXD8 and AXIN2).Hypoxia-related genes and angiogenesis related genes were highly expressed in MIBC-derived endothelial cells and pericytes.Conclusions:NMIBC and MIBC have different tumor microenvironment,which is reflected in different stages of tumor microenvironment remodeling,including the synthesis of extracellular matrix,destruction of extracellular matrix,formation of new blood vessels and cell recruitment.Different structural cells play different roles in shaping different tumor microenvironments,among which iCAF,myCAF,and tumor neovascularization endothelial cells play key roles in shaping the tumor microenvironment.ICAF may be related to the recruitment of immune cells and the synthesis of hyaluronic acid,while myCAF may be related to the destruction of basement membrane and extracellular matrix.The neovascular endothelial cells are related to tumor neovasculation,and Bec-c3-CXCL12 and Bec-c4-CXCL2 may have the role of recruiting immune cells.The tumor microenvironment remodeling activity of MIBC is more active and closely related to hypoxia factors.The tumor microenvironment is a process of constant remodeling and there is a certain transformation relationship between structural cells.Part 3:Analysis of the Interaction Between Tumor Cells and Structural Cells in Bladder CancerBackground and objective:Analysis of cell-to-cell interactions is helpful to understand complex relationships between cells in TME.Recently,the interaction between tumor cells and various structural cells in bladder cancer is still obscure,and whether these interactions are also different between NMIBC and MIBC needs further study.In this section,we will compare the interactions between tumor cells and structural cells in NMIBC and MIBC,with the aim of building a network of interactions between structural cells in the two types of bladder cancer respectively.In addition,we aim to further explore the role of these interactions in the progression and clinical application of bladder cancer.Methods:Known pairs of ligands and receptors were listed as described and ligand-receptor(L-R)interaction scores were calculated according to a previously reported method.L-R pairs with an interaction score of greater than 1 were selected.A circle plot for cell clusters with corresponding L-R pairs was generated with the R package circlize.Results:CAF plays an important role in the tumor microenvironment of both NMIBC and MIBC.The ligands of CAF in both types of bladder cancer predominantly interact with the receptors of tumor cells,endothelial cells and pericytes.In addition,there are some differences in the cell interaction between NMIBC and MIBC.In MIBC,CAF interacts with ITGB1 of tumor cells mainly through a variety of collagens,in addition,CAF interacts with tumors through APP_CD74 and TIMP1_CD63.CAF interacts with tumor cells in NMIBC mainly through C3_CD81 and TIMP1_CD63.In MIBC,CAF interacts with endothelial cell receptor ITGA6 mainly through ligand COL6A1,THBS1,THBS2 and FN1.In addition to the interaction between CAF and endothelial cells in NMIBC through COL6A1_ITGA6 and FN1_ITGA6,CAF also interacts with the endothelial cell receptor CAV1 through ligand APP and CYR61.CAF in MIBC interacts with pericyte receptor CD44 through ligand COL1A1,COL14A1,COL1A2,FN1,PKM,VCAN and VIM,and also interacts with pericyte receptor ITGA1 through collagens.CAF in NMIBC not only interacts with pericyte receptor ITGA1 through collagens,but also interacts with pericyte receptor SDC2 through ligand MMP2 and FN1.In MIBC,endothelial cells mainly interact with tumor cell receptor ITGB1 through ligand ADAM 15,COL4A1,HSPG2 and TGM2.In NMIBC,endothelial cells mainly interact with tumor cell receptor SDC1 through ligand HSPG2.Endothelial cells in both NMIBC and MIBC interact with pericyte receptor ITGA1 via ligand COL4A1.In MIBC,pericyte mainly interact with tumor cell receptor ITGB1 through ligands COL18A1 and LAMA4.In NMIBC,there was no significant interaction between pericyts and tumor cells.Conclusions:There are different interaction relationships between NMIBC and MIBC cells.CAF in tumor microenvironment plays an important role in bridging these complex interactions.Targeting CAF therapy will be a great application prospect for the treatment of bladder cancer.