| Objective: Bladder cancer(BC)is one of the most common malignant tumors of the genitourinary system.,with the high mortality,recurrence rate and treatment costs.Although substantial progress has been made in the treatment of Immune checkpoint inhibitors(ICIs),the clinical efficacy of bladder cancer patients was still unsatisfactory.Tumor microenvironment(TME)is a potential target.Therefore,this study aims to further explore the composition of infiltrated cells in the tumor microenvironment of bladder cancer and the molecular characteristics of different phenotypes,and identify potentially predictive biomarkers through single cell RNA sequencing analysis of BC.Methods:1.The gene expression profiles of bladder cancer and adjacent tissues in 3 cases of NMIBC and 4 cases of MIBC were detected by 10 X single cell transcriptome sequencing.2.Single cell clustering and subgroup identification for high-quality single cells captured based on Singer and selected marker genes with significant differences.3.Screening out myeloid cells,re-cluster and subdivide subgroups of myeloid cells,and compare the difference in quantity and proportion between different myeloid cell subgroups between MIBC and NMIBC,as well as between BC and NC.4.GO annotation,KEGG pathway enrichment,and GSEA enrichment analysis were performed on differentially expressed genes in the TAM-C1 QC subgroup.5.Further subdividing the tumor epithelial cells in the BC group into different subgroups,and performing hierarchical clustering based on marker substrates,luminal markers,squamous cell surface markers,in situ cancer related expression patterns,Claudin expression patterns,cell cycle characteristics,EMT,immune function markers,neuronal differentiation signaling proteins,and P53 signaling pathway related markers.6.CNV analysis of tumor epithelial cells in BC was performed with endothelial cell colonies as the control group.7.GO annotation and KEGG pathway enrichment analysis were performed on differentially expressed genes of tumor epithelial cell subgroups in BC group.WGCNA and PPI network analysis were used to compare the differences between MIBC and NMIBC groups,in which 6 prognostic genes related to the progression and transformation of bladder cancer were screened.Kaplan-Meier survival prognosis analysis was performed on the screened genes based on GEPIA2 platform,The dynamic changes of 6 genes in the progression of bladder cancer were simulated by Pseudo-timing analysis.Results:1.The tumor microenvironment of bladder cancer consists of 8 basic cell types: T cells,myeloid cells,epithelial cells,B cells,fibroblasts,NK cells,endothelial cells and mast cells.2.Myeloid cells can be re-clustered and subdivided into 9 subgroups.Most monocytes come from normal mucosal tissue,while TAMS is mainly enriched in BC tissue.LAMP3+DC expresses various genes encoding various chemokines and has a strong chemotactic effect on Tregs.Tumor associated macrophages(TAM-C1QC)mainly play an immunosuppressive role in the TME.Pathway enrichment analysis showed that the differential genes in TAM-C1 QC subgroup were significantly enriched in Th17 cell differentiation and IL-17 signaling pathways.The Proliferating TAMs subtype is associated with cell proliferation,differentiation,and migration.3.Based on the expression of marker genes in basal/luminal bladder cancer epithelial cells,14 tumor epithelial cell subtypes can be further clustered into 4 expression subtypes: luminal-papillary,luminal-infiltrated,luminal,and basal-squamous.The luminal subtype expresses high levels of urinary tract proteins and genes related to umbrella cells,with high cell cycle characteristic scores,high proliferative ability,strong invasiveness,and a certain level of neuronal cell secretion characteristics,as well as high expression of claudins genes.The luminal-papillary type is a subgroup with papillary morphological characteristics,which is rich in FGFR3 mutations,amplification,and overexpression,with lower levels of in situ carcinoma(CIS)gene expression characteristics,and constitutes the main epithelial cell component of the NMIBC group.The basal-squamous subgroup highly expresses basal layer keratin marker genes,which have the characteristics of squamous epithelial cell differentiation and also exhibit strong expression of CIS signaling genes.The proportion of luminal-infiltrated type has the lowest proportion of tumor epithelial cells,with high expression of EMT related markers,which is related to poor prognosis.4.Compared to the NMIBC group,the MIBC group has a higher incidence of CNV events and exhibits high heterogeneity among different patients,exhibiting individual specific CNV expression patterns.5.Compared with the NMIBC group,the genes ANXA1,HSP90B1,PSMA7,PRDX6,and PPP1 CB were highly expressed in the MIBC group,while the VEGFA gene was low expressed in the MIBC group.Pseudo-timing analysis showed that the expression of ANXA1,HSP90B1,PSMA7,PRDX6,and PPP1 CB genes gradually increased with the progress of the clinical stage in the simulated time trajectory,while the expression of VEGFA gene gradually decreased.Conclusions:1.The composition of tumor infiltrated cells shows high heterogeneity in different bladder cancer.2.Tumor associated macrophages(TAM-C1QC)tend to M2 polarization in the tumor microenvironment,which mainly plays the role of immunosuppression.And their immunosuppressive function is related to Th17 cell differentiation and IL-17 signaling pathway.3.4 different subtypes of tumor epithelial cells in BC exhibit different gene expression patterns.4.High expression of ANXA1,HSP90B1,PSMA7,PRDX6,PPP1 CB genes is associated with poor prognosis of bladder cancer.Simultaneously overexpressing ANXA1,HSP90B1,PSMA7,PRDX6,and PPP1 CB genes may promote the progression of NMIBC towards MIBC,while overexpression of VEGFA genes may inhibit this progression.MIBC,while overexpression of VEGFA genes may inhibit the progression of NMIBC to MIBC. |
