Dissecting NK Cells And Dendritic Cells Heterogeneity In The Bladder Tumor Microenvironment At The Single-Cell Level

Objective:Bladder cancer is a common tumor of urinary system with the characteristics of easy recurrence and poor prognosis.The tumor microenvironment is widely heterogeneous and plays a key role in tumor progression and therapeutic response.In order to investigate the characteristics of natural killer（NK）cells and dendritic cells（DC）in the immune microenvironment of bladder cancer,and to understand the heterogeneity of natural killer cells and dendritic cells and the interactions between these two types of cells in the tumor microenvironment,so as to provide a basis for improving the accuracy and efficacy of immunotherapy for bladder cancer.We used single cell RNA sequencing to analyze the heterogeneity of NK cells and DC in bladder tumor microenvironment at the single-cell level.Methods:Bladder cancer tissues,adjacent normal tissues,pelvic lymph nodes,and preoperative peripheral blood were collected from 12 patients diagnosed with bladder cancer.Single cells were isolated from the four tissue sites,single cell suspensions were prepared for single cell RNA sequencing,and NK cells and DCs were extracted after defining NK/T and dendritic cells from the total single cell transcriptome data.The extracted NK cells and DCs were then re-clustered separately,and each cell subpopulation was annotated and named according to the characteristic genes,then further bioinformatics analysis of each subpopulation,including gene function enrichment,transcription factor regulation,cell differentiation trajectory and RNA rate analysis.Differential genes between NK cells and DCs in tumor and normal tissues adjacent to cancer were compared separately.Then the Cell Chat package was used to explore the interaction between NK cells and DCs in the bladder tumor microenvironment.Finally,immunohistochemical experiments were used to verify the expression of signature genes（XCL1 and NCAM1）in bladder cancer tissues.Results:We studied 13,584 NK cells and 3,995 DCs from single-cell RNA sequencing data.NK cells were divided into six groups of NK cells and one group of ILC3s,and DCs were subdivided into five groups.（1）NK cells and DCs in tumor tissues differed significantly from those in normal tissues.NK cells in tumors expressed higher expression of inhibitory molecules and lower expression of cytotoxicity-related genes than those in normal tissues,suggesting that NK cells in tumors were mostly in a functionally inhibited state.（2）The pseudotime and RNA velocity analysis demonstrated the differentiation of CD56^brightNK cells to CD56^dimNK cells.（3）The proportion of DC4-LAMP3cells in bladder cancer tissues were significantly higher than that in normal tissue adjacent to cancer（p<0.05）.It belongs to mature DC and highly expresses PD-L1,which is involved in regulating T cell activation and IFN-γresponse.（4）Two groups of c DC2s were identified,in which DC3-FCGR3A resembled inflammatory DCs and highly expressed chemokines that chemotactic neutrophils,suggesting the ability to chemotactic neutrophils to the site of inflammation to mediate the inflammatory response.（5）NK4-XCL1(CD56^brightNK),which is more predominant in tumor tissues,exhibits the characteristic of tissue-resident NK cells and with high expression of XCL1 and chemokine CCL5,while DC5-CLEC9A（c DC1）has high expression of XCR1.NK4-XCL1and DC5-CLEC9A were found to have a strong interaction in the XCL pathway interaction.Conclusion:We took advantage of single-cell RNA sequencing technology to reveal the heterogeneity and differentiation trajectory of NK cells and dendritic cells in the tumor microenvironment of bladder cancer at an unprecedented resolution level.We not only made fine subpopulation of NK cells and dendritic cells,identified CD56^brightNK cells may be the tissue resident NK cells in bladder tissues,confirmed the direction of differentiation among NK cell subpopulations,but also discovered the inhibitory functional state of NK cells in tumors.LAMP3⁺DCs,which were significantly represented in bladder cancer tissues,were also found to express PD-L1 and could directly suppress CD8⁺T cells or form an immunosuppressive environment by recruiting Treg into the tumor region.What’s more,the interaction between NK cells and dendritic cells in the bladder tumor microenvironment was found,where NK cells mediated adaptive immune responses by recruiting dendritic cells through ligand receptors and secreted cytokines.Characteristic genes associated with survival prognosis of bladder tumors were also identified.These findings provide the basis for immunotherapy of bladder cancer.