| Background and purpose: Upper tract urothelial carcinoma(UTUC),including renal pelvis and ureter tumors,has the characteristics of insidious onset,rapid clinical progress,high recurrence rate and poor survival prognosis.Because of the low incidence of UTUC,the same clinical feature and histologic appearance with bladder cancer,the diagnosis and treatment of UTUC are almost referring to the bladder cancer.However,a growing number of evidences suggest that UTUC is a unique disease entity and should be differentiated from bladder cancer.With the rapid development of medical technology,the early detection rate of UTUC has been greatly increased,and surgical operation techniques have also improved from traditional open surgery to laparoscopic and even robotic surgery or intravitary minimally invasive surgery.At the same time,as the conception of precise treatment,new tumor molecular typing,including urothelial carcinoma,can not only provide individualized treatment plan for patients,but also accurately evaluate the prognosis.Nevertheless,the therapeutic effect of urothelial carcinoma has not been substantially improved,such as high recurrence rate of patients after surgery,the limited treatment methods for advanced patients,low overall response rate of platinum-based chemotherapy and immunotherapy,and short effective duration of treatment.Therefore,it is urgent to find a new breakthrough point for the treatment of UTUC.The development of Next Generation High-throughput Sequencing Technology has made it possible to analyze the whole transcriptome and genome of a disease.But there also has certain limitation,such as submerged information of a small number of cells with important function,because this method reflect the average expression level of all cells,and cannot accurately locate specific cell groups,The emergence of single-cell RNA sequencing(sc RNA-seq)technology makes up for the deficiency,which turns the traditional batch histology study to the single cell level.By sc RNA-seq analysis,not only the unique changes of each cell can be detected,but also new cell types and new marker genes of cell types can be found.The cell types in complex tissues can be accurately identified,and the cells with the same functional status can be clustered with a single cell as a unit,so as to conduct more accurate molecular mechanism research at the singlecell level.In this study,the advantages of sc RNA-seq technology were utilized to map the landscape of ureteral cancer cells and conduct in-depth analysis of the functions of each cell group.At the same time,by comparing the transcriptome sequencing data of bladder cancer,the causes of obvious malignant phenotype and poor prognosis of ureteral cancer were found,and further provide single-cell basis and potential new therapeutic targets for the treatment of ureteral cancer.Methods: Three ureteral carcinoma cases were enzymatically digested into single cell suspension.The cell suspension was prepared into a single cell Gel Beads-in-emulsion structure by 10 X Genomics equipment,and the c DNA library was made and sequenced.A preliminary analysis of the sequencing data was performed using the Cellranger software provided by 10 X Genomics,and also single cell correlation analysis software,such as R package Seurat and Monocle2,was used for quality control and in-depth analysis of human ureteral cancer s RNA-Seq data.After preliminary analysis of the sequencing data,the Harmony package was used to integrate the data of ureteral cancer and bladder cancer.Unbiased cluster analysis of combined data was performed using the Find Clusters and Find All Marker functions to calculate the differentially expressed genes(DEGs)between different types of cells.The expression level of PPP4 C in ureteral cancer tissues and normal ureteral tissues was verified by q PCR,Western Blot and immunohistochemical staining.Recombinant plasmid of PPP4 C was constructed and transfected into normal urothelial cells.The biological effects of overexpression of PPP4 C gene on the normal urothelial cells were observed by cell proliferation analysis,clone formation and cell migration assay.Three bladder cancer cases were also enzymatically digested into single cell suspension,10 X Genomics equipment and c DNA library were constructed for sequencing.After preliminary analysis of the sequencing data,the Harmony package was used to integrate the data of ureteral cancer and bladder cancer.Unbiased cluster analysis of combined human ureteral and bladder cancer cells was performed using the find Clusters function.At the same time,the Find All Marker function was used to calculate the differentially expressed genes(DEGs)between different types of cells.Results: The landscape of ureteral cancer cell atlas was described.Through principal component analysis and unbiased clustering analysis,the human ureteral cancer cell map was divided into 17 groups of different cell types.In 7 different tumor cell subpopulations,the enrichment pathways of highly expressed genes in each subpopulation were different.The development trajectory of human ureteral cancer cells was reconstructed by pseudotime analysis and the key genes involved in the differentiation and development of ureteral cancer were identified.Tumor associated fibroblasts in ureteral cancer expressed high levels of ACTA2 and TAGLN,as well as PDGFRB and TGF-β.While it has been verified that ureteral cancer has high FGFR expression,it also found that there is a high expression level of PPP4 C in ureteral cancer,especially the significantly increased expression in tumor nucleus.The overexpression of PPP4 C in normal urothelial cells could promote the proliferation and migration of urothelial cells.We successfully captured 46,180 high-quality single-cell transcriptome data,including ureteral and bladder cancer samples,to create a comprehensive landscape of urothelial cancer cells.The human urothelial carcinoma cell map was divided into 27 groups of different cell types by principal component analysis and unbiased clustering analysis.By comparison of ureteral cancer and bladder cancer samples,we found several distinct features.Firstly,in the ureteral cancer and bladder cancer tumor microenvironment,the composition of cell subtypes is different.Cell mapping of ureteral carcinoma is given priority to tumor cells,accounted for 81.3%,with less stromal cells and immune cells,and in bladder cancer cell mapping immune cells accounted for the vast majority,with only 22.8%tumor cells.Secondly,the composition ratio of tumor cells in ureteral cancer and bladder cancer is different.In particular,the 10 th group of tumor cells which has active proliferation,obvious malignant phenotype and high expression of UBE2 C gene is mainly from ureteral cancer,very little in bladder cancer.Thirdly,although the proportion of umbrella cells in ureteral cancer and bladder cancer was similar(3.8% and 4.0%,respectively),the 12 th group of umbrella cells were dominant in ureteral cancer,and the 16 th group of umbrella cells were in bladder cancer.Finally,the proportion of fibroblasts in bladder cancer was significantly higher than ureteral cancer.Conclusion: For the first time,we comprehensively and systematically delineated the landscape of ureteral cancer cell atlas and conducted in-depth analysis on the functions of each cell group,which provide single-cell reference for further research on ureteral cancer.PPP4 C is potential to be a new promising target in the treatment of urothelial carcinoma.There are some similarities and differences between ureteral cancer and bladder cancer.The ureteral cancer cells mainly compose of tumor cells,especially a group of tumor cells with active proliferation and obvious malignant phenotype,but less immune cells.Our study provides new evidence and clues for the precise treatment of urothelial carcinoma. |
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