Study On Cell Populations And Molecular Characteristics Of Metastatic Bladder Cancer By Single-Cell RNA Sequencing

Objective:Bladder cancer(BC)is the tenth most common tumor in the world and the ninth leading cause of cancer death.There are more than 570000 new cases and 210000 new deaths every year.The most common histological type of BC is urothelial carcinoma(UC),accounting for 90% of all BC.According to the depth of tumor invasion,it can be divided into non-muscle invasive bladder cancer(NMIBC)and muscle invasive bladder cancer(MIBC).NMIBC accounts for about 75% of BC.It’s survival rate of patients is high,but it is very easy to recur,requiring lifelong follow-up and surgical treatment.MIBC accounts for about 25% of BC.The tumor progresses rapidly,and it has a high degree of malignancy,and it is prone to early metastasis.It is also called metastatic BC,and has a high recurrence rate and mortality.Therefore,this study will discover the cell population and genome information of metastatic BC,providing a theoretical basis for accurate diagnosis and treatment of metastatic BC.Methods:Samples were obtained from three patients diagnosed as MIBC by medical imaging and histopathological examination,without preoperative chemotherapy and radiotherapy.All samples were collected from primary tumor(PT),paracancerous normal tissue(PNT),and lymph node metastasis(LNM)of each patient immediately after radical cystectomy and pelvic lymph node dissection.To study the mutant genes and cell types among the tumor cells and tumor microenvironment(TME),we performed single cell RNA sequencing(sc RNA-seq)on these samples,and then identified the total cell population and its subpopulations based on the original data.Through the analysis of infer CNV,cell trajectory,GSVA and cell communication,we studied the similarities and differences between primary and metastatic tumors in cell population and molecular characteristics.Finally,through TCGA database,we evaluated the prognostic index of the specific genes,and through immunohistochemistry to retrospectively study gene expression in 24 patients with MIBC and lymph node metastasis for clinical validation.Results:1.A total of 50158 effective cells were detected in all samples,and six main cell populations were identified,namely,epithelial cells,endothelial cells,fibroblasts,macrophages,B lymphocytes,and T lymphocytes.Epithelial cells,fibroblasts,macrophages,and T lymphocytes account for the majority.In PT,there are mainly epithelial cells,macrophages,and T lymphocytes.The main components of PNT are fibroblasts,macrophages,and T lymphocytes.The main components of LNM are macrophages and T lymphocytes.2.Next,we divided these cell populations into subgroups,including malignant epithelial cells,CD4+ regulatory T cells(Treg),CD8+ exhausted T cells(Tex),CD8+resident memory T cells(Trm)tumor associated macrophages(TAM)and tumor associated Myofibroblasts(Myo-CAF)are the main types of macrophages.These cell subsets overexpress genes related to the promotion of tumors such as SLITRK6,WNT5 A,LYVE1,and IDO1.3.PNT is mainly composed of inflammatory CAF(i CAF),TAM,CD4+ helper T cells(Th),and CD8+ cytotoxic T cells(CD8cyto).These cell subsets overexpress anti-tumor immune response genes(GZMA,IFNG)associated with Th and CD8 cyto,as well as tumor progression promoting genes(SFRP4,LYVE1 and IDO1)associated with i CAF and TAM.Stain images shows that TAM was present in the region adjacent to tumor cells in PNT.4.The cell subpopulation characteristics of LNM are the same with PT,but they do not share the same malignant epithelial cell subpopulations.Through cell trajectory analysis,PT and LNM differentiated into two different subgroup branches.The subgroups in PT are mainly characterized by high expression of SLITRK6,while the subgroups in LNM are mainly characterized by high expression of LMO3.Through the TCGA database,it was found that higher expression of LMO3 was associated with a poorer prognosis compared to SLITRK6,suggesting a higher degree of malignancy in metastatic cancer cells.Stain images shows that there were different tumor cell subsets between PT and LNM.5.In addition to malignant epithelial cells,the inhibitory immune cell populations(Treg,Tex and Trm)are also an important feature of PT and LNM.Cell communication analysis shows that i CAF and TAM are the centers of signal pathways that activate Tex.And there is a close signal relationship between these two.Immunohistochemical images shows high expression of LYVE1 and IDO1 in normal mesenchymal cells located near tumor cells,suggesting that these normal mesenchymal cells are likely to be i CAF and participate in mediating the accumulation of TAM into the tumor microenvironment(TME).Therefore,i CAF and TAM play an important role in the occurrence and development of tumors.6.Cell communication analysis shows that CAF involves almost all cell subpopulations in signal pathways,and is a major signal transmitter involved in promoting tumor cell proliferation and inhibiting immune cell formation.Where i CAF and TAM are the signal path centers that activate Tex.Both transmit signals directly to CD8 cyto or indirectly to CD8 cyto by activating downstream Treg,making it a depleted phenotype.This analysis illustrates the regulatory mechanism for the differential distribution of cell subpopulations in the above three groups.Conclusion:This study used the sc RNA-seq to analyze the cell population and molecular expression characteristics of matched PNT,PT,and LNM samples from patients with metastatic BC for the first time.Mainly including:1.The common feature of PT and LNM is the presence of tumor cells and inhibitory immune cells(Treg,Tex,and Trm).2.The difference between PT and LNM is that their tumor cells have different cell subsets.Compared to the subset with high expression of SLITRK6 in PT,the subset with high expression of LMO3 in LNM may be associated with higher malignancy and poorer prognosis.3.The common feature of PNT,PT,and LNM is the presence of TAM that expresses high levels of LYVE1 and IDO1,and their expression in PNT may be related to increasing the probability of tumor recurrence.The common and differential characteristics of three tissue derived samples have been revealed from the cell population and genome levels,providing new insights into the bioinformatics research and future treatment methods of metastatic BC.