Abstract: Objective To investigate the effect of CD45+CD326+ double positive cells (DPCs) on the prognosis of bladder cancer (BLCA), to construct a scoring system for bladder cancer DPCs⁃related genes, and to explore the characteristics of tumor microenvironment in bladder cancer patients with different DPCs scores. Methods  Single⁃cell sequencing technology was used to analyze the molecular characteristics of DPCs, and mass cytometry was used to analyze the heterogeneity of the tumor microenvironment in patients with high and low proportions of DPCs. Based on the TCGA⁃BLCA cohort, a scoring system was constructed according to the characteristic genes of DPCs and validated in the GSE13507 cohort and the GSE32894 cohort. Results  The prognosis of bladder cancer patients in the high DPCs proportion group was worse than that of patients in the low DPCs proportion group, the tumor microenvironment of patients in the high proportion and low proportion group was heterogeneous, and the immune cells and tumor cells of patients in the low DPCs proportion group had higher expression levels of immune checkpoints, co⁃inhibitors and coactivators (all P<0.05). A scoring system for bladder cancer with 9 DPCS⁃related genes was constructed based on TCGA⁃BLCA cohort (APOBEC3G, CD96, CLEC2D, GNG2, GNLY, IL32, PSMB9, RORA, SKAP1). The results of TCGA⁃BLCA cohort, GSE13507 cohort and GSE32894 cohort showed that the overall survival of bladder cancer patients in the high DPCs score group was significantly shorter than that of patients in the low DPCs score group (all P<0.01), and the signaling pathways such as immunosuppression and estrogen were significantly enriched in the high⁃risk score group. Conclusions  DPCs are closely related to the poor prognosis of bladder cancer, and the model based on the characteristic gene of DPCs can accurately predict the clinical prognosis of bladder cancer patients.

Key words: Bladder Cancer, Tumor microenvironment, Mass cytometry, Single?cell sequencing technology, Immunosuppression