Abstract: Objective  To investigate the effect of single-cell mass cytometry（CyTOF） analysis on the characteristics of infiltrating immune cell subsets in bladder cancer tissues and paracancerous tissues，providing a new strategy for the study of bladder cancer heterogeneity. Methods The cancer tissues and paracancerous tissues of 4 bladder cancer patients were collected and prepared into single-cell suspensions by enzymatic hydrolysis and mechanical dissociation. CyTOF was used to detect the expression of 26 immune-related markers in tumor-infiltrating immune cells. Results The bladder CD45⁺ immune cells were divided into 25 subgroups （7 CD4⁺ T cell clusters，8 CD8⁺T cell clusters，4 macrophage clusters，2 B cell clusters，1 dendritic cell cluster，and 3 other types immune cell clusters） by cell similarity analysis. The contents of infiltrating immune cell subpopulations in bladder cancer tissues and paracancerous tissues were different. T cells were more abundant in bladder cancer tissues at 76.73%，whereas B cells and macrophages were more abundant in paracancerous tissues，accounting for 15.20% and 23.82%，respectively. The average proportion of CD4⁺ Treg cells in bladder cancer tissues was higher than that in paracancerous tissues（2.20% vs 0.81%，P=0.016）. Conclusions CyTOF can effectively distinguish the immune cell subsets in the bladder tumor microenvironment and detect tumor heterogeneity，providing a basis for the accurate treatment of bladder cancer.

Key words: Bladder cancer, Single-cell mass cytometry, Tumor microenvironment, Immune characteristics