INTRODUCTION AND OBJECTIVE: Tumor infiltrating B cells in bladder cancer (BCa) have been associated with cancer invasion, but little is understood regarding their role in the tumor microenvironment. In this study, we analyzed single cell sequencing data from tumor (n[19) and PBMC (n[20) samples from 26 BCa patients to profile the heterogeneity of B cells and plasma cells (PCs).METHODS: Single cell data was imported into R (v4.03) using the SingleCellExperiment package, and outliers in mitochondrial gene percentage, library size, or detected genes were discarded. Normalization, deconvolution, and library size adjustment were done using the scran and batchelor R packages. Trajectory analysis was done using the Monocle R package.RESULTS: We identified 3,158 CD45þ naïve, non-switched memory (NSM), and switched memory (SM) B cells and PCs according to their marker gene expression (Fig 1). The majority of naïve and memory B cells were from PBMC, whereas plasma cells were primarily from tumor. To understand the heterogeneity of PCs, we performed clustering and trajectory analysis on 1,411 PCs and identified eight clusters (Fig 2A). Though most clusters consisted mainly of tumor cells, clusters 7 is almost exclusively from PBMC and is at the beginning of the pseudotime trajectory (Fig 2A). Its lower expression of PC markers such as MZB1, XBP1, and immunoglobulin genes suggest that it's at an earlier stage of PC maturation (Fig 2A). Clusters 1, 6, and 8 contain PCs likely representative of mature plasmablasts, as they are at the end of the pseudotime trajectory and express high levels PC markers and immunoglobulin genes (Fig 2A). Lastly, cluster 4 upregulates expression of HLA-DP, HLA-DQ, and HLA-DR genes further on the pseudotime trajectory (Fig 2B).CONCLUSIONS: From our survey of B cells and PCs from a large single cell BCa cohort, we observed the majority of PCs to be found in the tumor. Tumor PCs demonstrate a high degree of heterogeneity, encompassing at least an early stage subset, a more mature plasmablast-like subset, and another subset with upregulated HLA expression with potential for T-cell stimulatory function.