Tumor-derived extracellular vesicles (TEVs) are membrane-bound, nanosized vesicles released by cancer cells and taken up by cells in the tumor microenvironment to modulate the molecular makeup and behavior of recipient cells. In this report, we summarize the pivotal roles of TEVs involved in bladder cancer (BC) development, progression and treatment resistance through transferring their bioactive cargos, including proteins and nucleic acids. We also report on the molecular profiling of TEV cargos derived from urine and blood of BC patients as non-invasive disease biomarkers. The current hurdles in EV research and plausible solutions are discussed.

Tumor-derived extracellular vesicles (TEVs) play crucial roles in mediating immune responses, as they carry and present functional MHC-peptide complexes that enable them to modulate antigen-specific CD8+ T-cell responses. However, the therapeutic potential and immunogenicity of TEV-based therapies against bladder cancer (BC) have not yet been tested. Here, we demonstrated that priming with immunogenic Extracellular Vesicles (EVs) derived from murine MB49 BC cells was sufficient to prevent MB49 tumor growth in mice. Importantly, antibody-mediated CD8+ T-cell depletion diminished the protective effect of MB49 EVs, suggesting that MB49 EVs elicit cytotoxic CD8+ T-cell-mediated protection against MB49 tumor growth. Such antitumor activity may be augmented by TEV-enhanced immune cell infiltration into the tumors. Interestingly, MB49 EV priming was unable to completely prevent, but significantly delayed, unrelated syngeneic murine colon MC-38 tumor growth. Cytokine array analyses revealed that MB49 EVs were enriched with pro-inflammatory factors that might contribute to increasing tumor-infiltrating immune cells in EV-primed MC-38 tumors. These results support the potential application of TEVs in personalized medicine, and open new avenues for the development of adjuvant therapies based on patient-derived EVs aimed at preventing disease progression.

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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.

Baseline titers were not associated with response. On-treatment titers >800 were seen in 89% and 59% of responders (R) and nonresponders (NR), respectively (p[0.001). Sensitivity of this test for detecting R was 89% (negative predictive value, 78%). Ontreatment Ab fold change >8 from baseline was seen in 51% and 27% of R and NR, respectively (p[0.02). Elevated on-treatment Ab titers and fold change from baseline were noted in 47% and 18% of R and NR, respectively (p[0.004). Specificity of the combined test for detecting R was 82% (positive predictive value, 73%). These findings were also corroborated in the CISAETa/T1 subcohort.CONCLUSIONS: Secondary analysis of the prospective, multicenter phase 3 nadofaragene firadenovec trial in BCGunresponsive NMIBC indicates a role for assaying baseline and ontreatment Ab titers. A combination of Ab titer and fold-change levels can potentially predict response to this novel therapeutic in a patient population with urgent unmet clinical need.