Single-cell RNA sequencing analysis reveals the dynamic changes in the tumor microenvironment during NMIBC recurrence.

Background: Due to the clinical characteristic of frequent recurrence in urothelial bladder cancer (UBC), patients face significant health impacts and economic burdens. Therefore, understanding the molecular mechanisms involved in UBC recurrence is crucial for reducing its recurrence rate. The aim of our study is to help urologists and clinical researchers gain a deeper understanding of the changes in the tumor microenvironment (TME) during UBC recurrence.

Methods: We collected 10 samples from primary and recurrent non-muscle-invasive bladder cancer (NMIBC) and performed single-cell RNA sequencing. By distinguishing and annotating cell subpopulations, we identified tissue preferences of some novel cell subgroups. Next, pseudotime trajectory analysis, cell-cell communication analysis, and function enrichment analysis were applied to evaluate the dynamic changes in the TME and biological functions. Finally, we validated the distribution of some of these cell subgroups using multiplex immunofluorescence experiments.

Results: We identified a tumor-associated fibroblast (CAF) subtype with high COL18A1 expression that is highly expressed in recurrent NMIBC, suggesting that the stromal component of the tumor may play a crucial role in the recurrence process. Additionally, pseudotime trajectory analysis revealed a macrophage subtype with high IL-6 expression at the terminal stage of macrophage differentiation, exhibiting significant immunosuppressive features. This indicated the presence of immune exhaustion during NMIBC recurrence. Lastly, we found an upregulation of estrogen in recurrent urothelial cancer cells, which may partially explain the gender disparity observed in UBC.

Conclusion: This study identified several cell subpopulations influencing NMIBC recurrence, which were heavily infiltrated in the TME of recurrent NMIBC. Additionally, the enrichment of estrogen in urothelial cancer cells from various sources suggested a role of sex hormones in NMIBC recurrence.