Immune profiling at the single-cell level to evaluate the immune microenvironment in bladder cancer: Insights from non-muscle invasive and muscle-invasive disease.

842 Background: Advanced Bladder Cancer (BC) presents significant challenges due to its high incidence and limited treatment options. Most patients initially present with non-muscle invasive bladder cancer (NMIBC), managed with transurethral resection (TURBT) and Bacillus Calmette–Guerin (BCG) therapy. However, some cancers recur or develop resistance, progressing to muscle-invasive bladder cancer (MIBC), which is associated with high mortality and limited curative options. While radical cystectomy (RC) is the standard treatment for MIBC, it has considerable morbidity. Thus, there is a critical need for predictive tools to guide treatment decisions and novel therapeutic approaches. Immunotherapies show promise, but their efficacy remains constrained. Analyzing the immune infiltrate in BC may uncover mechanisms of resistance and identify new targets. Methods: We investigated the immune microenvironment in NMIBC and MIBC using high-dimensional single-cell analysis, including scRNAseq and Imaging Mass Cytometry (Cytomass). Tumor tissues were obtained from BC patients who underwent cystectomy or Trans urethral removal of bladder tumour (TURBT). We enrolled 7 NMIBC and 8 MIBC patients for scRNAseq, isolating CD45+ immune cells and CD45- epithelial and stromal cells using FACS. The samples were processed with 10X technology to reveal transcriptional landscapes. Additionally, we enrolled 5 NMIBC and 5 MIBC patients for Cytomass analysis, where tissue sections were snap-frozen, stained, and analyzed with the Hyperion system. Our primary endpoint was to identify differences in the composition of tumor and immune cells between NMIBC and MIBC. The study was funded by the Next Generation EU Plan - Piano Nazionale di Ripresa e Resilienza (PNRR-MAD-2022-12376756). Results: The cohort included 15 patients with clinically significant BC who underwent radical cystectomy or TURBT. scRNAseq analysis of patients revealed differences in immune cell abundance and activation states between NMIBC and MIBC samples, identifying distinct cell types and high intra-tumoral heterogeneity. Notable changes were observed tumor-infiltrating B cell lymphocytes, with different abundance in tumors depending on the cell subsets. Immature B cells were enriched in MIBC when compared to NMIBC, indicating a potential role in tumor progression. Cytomass analysis further highlighted modulations in immune cell activation and spatial localization between NMIBC and MIBC. Conclusions: Our findings demonstrate the utility of single-cell RNA sequencing in elucidating the immune landscape of BC and highlight significant immune changes between MIBC and NMIBC. This data enhances our understanding of immune composition and the role of specific immune subsets in BC, potentially informing clinical risk stratification and treatment decision-making.