INTRODUCTION:Muscle-invasive bladder cancer (MIBC) represents a genetically heterogeneous disease with limited prognostic markers. This study aimed to validate the prognostic relevance of combined alterations in cell cycle regulators RB1, p53, and p21 in a broad cohort of MIBC patients undergoing radical cystectomy (RC). MATERIAL AND METHODS:We analyzed formalin-fixed paraffin-embedded material from MIBC patients who underwent RC at the Department of Urology, University Hospital, Ludwig-Maximilians-University Munich. Tissue microarrays (TMAs) from 251 MIBC patients (pT2-pT4) were constructed, incorporating triplicate cores from tumor center and front. Immunohistochemical expression of RB1, p53, and p21 was assessed using a four-grade scoring system. Prognostic associations with overall survival (OS) and cancer-specific survival (CSS) were evaluated using multivariable Cox regression, Kaplan-Meier curves, and log-rank tests. RESULTS:We assessed 4518 stainings from 251 patients. Single marker analysis revealed no significant association between the loss of RB1, p53, or p21 and OS or CSS. However, the loss of two or three markers was significantly associated with worse OS (HR 3.49, 95 % CI 1.28-9.50; p = 0.01) and CSS (HR 11.2, 95 % CI 1.46-86.04; p = 0.02). CONCLUSIONS:RB1, p53, and p21 are insufficient as single prognostic markers in MIBC but demonstrate significant prognostic relevance when analyzed in combination. These findings underscore the need for multi-marker approaches in prognostic modeling and personalized treatment strategies for MIBC.

Cancer invasion is driven by complex interactions between tumor cells and tumor microenvironment (TME), which promote cancer cell plasticity and remodel the TME to support invasive behavior. In bladder cancer, invasion into the muscularis propria reduces the five-year survival rate to below 30%. Despite this clinical significance, the molecular mechanisms underlying bladder cancer invasion remain poorly understood. Current studies predominantly focus on the comparison between non-invasive and invasive tumor tissue, leaving dynamic TME variation largely unexplored. Leveraging the capabilities of spatial information, we integrated Stereo-seq spatial transcriptomics with single-nucleus RNA sequencing (snRNA-seq) on whole-layers bladder cancer specimens. These integrated datasets delineated a spatially resolved whole-layers landscape of bladder cancer at single cell resolution, elucidating the localization and function of principle cell types during the invasive process. We discovered a bladder cancer invasive leading structure characterized by EPCAM and KRT17 co-expressing, whose plasticity is induced and maintained through interactions with POSTN+ cancer associated fibroblasts (CAFs) and APOE+ macrophages. Additionally, we uncovered the specific spatial distribution of different CAF subtypes during bladder cancer progression, highlighting their roles in shaping distinct TME. Notably, we revealed a progressive increase in immunosuppressive states from superficial to muscle-invading bladder tumors. Our findings underscore the orchestrated dynamics of bladder cancer progression driven by intricate tumor-stroma interactions within the TME. These insights provide a framework for understanding invasive behavior in other muscle-invasive cancers, guiding future research into shared mechanisms of tumor progression and microenvironmental remodeling.