Bidirectional Crosstalk Between Bladder Cancer Cells and Normal Fibroblasts Drives Phenotypic Reprogramming and Modulates Chemosensitivity

During early bladder cancer progression, invading tumour cells first encounter normal fibroblasts (NFs) residing in the lamina propria. Whether and how this interaction shapes tumour behaviour and treatment response, however, has not been systematically examined. While cancer-associated fibroblasts (CAFs) are established drivers of tumour progression and therapy resistance, the role of their precursor NFs in shaping bladder cancer behaviour has been largely overlooked. Here we dissect bidirectional interactions between bladder cancer cells and NFs using indirect (conditioned media) and direct co-culture systems. Fibroblast-conditioned media (FCM) significantly reduced proliferation while accelerating migration in both RT112 and T24 bladder cancer cells, consistent with a pro-migratory "go-or-grow" phenotypic shift. Immunofluorescence revealed FCM-induced cadherin switching reduced E-cadherin and elevated N-cadherin, indicative of an epithelial-to-mesenchymal transition (EMT)-like state. Reciprocally, NFs acquired CAF-like features within 48 hours of tumour cell exposure, with significant upregulation of SMA and FAP confirmed by both immunofluorescence and flow cytometry. Functionally, increasing fibroblast-to-cancer cell ratio progressively attenuated mitomycin C (MMC)-induced cytotoxicity, demonstrating a stromal-mediated, ratio-dependent protective effect against intravesical chemotherapy. Data from the Cancer Genome Atlas (TCGA) for Urothelial Bladder Carcinoma provided corroboratory evidence that fibroblast-enriched tumours exhibited a strong transcriptional correlation with EMT, elevated resistance-associated transcriptional signatures, and significantly poorer overall survival. Collectively, these findings establish tumour-NF crosstalk as a critical and bidirectional regulator of bladder cancer phenotypic plasticity and chemoresistance, and suggest that fibroblast-targeting strategies combined with intravesical chemotherapy may offer a rational approach to overcoming stromal-mediated treatment failure.