Rapid normal fibroblast‐tumor crosstalk promotes cancer‐associated fibroblast‐like activation and attenuates mitomycin C cytotoxicity in bladder cancer

Normal fibroblasts (NFs) are among the earliest stromal cells encountered by bladder cancer cells during invasion into the lamina propria, yet their contribution to tumor progression and intravesical chemotherapy response remains poorly defined. Here, we investigated whether NFs are passive stromal bystanders or active participants in early bladder cancer microenvironmental remodeling. Analysis of an early-stage non-muscle-invasive bladder cancer transcriptomic cohort revealed that fibroblast-associated scores were higher in T1 than Ta tumors, enriched in European Organisation for Research and Treatment of Cancer high-risk disease, positively correlated with epithelial-mesenchymal transition (EMT) scores, and associated with increased expression of resistance-linked genes. Experimentally, fibroblast-conditioned medium reduced bladder cancer cell proliferation while accelerating wound closure, indicating a less proliferative but more motile phenotype. This was accompanied by EMT-like cadherin remodeling, including reduced E-cadherin and increased N-cadherin staining. Reciprocally, tumor-derived signals rapidly induced cancer-associated fibroblast (CAF)-like activation features in NFs within 48 h, including increased α-smooth muscle actin, fibroblast activation protein, and PDGFRβ expression. Functionally, increasing fibroblast-to-tumor cell ratios progressively attenuated mitomycin C (MMC)-induced cytotoxicity, demonstrating that stromal context can modify chemotherapy response in vitro. Together, these findings show that NF-tumor crosstalk is rapid, bidirectional and functionally relevant to MMC response in vitro. Rather than acting only after stable CAF formation, NFs can rapidly acquire CAF-like activation features following tumor-derived stimulation and contribute to a stromal context associated with EMT-like plasticity and reduced MMC-induced cytotoxicity in vitro.