Targeting neuropilin-1 and neutralizing interleukin-6 inhibits cancer stem cell formation in bladder cancer cells.

BACKGROUND: Bladder urothelial carcinoma (BLCA) remains a clinical challenge because of its high recurrence rate and the persistence of cancer stem cells (CSCs) within the tumor microenvironment (TME). Neuropilin-1 (NRP1) is recognized as a key orchestrator of epithelial–mesenchymal transition (EMT) and stemness in several malignancies; however, its specific mechanism in driving BLCA aggressiveness and CSC maintenance has not been fully elucidated. This study aimed to characterize the regulatory role of NRP1 in bladder cancer progression and identify potential therapeutic options. METHODS: The clinical significance of NRP1 was first assessed using TCGA patient datasets and tissue microarrays. The functional roles of NRP1 were determined through CRISPR/Cas9-mediated knockout, shRNA-mediated knockdown, and overexpression of NRP1 in the T24 and 5637 cell lines. Bulk RNA sequencing and GSEA were utilized to identify downstream pathways, and ELISA and Western blotting were used to validate signaling interactions. Finally, a synergistic therapeutic strategy was tested using a subcutaneous xenograft nude mouse model. RESULTS: High NRP1 expression was significantly associated with advanced tumor stage, chemoresistance, and poor overall survival in patients with BLCA. Mechanistically, NRP1 regulated the IL-6–STAT3 signaling axis; NRP1 depletion significantly reduced IL-6 secretion and phosphorylated STAT3 levels, thereby impairing sphere formation and EMT features. Conversely, exogenous IL-6 treatment rescued the CSC phenotype in NRP1-deficient cells. Clinically, NRP1 expression strongly correlated with IL-6 and STAT3 levels, and patients with high expression of NRP1 and IL-6 had significantly worse survival outcomes, identifying NRP1–IL-6 coexpression as a novel prognostic biomarker in BLCA. Based on these findings, we demonstrated that dual targeting with the NRP1 inhibitor EG00229 and the IL-6-neutralizing antibody siltuximab synergistically suppressed CSC self-renewal and inhibited tumor growth in vivo. CONCLUSIONS: This study provides a comprehensive characterization of the NRP1–IL-6–STAT3 axis as a fundamental driver of bladder cancer stemness. Our findings establish a foundational mechanistic proof-of-concept for a dual-targeting approach. By simultaneously inhibiting NRP1 and neutralizing IL-6, we demonstrated profound suppression of tumor-initiating capacity and CSC self-renewal. This strategy offers significant translational potential for improving clinical outcomes and overcoming therapeutic resistance in patients with aggressive BLCA.