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

Background Bladder urothelial carcinoma (BLCA) recurrence is frequently driven by an aggressive tumor microenvironment and the persistence of cancer stem cells (CSCs), both of which contribute to therapeutic resistance and metastasis. Neuropilin-1 (NRP1) is increasingly recognized as a key orchestrator of epithelial-mesenchymal transition (EMT), endowing tumor cells with enhanced chemoresistance, metastatic potential, and stemness. However, the precise mechanisms by which NRP1 exacerbates BLCA aggressiveness remain unclear. Methods Gene expression in BLCA patients was analyzed via the TCGA dataset. In vitro studies utilized genetic modulation of NRP1 to dissect its role in BLCA progression. Functional assays were used to assess the impact of NRP1 on proliferation, migration, invasion, and stemness. Bulk RNA-sequencing was used to investigate transcriptomic consequences of NRP1 modulation. The role of NRP1 in regulating interleukin-6 (IL-6) was examined using RT-qPCR, ELISA, and immunohistochemistry. A subcutaneous nude mouse model using T24 CSCs was used to validate in vivo therapeutic strategies. Results High NRP1 expression was significantly associated with tumor drug resistance, stemness, EMT features, and poor overall survival in BLCA patients. Genetic ablation of NRP1 markedly suppressed BLCA cell proliferation, migration, invasion, and CSC properties. Mechanistically, NRP1 depletion profoundly reduced IL-6 secretion, leading to a downstream decrease in p-STAT3 activity. Importantly, exogenous IL-6 rescued stemness traits in NRP1-deficient CSCs, establishing IL-6 as a critical effector of the protumorigenic functions of NRP1. A novel combinatorial therapeutic strategy in which the NRP1 inhibitor EG00229 was coadministered with the neutralizing IL-6 antibody siltuximab significantly attenuated spheroid invasion in vitro and abrogated CSC maintenance both in vitro and in vivo. Conclusions The NRP1-IL-6-STAT3 signaling axis promotes CSC maintenance and aggressive tumor phenotypes in BLCA. This study identifies a promising dual-targeting strategy against NRP1 and IL-6 with significant translational potential for improving outcomes in BLCA patients. Keywords Bladder urothelial carcinoma, Epithelial-mesenchymal transition, Cancer stem cells, Interleukin-6, Neuropilin-1, Tumor microenvironment