Oncolytic virus OHSV2 induces pyroptosis in bladder cancer cells via the TLR4/NLRP3/Caspase-1/GSDMD pathway.

Bladder cancer remains a significant clinical challenge due to high recurrence and progression rates, necessitating novel therapeutic strategies. Oncolytic viruses, such as the herpes simplex virus type 2-based OHSV2, have demonstrated promising antitumor effects through direct oncolysis and immune activation. This study investigated the efficacy, safety, and molecular mechanisms of OHSV2 in the treatment of bladder cancer. In vitro and in vivo experiments demonstrated that OHSV2 potently inhibited bladder cancer cell proliferation, migration, and clonogenicity in a dose-dependent manner, while exhibiting a favorable safety profile. Critically, OHSV2 treatment triggered a pro-inflammatory tumor immune microenvironment, characterized by increased infiltration and activation of CD8+ T cells. Mechanistically, OHSV2 induced pyroptosis in bladder cancer cells via the canonical Caspase-1/Gasdermin D (GSDMD) pathway, accompanied by increased interleukin-18 (IL-18), interleukin-1β (IL-1β), and lactate dehydrogenase (LDH) release. Further analysis identified NOD-like receptor family pyrin domain containing 3 (NLRP3) as the key upstream pattern recognition receptor for Caspase-1/GSDMD activation, and Toll-like receptor 4 (TLR4) as a critical mediator of NLRP3-dependent pyroptosis. Inhibition of TLR4 or NLRP3 partially reversed OHSV2-induced cytotoxicity, confirming their functional roles. Additionally, combining OHSV2 with the TLR4 agonist enhanced pyroptosis in a subcutaneous xenograft model, amplified antitumor immunity, and improved tumor control in vivo, suggesting potential synergism for clinical translation. These findings elucidate a novel TLR4/NLRP3/Caspase-1/GSDMD axis as the core mechanism behind the antitumor effect of OHSV2 and propose a rationale for its combination with immunomodulators to improve outcomes in bladder cancer.