Improved oncolytic and immunostimulatory activity of the spontaneous jin-3 reovirus mutant in preclinical bladder cancer models

Immunotherapy has emerged as a promising strategy for treatment of urothelial carcinoma of the bladder (UCB) but not all patients show clinically desirable responses upon treatment with immune-checkpoint inhibition. Oncolytic viruses may unleash the full potential of immunotherapy. Besides tumor-lysing properties, oncolytic viruses can induce durable and systemic anti-viral and anti-tumor immune responses. Here, we evaluated and compared oncolytic and immunostimulatory properties of wild-type reovirus (R124) and the junction-adhesion molecule-independent mutant reovirus (<i>jin-3</i>) in preclinical models of human UCB. Both reovirus variants effectively infected and replicated in human UCB cells and induced cell lysis in flatbottom or 3D cultures and in <i>ex vivo</i> cultured human tumor tissue slices. However, <i>jin-3</i> reovirus demonstrated greater efficacy in a dose-dependent manner, effectively inducing expression of immunogenic cell death markers, interferon (IFN)-stimulated genes, and inflammatory cytokines. To study interactions between tumor and immune cells, we established a co-culture system. In this context, co-culturing reovirus-infected bladder tumoroids with peripheral blood mononuclear cells was found to significantly enhance cancer death dose-dependently, alongside induction of key immune mediators such as CXCL10 and IFN-γ. Together, reoviruses display strong oncolytic properties in preclinical UCB models. <i>jin-3</i> reoviruses elicit robust immunostimulatory responses, highlighting their potential as candidate agents for clinical translation in UCB.