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

## TL;DR

A mutant reovirus called jin-3 shows stronger cancer-killing and immune-boosting effects in bladder cancer models compared to the wild-type virus.

## Contribution

The study introduces jin-3, a reovirus mutant with enhanced oncolytic and immunostimulatory activity in preclinical bladder cancer models.

## Key findings

- jin-3 reovirus effectively infects and kills human bladder cancer cells in 2D and 3D models.
- jin-3 induces stronger immunogenic cell death and immune mediator production compared to wild-type reovirus.
- Co-culture experiments show jin-3 enhances cancer cell death and immune activation in a dose-dependent manner.

## Abstract

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 (jin-3) 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 ex vivo cultured human tumor tissue slices. However, jin-3 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. jin-3 reoviruses elicit robust immunostimulatory responses, highlighting their potential as candidate agents for clinical translation in UCB.

This study describes a mutant reovirus jin-3 that enhances immune activation while effectively killing human bladder cancer cells in preclinical models. Oncolytic reoviruses, particularly jin-3, may boost immunotherapy effectiveness, offering a promising new strategy for bladder cancer patients who poorly respond to current immunotherapeutic approaches.