Mesenchymal stem cells delivering IL-33 oncolytic vaccinia virus enhance antitumor effects and overcome PD-1 inhibitor resistance in bladder cancerResistance in bladder cancer

Approximately 80% of patients with advanced bladder cancer (BCa) develop resistance to PD-1 inhibitors, presenting a substantial clinical challenge. This study seeks to develop an innovative therapeutic strategy utilizing mesenchymal stem cells (MSCs) to deliver an oncolytic vaccinia virus engineered to carry interleukin-33 (IL-33), with the objective of overcoming resistance to PD-1 inhibitors and elucidating the associated immune mechanisms. To create the oncolytic vaccinia virus variants OVV-hIL33(GVB-2030)and OVV-mIL33, human or mouse IL-33 cDNA was inserted into the vaccinia virus Copenhagen strain. Integration was confirmed via quantitative PCR and ELISA. The impact on human and mouse bladder cancer cell proliferation was measured using real-time cell analysis. In C57BL/6 mice with the MB49 model, OVV-mIL33's effectiveness with anti-PD-1 therapy was tested. A PD-1 inhibitor-resistant MB49/R cell line was developed, and MSCs were used to deliver OVV-mIL33, forming MSC-OVV-mIL33. Its efficacy with PD-1 inhibitors was evaluated against uncoated OVV-mIL33 by comparing tumor size and survival. Single-cell sequencing, spectral flow cytometry, and multiplex immunofluorescence analyzed cytokines and immune cells in the tumor environment. The findings indicate that OVV-hIL33 (GVB-2030) and OVV-mIL33 demonstrate selective cytotoxicity towards BCa cells in vitro, sparing normal cells. In murine models, intraperitoneal administration of OVV-mIL33 in conjunction with a PD-1 inhibitor effectively inhibited tumor progression and extended survival, although tumor recurrence was observed. Notably, the combination therapy involving MSC-OVV-mIL33 and PD-1 inhibitors demonstrated enhanced efficacy. This regimen not only suppressed both primary and recurrent tumors, achieving a 90% cure rate in mice, but also significantly reduced tumor formation by 60% upon re-challenge with MB49/R, thereby establishing an anti-BCa immune response. The combination therapy effectively remodeled the tumor microenvironment by augmenting the infiltration of cytotoxic CD8+ T cells, memory B cells, and type 2 innate lymphoid cells, while concurrently decreasing the presence of regulatory T cells and terminally exhausted CD8+ T cells. Additionally, the MSC coating conferred protection to the virus against neutralization, thereby ensuring efficient tumor targeting and proliferation. Consequently, we propose that the MSC-mediated delivery of OVV-mIL33, combined with PD-1 blockade therapy, presents significant potential to overcome resistance to PD-1 inhibitors in BCa. This synergistic interaction is principally attributed to the reversal of T cell exhaustion and the enhancement of anti-tumor immune memory formation.