Viral expression of NE/PPE enhances anti-colorectal cancer efficacy of oncolytic adenovirus by promoting TAM M1 polarization to reverse insufficient effector memory/effector CD8+ T cell infiltration.

Background: Oncolytic adenoviruses are among the most widely utilized oncolytic viruses due to their notable anti-tumor and gene expression capabilities, and modification of ADVs to create armed adenoviruses remains a popular research direction. Nonetheless, immune suppression triggered by ADV and targeted enhancements based on this limitation have been relatively unexplored.

Methods: Flow cytometry was employed to assess immune infiltration in the tumor microenvironment following ADV therapy. Targeted novel recombinant oncolytic viruses, ADV^NE and ADV^PPE, were designed, and their antitumor efficacy, safety, and ability to reshape immune infiltration were evaluated in both subcutaneous tumor models in mice and in vitro experiments. Immune cell depletion assays confirmed the critical role of macrophages. The impact of HMGB1 on macrophage polarization was investigated using shRNA, qRT-PCR, ELISA, and flow cytometry. Furthermore, the importance of TLR4 and its downstream pathways was validated through immunoprecipitation, Western blotting, homozygous knockout mice, and TLR4 inhibitors.

Results: We demonstrated that ADV limits the infiltration of effector memory/effector CD8 + T cells (T_EM/T_E) within the tumor microenvironment. To address this, we leveraged the strong capacity of NE or PPE to recruit T_EM/T_E by constructing novel recombinant oncolytic adenoviruses, ADV^NE or ADV^PPE, armed with NE or PPE. These recombinant viruses induce pyroptosis in colorectal cancer cells accompanied by the release of HMGB1. HMGB1 binds to TLR4 on the surface of macrophages, activating the MyD88-NFκB-NLRP3 (ASC) pathway and promoting M1 polarization of TAMs, thereby increasing T_EM/T_E cell infiltration and enhancing antitumor efficacy.

Conclusions: In summary, this study presents the development of the novel oncolytic adenoviruses ADV^NE and ADV^PPE with enhanced anti-tumor efficacy and provides an in-depth exploration of their specific anti-tumor mechanisms. These findings indicate promising clinical therapeutic prospects and offer new insights for advancing oncolytic adenovirus therapies.