Abstract LB278: IL-12 immunotherapy prevents hepatocellular carcinoma in a murine NAFLD induced cirrhosis model
Authors: Sojoodi Mozhdeh , Barrett Stephen C. , Erstad Derek J. , Jordan Veronica Clavijo , Gale Eric M. , Salloum Shadi , Wang Yongtao , Lanuti Michael , Zukerberg Lawrence , Caravan Peter , Lauer Georg M. , Tanabe Kenneth K. 
Published: April 2023

Abstract
Introduction: Interleukin 12 (IL-12) is an important bridge between the innate and adaptive immune systems. It is mainly produced by macrophages and can bind to its receptor (IL12R) on T cells. It affects several aspects of T cell activation, including IFN-γ and perforin production. Tumor-infiltrating CD8+ T cells perform important anti-tumoral actions, which need to be overcome to allow cancer cell growth. This work studied IL-12 administration as an HCC preventive strategy in cirrhotic mice. We investigated whether IL-12 can boost the recruitment of macrophages to the liver, increase their antigen-presenting ability, and activate cytolytic T cells to inhibit HCC development in cirrhotic livers.
Method: 8 weeks old mice were fed a choline-deficient High fat diet (CDAHFD) for 16 weeks to create NAFLD-induced HCC. A subset of CDAHFD mice was injected intravenously with IL-12 (0.5mg/gr) weekly from 12 to 16 weeks. Mice were euthanized after 16 weeks, and liver and serum were collected for further analysis.
Results: After tissue collection, we observed that 50% of untreated animals developed HCC lesions, while IL-12-treated mice did not have any HCC. After treatment with IL12, qPCR and western blot on total liver tissue showed up-regulation in the IL12 receptor and activation of the IL-12 pathway, evidenced by the increase in Stat4 expression and phosphorylation. Additionally, we detected an increase in IFN-γ expression by qPCR in IL-12-treated mice. Histological analysis showed a significant decrease in fat and collagen deposition in IL-12-treated livers compared to untreated livers. IL-12-treated mice had lower serum aminotransferase enzymes (AST & ALT), indicating improved liver function. Immunostaining and flow cytometry of F4/80 (macrophage marker) showed an increase in the number of macrophages in the livers treated with IL-12. Flow cytometry data showed that the majority of macrophages present in the IL-12 livers were CCR2+, a marker for recruited macrophages. Notably, IL-12-treated macrophages expressed more MHCII (antigen presentation marker) and less B7H4 (inhibitory marker). In addition, we observed a significant increase in the number of cytolytic T cells (CD8+) in the IL-12 treated livers, which had higher expression of the T cell activation marker (CD25) and elevated levels of perforin and IFN-γ that are nessacery for their cytotoxic activity.
Conclusion: There is a lack of substantial evidence to describe the underlying mechanisms supporting the idea of cirrhosis regression and HCC prevention. Our study shows that the exogenous administration of IL-12 causes an increase in migrating profibrolysis macrophages to the liver and elevates cytolytic T-cell activity. IL-12 immunotherapy can trigger cirrhosis regression and prevent HCC development.
Citation Format: Mozhdeh Sojoodi, Stephen C. Barrett, Derek J. Erstad, Veronica Clavijo Jordan, Eric M. Gale, Shadi Salloum, Yongtao Wang, Michael Lanuti, Lawrence Zukerberg, Peter Caravan, Georg M. Lauer, Kenneth K. Tanabe. IL-12 immunotherapy prevents hepatocellular carcinoma in a murine NAFLD induced cirrhosis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB278.