Abstract 1636: Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B)
Authors: Tijeras-Raballand Annemilaï , Hobeika Christian , Bonnin Philippe , Rousseau Benoit , Rodrigues Aurélie , Ladfil Fouad , Pocard Marc , de Gramont Armand , Raymond Eric , Faivre Sandrine , Paradis Valérie , Eveno Clarisse 
Published: September 2020

Abstract
Background: Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). Due to western way of life, NASH incidence is rising and predicted to become the leading cause of liver transplant in 2020 and of HCC in the next decades. There is an urgent need for robust animal models fully recapitulating the NASH-related HCC carcinogenesis due to this changing in HCC etiology. In this study, we develop and characterize specific diet-induced variants from our transgenic HCC mouse model, focusing on immune landscape.
Methods: We used in the whole study ASV-B mice: a transgenic mouse model (C57BL/6J) spontaneously developing a reproducible stage-defined HCC (hyperplasia at week(W)8, nodular stage at W12, and diffuse carcinoma at W16-20). Livers were characterized for angiogenesis and immune populations using immunostaining (IHC and IF), and qRT-PCR (LightCycler, Roche). To mimic NASH, ASV-B mice were exposed to 5 different diets. Ten ASV-B and 5 control mice were fed as follows: classic diet as control (yellow), or a high-fat diet (blue), a diet enriched with saturated fatty acids + 1.25% cholesterol (green), a diet containing 22% of vegetal oil + 0.2% cholesterol (orange), and a 1.25% cholesterol diet containing 21% of milkfat (red). All mice fed with special diets also received 30% fructose in the drink water. A second experiment was performed on ASV-B and C57BL/6J wild type mice, using control and Nash-inducing regimen, to confirm our first results.
Results: ASV-B model showed an increase in liver volume and angiogenesis, HCC livers harboring marked arterialization and capillarization as compared to control. Assessing immune markers on 7 evaluable tumor specimens, we observed an increase in CD8, Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A and IL17F mRNA expressions, as frequently observed in human inflammatory HCC. In addition, IHC staining showed intratumoral infiltration of lymphocytes (CD8+) and macrophages (F4/80+, a well-characterized and extensively referenced mouse macrophage marker). ASV-B mice receiving yellow, blue, and green regimens showed similar liver volumes and weights. By macroscopic analysis, we observed increased liver steatosis, and fibrosis in the red and orange regimen compared to others. Moreover, we observed a 40% mortality rate in the orange regimen, and a 20% mortality rate in the blue and green regimens. Interestingly, by microscopic analysis, we observed liver steatosis and inflammation, in 100%, and 75% of the mice, respectively. These results indicate that the green regimen is the most suitable to induce NASH-underlyning disease in our transgenic HCC model. At the conference, we will show in the diet-variants, the immune landscape of the livers and the results of our ongoing second experiment.
Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including inflammatory reaction and immune cell infiltration. In the ASV-B model, we have been able to develop specific-diets variants mimicking NASH characteristics that could be used for drug testing.
Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Philippe Bonnin, Benoit Rousseau, Aurélie Rodrigues, Fouad Ladfil, Marc Pocard, Armand de Gramont, Eric Raymond, Sandrine Faivre, Valérie Paradis, Clarisse Eveno. Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1636.