Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has a high mortality rate due to limited treatment options. Hence, the response of HCC to different cancer immunotherapies is being intensively investigated in clinical trials. Immune checkpoint blockers (ICB) show promising results, albeit for a minority of HCC patients. Mouse models are commonly used to evaluate new therapeutic agents or regimens. However, to make clinical translation more successful, better characterized preclinical models are required. We therefore extensively investigated two immune-competent orthotopic HCC mouse models, namely transplanted Hep-55.1c and transgenic iAST, with respect to morphological, immunological and genetic traits and evaluated both models’ responsiveness to immunotherapies. Hep-55.1c tumors were characterized by rich fibrous stroma, high mutational load and pronounced immune cell infiltrates, all of which are features of immune-responsive tumors. These characteristics were less distinct in iAST tumors, though these were highly vascularized. Cell depletion revealed that CD8 + T cells from iAST mice do not affect tumor growth and are tumor tolerant. This corresponds to the failure of single and combined ICB targeting PD-1 and CTLA-4. In contrast, combining anti-PD-1 and anti-CTLA-4 showed significant antitumor efficacy in the Hep-55.1c mouse model. Collectively, our data comprehensively characterize two immune-competent HCC mouse models representing ICB responsive and refractory characteristics. Our characterization confirms these models to be suitable for preclinical investigation of novel cancer immunotherapy approaches that aim to either deepen preexisting immune responses or generate de novo immunity against the tumor.

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Introduction Immunotherapy is a promising treatment strategy for hepatocellular carcinoma (HCC). A phase III trial for advanced HCC shows favorable results for nivolumab compared to sorafenib. In order to evaluate the outcome of different therapeutic strategies, we compared two HCC mouse models. The orthotopic transplanted HCC fragment Hep-55.1c model and the inducible HCC mouse model (iAST) were extensively characterized ex vivo and in vivo in response to immunotherapies, and were compared to the clinical situation of HCC patients. Experimental Procedures Fragments of mouse Hep-55.1c tumors were implanted in the left lateral liver lobe of C57/Bl6 mice and tumor growth kinetic was monitored by µ-CT. HCC tumor growth in iAST mice was induced via intravenous injection of the adenovirus expressing Cre recombinase. iAST mice express a loxP flanked stop cassette and the SV40 large T-antigen under control of a hepatocyte-specific albumin promoter. Both tumor models were characterized with respect to immune infiltration, cytokine release, somatic mutational load and histopathological characteristics. Mice were treated using different immunotherapeutic agents in monotherapy or in combination such as anti-PD-1, anti-CTLA-4 or the TLR7/8 agonist R848. Human HCC samples were analyzed for mutational load by sequencing, while tumor architecture and immune infiltrate were analyzed by histology. Results Hep-55.1c tumors showed high stroma content accompanied by a low and disorganized vasculature whereas multinodular iAST tumors were highly vascularized lacking stroma content. Furthermore, a high mutational load and a strong immune cell infiltrate including cytotoxic T cells, NK cells and myeloid cells were found in Hep-55.1c tumors. The iAST tumors were characterized by a relatively low immune infiltrate and a small number of mutations. Comparison of these baseline results with data obtained from immune-histopathological analysis and sequencing of human HCC samples confirmed this differentiated picture in the clinics. Following treatment, iAST mice showed no response to immune checkpoint monotherapies and only a marginal number of reactive T cells was found within the tumor. In contrast, tyrosine kinase inhibitor sorafenib led to tumor growth inhibition in iAST model. Treatment of mice bearing Hep-55.1c tumors with immunotherapy e.g. anti-PD-1 showed good response. Combination of anti-PD-1 with R848 increased therapeutic efficacy compared to monotherapies. Conclusions In this study, we have established two orthotopic HCC mouse models that reflect the diverse clinical situation of human HCC. Our findings demonstrate that the composition of the tumor microenvironment has a tremendous influence on the outcome of therapeutic strategies for HCC. Hence, thorough characterization of tumor patients is indispensable in order to predict response to immunotherapy. Citation Format: Carina Hage, Sabine Hoves, Mailin Ashoff, Leanne Strauss, Mario Perro, Frank Herting, Fabian Kiessling, Thomas Pöschinger. Identifying characteristics of orthotopic HCC mouse models to predict response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4084.