Cooperation Between Distinct Cancer Driver Genes Underlies Intertumor Heterogeneity in Hepatocellular Carcinoma

Abstract

See Covering the Cover synopsis on page 1997. BACKGROUND AND AIMS: The pattern of genetic alterations in cancer driver genes in patients with hepatocellular carcinoma (HCC) is highly diverse, which partially explains the low efficacy of available therapies. In spite of this, the existing mouse models only recapitulate a small portion of HCC intertumor heterogeneity, limiting the understanding of the disease and the nomination of personalized therapies. Here, we aimed at establishing a novel collection of HCC mouse models that captured human HCC diversity. METHODS: By performing hydrodynamic tail-vein injections, we tested the impact of altering a well-established HCC oncogene (either MYC or bcatenin) in combination with an additional alteration in one of eleven other genes frequently mutated in HCC. Of the 23 unique Gastroenterology 2020;159:2203-2220 BASIC AND TRANSLATIONAL LIVER pairs of genetic alterations that we interrogated, 9 were able to induce HCC. The established HCC mouse models were characterized at histopathological, immune, and transcriptomic level to identify the unique features of each model. Murine HCC cell lines were generated from each tumor model, characterized transcriptionally, and used to identify specific therapies that were validated in vivo. RESULTS: Cooperation between pairs of driver genes produced HCCs with diverse histopathology, immune microenvironments, transcriptomes, and drug responses. Interestingly, MYC expression levels strongly influenced b-catenin activity, indicating that inter-tumor heterogeneity emerges not only from specific combinations of genetic alterations but also from the acquisition of expression-dependent phenotypes. CONCLUSIONS: This novel collection of murine HCC models and corresponding cell lines establishes the role of driver genes in diverse contexts and enables mechanistic and translational studies.

Multiparametric Analyses of Hepatocellular Carcinoma Somatic Mouse Models and Their Associated Tumor Microenvironment

“…In generating HCC models (Basic Protocol 1), careful attention should be paid on the age, strain, and gender of the animals used, as these may influence the phenotype of liver tumors and associated immune landscape. We recommend using 6‐ to 8‐week‐old C57/BL6J mice to enable the comparison of different HCC genetic backgrounds to what has been previously reported in the literature (Molina‐Sánchez et al., 2020; Wang et al., 2019), and working in sterile conditions during the preparation of the plasmid mix in endotoxin‐free solutions, and the subsequent HDTVi, using an IVC flow.…”

“…This protocol contains a brief description of the HDTVi procedure for generating HCC mouse models (Zhang et al, 1999). Examples of HCC mouse models obtained by this procedure and further applications of this technique have been reported in the literature (Molina-Sánchez et al, 2020;Wang et al, 2019). This approach allows the stable transduction of mouse hepatocytes to induce HCC by tail vein injection of a large volume of saline solution containing a combination of a Sleeping Beauty transposon for oncogene somatic integration, a vector encoding the Sleeping Beauty transposase, and a third vector expressing a single guide RNA (sgRNA) targeting the tumor-suppressor gene of interest, depending on the HCC oncogenic drivers of interest.…”

The Endless Sources of Hepatocellular Carcinoma Heterogeneity

“…As an example, our laboratory has contributed to this field by functionally validating the role of β-catenin activation promoting immune escape and resistance to immunotherapy in HCC. Using genetic customizable mouse models of HCC [ 58 ], Ruiz de Galarreta et al demonstrated that activated β-catenin pathway promotes immune escape by defective recruitment of dendritic cells and consequent impaired T-cell activity [ 59 ]. This study suggested that specific genetic alterations found in HCC might be able to trigger different mechanisms to avoid anti-tumor immune responses.…”

“…Despite the advances in the stratification of HCC patients, above summarized and supported by the implementation of high-throughput analysis [ 104 ], the integration of data obtained from human studies and preclinical models remains necessary to accelerate the identification of robust predictive biomarkers and classification of candidate patients to increase the efficiency of targeted and immune-based therapies. In that regard, our laboratory has recently used the hydrodynamic delivery of genetic elements to study how different genetic alterations cooperate with each other to contribute to different expression patterns, inter-tumor heterogeneity, and distinct patient responses [ 58 ].…”

Genetic tools for the stable overexpression of circular RNAs

“…Specifically, we examined whether a circRNA construct can be delivered to the liver of mice via hydrodynamic tail vein injection. To enrich for cells that contain the circRNA construct, we used a hepatocellular carcinoma model in which tumorigenesis is driven by delivering a Sleeping Beauty transposon harboring a c-Myc cDNA 32 . We combined the spGFP transposon plasmid with a Sleeping Beauty transposase expression plasmid, the c-Myc transposon, and a plasmid encoding Cas9 and a sgRNA targeting p53 32 and performed hydrodynamic tail vein injections in wildtype C57BL/6 mice (Fig.…”