Development and comprehensive characterization of porcine hepatocellular carcinoma for translational liver cancer investigation.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. New animal models that faithfully recapitulate human HCC phenotypes are required to address unmet clinical needs and advance standard-of-care therapeutics. This study utilized the Oncopig Cancer Model to develop a translational porcine HCC model which can serve as a bridge between murine studies and human clinical practice. Reliable development of Oncopig HCC cell lines was demonstrated through hepatocyte isolation and Cre recombinase exposure across 15 Oncopigs. Oncopig and human HCC cell lines displayed similar cell cycle lengths, alpha-fetoprotein production, arginase-1 staining, chemosusceptibility, and drug metabolizing enzyme expression. The ability of Oncopig HCC cells to consistently produce tumors in vivo was confirmed via subcutaneous (SQ) injection into immunodeficient mice and Oncopigs. Reproducible development of intrahepatic tumors in an alcohol-induced fibrotic microenvironment was achieved via engraftment of SQ tumors into fibrotic Oncopig livers. Whole-genome sequencing demontrated intrahepatic tumor tissue resembled human HCC at the genomic level. Finally, Oncopig HCC cells are amenable to gene editing for development of personalized HCC tumors. This study provides a novel, clinically-relevant porcine HCC model which holds great promise for improving HCC outcomes through testing of novel therapeutic approaches to accelerate and enhance clinical trials.

Characterization of an Inducible Alcoholic Liver Fibrosis Model for Hepatocellular Carcinoma Investigation in a Transgenic Porcine Tumorigenic Platform.

PURPOSE: This study used the Oncopig Cancer Model (OCM) to develop alcohol-induced fibrosis in a porcine model capable of developing hepatocellular carcinoma. MATERIALS AND METHODS: Liver injury was induced in 8-week-old Oncopigs (n = 10) via hepatic transarterial infusion of 0.75 mL/kg ethanol-ethiodized oil (1:3 v/v). Feasibility was assessed in an initial Oncopig cohort (n = 5) by histologic analysis at 8 weeks after induction, and METAVIR results were compared to age- and sex-matched healthy controls (n = 5). Liver injury was then induced in a second OCM cohort (n = 5) for a time-course study, with post-induction disease surveillance via biweekly physical exam, lab analysis, and liver biopsies until 20 weeks after induction. RESULTS: In Cohort 1, 8-week post-induction liver histologic analysis revealed median METAVIR F3 (range, F3-F4) fibrosis, A2 (range, A2-A3) inflammation, and 15.3% (range, 5.0%-22.9%) fibrosis. METAVIR and inflammation scores were generally elevated compared to healthy controls (F0-F1, P = 0.0013; A0-A1, P = .0013; median percent fibrosis 8.7%, range, 5.8%-12.1%, P = .064). In Cohort 2, histologic analysis revealed peak fibrosis severity of median METAVIR F3 (range, F2-F3). However, lack of persistent alcohol exposure resulted in liver recovery, with median METAVIR F2 (range, F1-F2) fibrosis at 20 weeks after induction. No behavioral or biochemical abnormalities were observed to indicate liver decompensation. CONCLUSIONS: This study successfully validated a protocol to develop METAVIR F3-F4 fibrosis within 8 weeks in the OCM, supporting its potential to serve as a model for hepatocellular carcinoma in a fibrotic liver background. Further investigation is required to determine if repeated alcohol liver injury is required to develop an irreversible METAVIR grade F4 porcine cirrhosis model.

Mechanism of Action, Pharmacokinetics, Efficacy, and Safety of Transarterial Therapies Using Ethiodized Oil: Preclinical Review in Liver Cancer Models.

PURPOSE: To systematically review mechanism of action, pharmacokinetics (PKs), efficacy, and safety of ethiodized oil-based locoregional therapy (LRT) for liver cancer in preclinical models. MATERIALS AND METHODS: A MEDLINE search was performed from 1988 to 2016. Search terms included hepatocellular carcinoma (HCC), HCC, liver-cell carcinoma, liver, hepatic, hepatocarcinoma, transarterial or chemoembolization, TACE, animal, Lipiodol, Ethiodol, iodized oil, and/or poppy-seed oil. Inclusion criteria were: publication in a peer-reviewed journal, an accepted animal model, and PK/safety/efficacy data reported. Exclusion criteria were: inadequate PK, safety, or efficacy data; anticancer drug name/dose not available; and article not in English. Outcomes included intratumoral anticancer drug uptake, PKs, tolerance, tumor response, and survival. RESULTS: Of 102 identified articles, 49 (49%) met the inclusion criteria. Seventeen, 35, and 2 articles used rat, rabbit, and pig models. Mechanism of action was investigated in 11 articles. Eleven articles reported drug uptake, PK, and tolerance data, showing 0.5%-9.5% of injected chemotherapy dose in tumor. Tumor-to-liver drug distribution ratios were 2-157. Toxicology data across 6 articles showed transient liver laboratory level elevations 1 day after LRT. There was no noteworthy liver or extrahepatic histologic damage. Nine articles reported tumor response, with 0%-30% viable tumor and -10% to -38% tumor growth at 7 days after LRT. Two articles reported survival, showing significantly longer survival after LRT vs untreated controls (56/60 d vs 33/28 d). Several articles described ethiodized oil mixed with radiopharmaceutical (n = 7), antiangiogenic (n = 6), gene (n = 6), nanoembolic (n = 5), immune (n = 2), or other novel (n = 1) agents. CONCLUSIONS: Animal studies show preferential tumor uptake of anticancer agent, good hepatic/systemic tolerance, high tumor response, and enhanced survival after ethiodized oil-based LRT.

A validated, transitional and translational porcine model of hepatocellular carcinoma.

Difficult questions are confronting clinicians attempting to improve hepatocellular carcinoma (HCC) outcomes. A large animal model with genetic, anatomical, and physiological similarities to humans is required to transition from mouse models to human clinical trials to address unmet clinical needs. To validate our previously reported inducible porcine cancer model (Oncopig) as a transitional HCC model, Oncopig hepatocyte cultures were transformed using Cre recombinase. The resulting porcine HCC cells (pHCC) expressed oncogenic TP53R167H and KRASG12D, and displayed nuclear pleomorphisms with pale to granular cytoplasm arranged in expanded plates similar to human HCC histopathology. Human HCC transcriptional hallmarks were detected in pHCC cells using RNA-seq, including TERT reactivation, apoptosis evasion, angiogenesis activation, and Wnt signaling activation. Master regulators of gene expression were conserved across Oncopig and 18 human HCC cell lines. pHCC injection into SCID mice resulted in tumors recapitulating human HCC characteristics, including thick trabeculae formation, pseudoacini patterning, and sheets of well-vascularized stroma. Finally, autologous injection of pHCC cells subcutaneously yielded a tumor histologically characterized as Edmondson Steiner (HCC nuclear grade assessment system) grade 2 HCC with trabecular patterning and T-lymphocyte infiltration. These data demonstrate the Oncopig HCC model's utility for improving detection, treatment, and biomarker discovery relevant to human HCC.

The Oncopig Cancer Model: An Innovative Large Animal Translational Oncology Platform.

Despite an improved understanding of cancer molecular biology, immune landscapes, and advancements in cytotoxic, biologic, and immunologic anti-cancer therapeutics, cancer remains a leading cause of death worldwide. More than 8.2 million deaths were attributed to cancer in 2012, and it is anticipated that cancer incidence will continue to rise, with 19.3 million cases expected by 2025. The development and investigation of new diagnostic modalities and innovative therapeutic tools is critical for reducing the global cancer burden. Toward this end, transitional animal models serve a crucial role in bridging the gap between fundamental diagnostic and therapeutic discoveries and human clinical trials. Such animal models offer insights into all aspects of the basic science-clinical translational cancer research continuum (screening, detection, oncogenesis, tumor biology, immunogenicity, therapeutics, and outcomes). To date, however, cancer research progress has been markedly hampered by lack of a genotypically, anatomically, and physiologically relevant large animal model. Without progressive cancer models, discoveries are hindered and cures are improbable. Herein, we describe a transgenic porcine model-the Oncopig Cancer Model (OCM)-as a next-generation large animal platform for the study of hematologic and solid tumor oncology. With mutations in key tumor suppressor and oncogenes, TP53R167H and KRASG12D , the OCM recapitulates transcriptional hallmarks of human disease while also exhibiting clinically relevant histologic and genotypic tumor phenotypes. Moreover, as obesity rates increase across the global population, cancer patients commonly present clinically with multiple comorbid conditions. Due to the effects of these comorbidities on patient management, therapeutic strategies, and clinical outcomes, an ideal animal model should develop cancer on the background of representative comorbid conditions (tumor macro- and microenvironments). As observed in clinical practice, liver cirrhosis frequently precedes development of primary liver cancer or hepatocellular carcinoma. The OCM has the capacity to develop tumors in combination with such relevant comorbidities. Furthermore, studies on the tumor microenvironment demonstrate similarities between OCM and human cancer genomic landscapes. This review highlights the potential of this and other large animal platforms as transitional models to bridge the gap between basic research and clinical practice.