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.
SummaryThe relationship between diabetes and endothelial dysfunction remains unclear, particularly the association with pathological activation of calpain, an intracellular cysteine protease. Here, we used human induced pluripotent stem cells-derived endothelial cells (iPSC-ECs) to investigate the effects of diabetes on vascular health. Our results indicate that iPSC-ECs exposed to hyperglycemia had impaired autophagy, increased mitochondria fragmentation, and was associated with increased calpain activity. In addition, hyperglycemic iPSC-ECs had increased susceptibility to cell death when subjected to a secondary insult—simulated ischemia-reperfusion injury (sIRI). Importantly, calpain inhibition restored autophagy and reduced mitochondrial fragmentation, concurrent with maintenance of ATP production, normalized reactive oxygen species levels and reduced susceptibility to sIRI. Using a human iPSC model of diabetic endotheliopathy, we demonstrated that restoration of autophagy and prevention of mitochondrial fragmentation via calpain inhibition improves vascular integrity. Our human iPSC-EC model thus represents a valuable platform to explore biological mechanisms and new treatments for diabetes-induced endothelial dysfunction.
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.
Hepatocellular carcinoma (HCC) spans more than 780,000 new annual diagnoses, and causes 750,000 yearly mortalities. This deadly malignancy is expected to become the third leading cause of cancer death by 2030, highlighting the urgent need for new treatment strategies. While human clinical trials are the benchmark for advancing standard-of-care cancer therapeutics, preclinical animal models represent pivotal tools for translational investigations to develop and test novel therapeutics both in vitro and in vivo. The development of clinically relevant systems to serve as a bridge between preclinical murine studies and human clinical practice is thus of vital importance. The Oncopig Cancer Model (OCM) is a novel transgenic swine platform that recapitulates human cancer through development of site/cell specific tumors after Cre recombinase induced expression of heterozygous KRASG12D and TP53R167H transgenes. In this study, we tested the hypothesis that isolation and transformation of OCM hepatocytes from multiple individuals results in development of phenotypically consistent porcine HCC (pHCC) cell lines which faithfully recapitulate the in vitro and in vivo features of human HCC. Eight pHCC lines were established from primary hepatocytes isolated from resected liver specimens (median 9.4, range 4.9-15.0 g) of 4- to 8-week-old OCMs (n = 8), with a median yield of 2.5 x 106 (range 1.5-5.5 x 106) cells/g and 52% (range 30-95%) viability. At 24-hours post-isolation, porcine hepatocytes were transformed into pHCC with median 95% (range 82-98%) efficiency, and were maintained in culture for median 9 (range 6-15) passages. Morphological and behavioral phenotyping of pHCC cells performed using qualitative and quantitative assays were compared to the most widely used human HCC cell line for in vitro investigations (HepG2). Similar to human HCC, all pHCC cell lines exhibited Arginase-1 immunohistochemical positivity—indicating hepatocellular origin—and 100% purity. Eight of 8 (100%) pHCC cell lines showed RT-PCR proven transgene expression, confirming malignant transformation. Flow cytometry demonstrated a median pHCC cell cycle length of 13.0 (range 12.0-16.9) hours, similar to human HCC (15.1 hours). pHCC migration assay showed a median time to half gap closure of 6.0 (range 4.3-9.0) hours, comparable to HepG2 (3 hours). In vivo malignant capability was confirmed by subcutaneous xenograft growth in both SCID mice and Oncopigs, resulting in biopsy proven malignant masses within 7-14 days. The results of the current work indicate that pHCC cell lines may be consistently developed from OCMs, and validates OCM pHCC as a platform which accurately replicates human cancer for translational research. Citation Format: Hanna H. Chen, Sulalita Chaki, Jordan L. Newson, Michele Obeid, Ramzy C. Khabbaz, Alvi Yasmin, Lauretta A. Rund, Mario F. Neto, Lawrence B. Schook, Kyle M. Schachtschneider, Ron C. Gaba. Development of porcine hepatocellular carcinoma cell lines: Comprehensive in vitro and in vivo characterization [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 4085.
necropsy. As a negative control, selective hepatic artery embolization using 100-300 micron Embospheres was performed in a different pig. Results: After selective hepatic artery immunoembolization using the lipiodol/saponin emulsion, CT performed 3 days later showed 19% decrease in size of the embolized liver tumors (n¼2), 71% decrease in size of an unembolized liver tumor (n¼1), and 25% decrease in size of peripancreatic tumors (n¼2). On histology, tumor-infiltrating lymphocytes were seen in the necrotic liver tumors, and no viable tumor cells were seen in the liver or peripancreatic tumors. In the negative control pig (n¼1), selective hepatic artery embolization using Embospheres resulted in tissue necrosis without a detectable local immune response on histology. Conclusions: In a pig model, intra-arterial tumor vaccination using lipiodol/saponin emulsion resulted in tumor-infiltrating lymphocytes, tumor necrosis, and regression of both intrahepatic and extrahepatic tumors. Additional experiments are necessary to confirm these encouraging preliminary results.
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