Oncopig Cancer Model Research Articles

Translational Relevance and Future Integration of the Oncopig Cancer Model in Preclinical Applications.

Porcine cancer models offer a valuable platform for evaluating interventions such as devices, surgeries, and locoregional therapies, which are often challenging to test in mouse models. In addition to size and anatomical similarities with humans, pigs share greater similarities in genetics, immunity, drug metabolism, and metabolic rate with humans as compared to mouse models, increasing their translational relevance. This review focuses on the Oncopig Cancer Model, a genetically engineered porcine model designed to recapitulate human cancer. Harboring a transgenic cassette that expresses oncogenic mutant KRAS and TP53 under control of a Cre-Lox system, the Oncopig allows temporal and spatial control of tumor induction. Its versatility has enabled the development of diverse cancer models including liver, pancreatic, lung, and bladder cancer. Serving as a clinically relevant model for human cancer, the Oncopig addresses unmet clinical needs and holds immense promise for advancing preclinical cancer research and therapeutic development.

Magnetic Resonance Elastography for Staging Liver Fibrosis in the Oncopig.

This pilot study investigated the feasibility of using magnetic resonance elastography (MRE) for the non-invasive detection and quantification of liver fibrosis in the Oncopig cancer model. Seven 8-week-old Oncopigs underwent alcoholic liver fibrosis induction and serial MRE imaging and liver biopsy at 1, 2, and 3 months post procedure. MRE was utilized to quantify liver stiffness, and liver fibrosis was histologically graded using the METAVIR system. The primary outcome measure was the capability to detect and quantify liver fibrosis using MRE with radiologic-pathologic correlation. Liver fibrosis induction, MRE imaging, and liver biopsy were successfully performed. MRE liver fibrosis was evident in 57% (4/7), 50% (3/6), and 40% (2/5) animal subjects 1, 2, and 3 months after fibrosis induction, with mean liver stiffness of 2.94, 3.25, and 2.91 kPa, respectively. Histological liver fibrosis was noted in 71% (5/7), 100% (5/5), and 100% (5/5) of animal subjects with available tissue samples. There was no significant statistical correlation between the MRE-measured liver stiffness and the METAVIR fibrosis scores. In conclusion, quantifiable liver fibrosis may be induced in the Oncopig. MRE has potential utility in non-invasively detecting liver stiffness in this large-animal preclinical model, though tissue biopsy was more sensitive in demonstrating disease.

Oncopig bladder cancer cells recapitulate human bladder cancer treatment responses in vitro.

Bladder cancer is a common neoplasia of the urinary tract that holds the highest cost of lifelong treatment per patient, highlighting the need for a continuous search for new therapies for the disease. Current bladder cancer models are either imperfect in their ability to translate results to clinical practice (mouse models), or rare and not inducible (canine models). Swine models are an attractive alternative to model the disease due to their similarities with humans on several levels. The Oncopig Cancer Model has been shown to develop tumors that closely resemble human tumors. However, urothelial carcinoma has not yet been studied in this platform. We aimed to develop novel Oncopig bladder cancer cell line (BCCL) and investigate whether these urothelial swine cells mimic human bladder cancer cell line (5637 and T24) treatment-responses to cisplatin, doxorubicin, and gemcitabine in vitro. Results demonstrated consistent treatment responses between Oncopig and human cells in most concentrations tested (p>0.05). Overall, Oncopig cells were more predictive of T24 than 5637 cell therapeutic responses. Microarray analysis also demonstrated similar alterations in expression of apoptotic (GADD45B and TP53INP1) and cytoskeleton-related genes (ZMYM6 and RND1) following gemcitabine exposure between 5637 (human) and Oncopig BCCL cells, indicating apoptosis may be triggered through similar signaling pathways. Molecular docking results indicated that swine and humans had similar Dg values between the chemotherapeutics and their target proteins. Taken together, these results suggest the Oncopig could be an attractive animal to model urothelial carcinoma due to similarities in in vitro therapeutic responses compared to human cells.

MP14-02 DEVELOPMENT OF A PORCINE MODEL OF BLADDER CANCER USING THE ONCOPIG

You have accessJournal of UrologyCME1 Apr 2023MP14-02 DEVELOPMENT OF A PORCINE MODEL OF BLADDER CANCER USING THE ONCOPIG Andreas Aulitzky, Rand Wilcox Vanden Berg, Kwanghee Kim, Hikmat Al-Ahmadie, Sebastien Monette, and Jonathan A. Coleman Andreas AulitzkyAndreas Aulitzky More articles by this author , Rand Wilcox Vanden BergRand Wilcox Vanden Berg More articles by this author , Kwanghee KimKwanghee Kim More articles by this author , Hikmat Al-AhmadieHikmat Al-Ahmadie More articles by this author , Sebastien MonetteSebastien Monette More articles by this author , and Jonathan A. ColemanJonathan A. Coleman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Large animal cancer models have potential to improve and accelerate the development of new treatment modalities but few exist. Oncopigs (OPG) are particularly suitable due to their similarities to humans. We sought to develop a urothelial tumor model using the oncopig cancer model (OCM: University of Illinois Cancer Center). The OCM carries two mutations (KRASG12D and TP53R167H) which can be induced by exposure to a buffered Adeno Virus Cre solution (bAdCre). Three procedures (P1, P2, P3) were devised and investigated. METHODS: Eight OPGs underwent tumor inoculation. In P1, bAdCre with plant starch (n=3) was injected submucosally at 3 distinct locations in the bladder wall. In P2 the urothelium was denuded in 3 locations followed by bAdCre instillation (n=4). In P3 bAdCre was instilled combined with N-Dodecyl- β-D-Maltoside (DDM), an alkyl disaccharide and polar surfactant that enhances virus transfection in the bladder, with intact urothelium (n=1). Serial imaging, cystoscopies and blood draws were performed until sacrifice at 3 timepoints (days: 14, 21, 28). Tumor characteristics were assessed by necropsy and histopathological examination of the entire tumor tissue. RESULTS: In total, 7/8 (87.5%) OPGs grew bladder tumors. Histopathological examination revealed 2 different tumor types: neoplastic tumors (NT) and inflammatory tumors (IT). Using P1, 3 OPGs developed IT (3/3). 2 OPGs in 2 locations (2/3) and 1 in 1 location (1/3). In P2, 3/4 OPGs grew tumors. 1 OPG in all 3 locations, all 3 were NTs (3/3) (Figure 1 a), 1 OPG in 2 locations (2/3), both NTs and 1 OPG in 1 location (1/3), an IT. In P3 1 NT developed (1/1). All tumors showed muscle invasive growth. P3 showed the most superficial lesion after 21 days (pT2a). In P1, 2 pigs had pT3 tumors after 21 and 28 days. No complications were observed. Based on the histological (Figure 1 b: H&E), multiple immunohistochemical findings (e.g.: Figure 1 c: high molecular cytokeratins 34Be12; Figure 1 d: P53) and the growth pattern, NT were diagnosed as urothelial cell carcinomas, sarcomatoid variant. CONCLUSIONS: This is the first description of bladder tumor development in a porcine model using the OCM. We were able to create neoplastic tumors in a short period of time. The model is reproducible and neoplastic lesions show urothelial origin with sarcomatoid transformation. Source of Funding: MSKCC Sidney Kimmel Center for Urologic Cancers © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e182 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Andreas Aulitzky More articles by this author Rand Wilcox Vanden Berg More articles by this author Kwanghee Kim More articles by this author Hikmat Al-Ahmadie More articles by this author Sebastien Monette More articles by this author Jonathan A. Coleman More articles by this author Expand All Advertisement PDF downloadLoading ...

The Oncopig as an Emerging Model to Investigate Copper Regulation in Cancer.

Emerging evidence points to several fundamental contributions that copper (Cu) has to promote the development of human pathologies such as cancer. These recent and increasing identification of the roles of Cu in cancer biology highlights a promising field in the development of novel strategies against cancer. Cu and its network of regulatory proteins are involved in many different contextual aspects of cancer from driving cell signaling, modulating cell cycle progression, establishing the epithelial-mesenchymal transition, and promoting tumor growth and metastasis. Human cancer research in general requires refined models to bridge the gap between basic science research and meaningful clinical trials. Classic studies in cultured cancer cell lines and animal models such as mice and rats often present caveats when extended to humans due to inherent genetic and physiological differences. However, larger animal models such as pigs are emerging as more appropriate tools for translational research as they present more similarities with humans in terms of genetics, anatomical structures, organ sizes, and pathological manifestations of diseases like cancer. These similarities make porcine models well-suited for addressing long standing questions in cancer biology as well as in the arena of novel drug and therapeutic development against human cancers. With the emergent roles of Cu in human health and pathology, the pig presents an emerging and valuable model to further investigate the contributions of this metal to human cancers. The Oncopig Cancer Model is a transgenic swine model that recapitulates human cancer through development of site and cell specific tumors. In this review, we briefly outline the relationship between Cu and cancer, and how the novel Oncopig Cancer Model may be used to provide a better understanding of the mechanisms and causal relationships between Cu and molecular targets involved in cancer.

Abstract 785: Multi-omics profiling identifies genetic and epigenetic regulation underlying alcohol induced liver fibrosis in a porcine hepatocellular carcinoma model

Abstract Background: Cirrhosis is a form of end-stage liver disease which manifests with a host of metabolic and hematologic derangements, and is commonly the result of long-term ethanol exposure. Large animal models of cirrhosis, and of one of its common long-term sequelae, hepatocellular carcinoma (HCC), are needed to study novel and emerging therapeutic interventions. Methods: Liver fibrosis was induced in the Oncopig cancer model, a large animal model of HCC, via intrahepatic, intra-arterial ethanol infusion. Post-mortem liver sections from five fibrosis induction and five age-matched controls were harvested for RNA-seq (mRNA and lncRNA), small RNA-seq (miRNA), and reduced representation bisulfite sequencing (RRBS; DNA methylation). Supervised, multi-omic analysis of the 5,000 genes and methylated regions demonstrating the largest inter-sample variance with non-zero counts along with all sequenced miRNA were analyzed using the DIABLO method within the mixOmics 6.8.5 R library. Results: Single component DIABLO latent variable analysis of mRNAs, lncRNAs, miRNAs, and methylated regions was performed to identify the genetic elements (variables) which most reliably discriminated between experimental arms. Analysis of the top 100 discriminatory variables from each ‘omics data set was performed to provide sufficient data for pathway analysis while maintaining separation of the experimental arms within the latent space. IPA pathway analysis of these genes revealed differential regulation of pathways related to hepatic fibrosis, adipogenesis, ketogenesis as well as several other liver-specific metabolic pathways. Identification of the five most discriminatory variables was also performed. Single component, five variable analysis revealed ELOVL2, LIPK, and LAMA3 most reliably differentiated control and fibrosis groups, along with two lncRNA transcripts, XLOC035153 and XLOC036409 which were predicted to target S1PR1 and UHRF1BP1L, respectively. LAMA3, XLOC036409, and its target UHRF1BP1L were overexpressed in Oncopig fibrotic liver samples. UHRF1, a potential binding target for UHRF1BP1L, has previously been implicated as an oncogene for hepatocellular carcinoma. S1PR1 is involved in vasculogenesis and cancer cell motility via the RAC-CDC42 and ERK pathways. Conclusions The differentially regulated genetic elements identified by the multi-omic analyses were largely concordant with known alterations in human alcoholic liver disease. This agreement lends validity to the Oncopig model as a model organism for human alcoholic liver disease. In addition, novel genes, UHRF1BP1L and S1PR1, were associated with porcine liver fibrosis and further work to characterize their precise mechanistic roles in the natural history of this disease is required. Citation Format: Mark Hieromnimon, Lawrence Schook, Ron Gaba, Kyle Schachtschneider. Multi-omics profiling identifies genetic and epigenetic regulation underlying alcohol induced liver fibrosis in a porcine hepatocellular carcinoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 785.

Transcriptional Profiling of Porcine HCC Xenografts Provides Insights Into Tumor Cell Microenvironment Signaling.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, representing the most common form of liver cancer. As HCC incidence and mortality continue to increase, there is a growing need for improved translational animal models to bridge the gap between basic HCC research and clinical practice to improve early detection and treatment strategies for this deadly disease. Recently the Oncopig cancer model—a novel transgenic swine model that recapitulates human cancer through Cre recombinase induced expression of KRASG12D and TP53R167H driver mutations—has been validated as a large animal translational model for human HCC. Due to the similar size, anatomy, physiology, immunology, genetics, and epigenetics between pigs and humans, the Oncopig has the potential to improve translation of novel diagnostic and therapeutic modalities into clinical practice. Recent studies have demonstrated the importance of tumor cells in shaping its surrounding microenvironment into one that is more proliferative, invasive, and metastatic; however, little is known about the impact of microenvironment signaling on HCC tumor biology and differential gene expression between HCC tumors and its tumor microenvironment (TME). In this study, transcriptional profiling was performed on Oncopig HCC xenograft tumors (n = 3) produced via subcutaneous injection of Oncopig HCC cells into severe combined immunodeficiency (SCID) mice. To differentiate between gene expression in the tumor and surrounding tumor microenvironment, RNA-seq reads originating from porcine (HCC tumor) and murine (microenvironment) cells were bioinformatically separated using Xenome. Principle component analysis (PCA) demonstrated clustering by group based on the expression of orthologous genes. Genes contributing to each principal component were extracted and subjected to functional analysis to identify alterations in pathway signaling between HCC cells and the microenvironment. Altered expression of genes associated with hepatic fibrosis deposition, immune response, and neo angiogenesis were observed. The results of this study provide insights into the interplay between HCC and microenvironment signaling in vivo, improving our understanding of the interplay between HCC tumor cells, the surrounding tumor microenvironment, and the impact on HCC development and progression.

Transcriptional regulation of alcohol induced liver fibrosis in a translational porcine hepatocellular carcinoma model

Hepatocellular carcinoma (HCC) is the 5th most common and 2nd deadliest cancer worldwide. HCC risk factors include alcohol induced liver cirrhosis, which prompts hepatic inflammation, cell necrosis, and fibrosis deposition. As 25% of HCC cases are associated with alcohol induced liver disease, understanding the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic responses are critical. This study utilized the Oncopig Cancer Model—a transgenic pig model that recapitulates human HCC through induced expression of KRASG12D and TP53R167H driver mutations—to investigate the molecular mechanisms underlying alcohol induced liver disease. Oncopigs (n = 5) underwent fibrosis induction via infusion of ethanol and ethiodized oil (1:3 v/v dosed at 0.75 mL/kg) into the hepatic arterial circulation. Eight-weeks post induction, liver tissue samples from fibrotic and age-matched control (n = 5) Oncopigs were collected for histological evaluation and transcriptional profiling. Increased hepatic inflammation and fibrosis was observed in fibrotic Oncopigs via pathological assessment. Transcriptional profiling (RNA-seq) resulted in the identification of 4387 differentially expressed genes between Oncopig fibrotic and control livers. GO term enrichment analysis identified pathway alterations associated with cirrhosis progression in humans, including cell proliferation, angiogenesis, extracellular matrix deposition, and oxidation-reduction. Key alterations include activation of hepatic stellate cells, increased matrix metalloproteinase production, and altered expression of ABC and SLC transporter genes involved in transport of anticancer drugs.These results demonstrate Oncopig liver fibrosis recapitulates transcriptional hallmarks of human cirrhosis, making the Oncopig an ideal model for studying the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic response.

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.

Abstract 3703: Development of oncopig urothelial carcinoma cell lines

Abstract Urothelial carcinoma (UC), more commonly known as bladder cancer, affects approximately 81,000 people a year in the United States and is the fourth most common type of cancer among men. Age also plays a tremendous factor, as 90% of people affected by bladder cancer are older than 55, and if the cancer has metastasized the 5-year survival rate is 35%. This requires the advancement of early detection and treatment methods for bladder cancer. The development of clinically relevant systems to serve as a bridge between preclinical murine studies and human clinical practice is 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 transgene. In this study, we standardized a protocol for the development and growth of Oncopig UC cell lines that faithfully recapitulates in vitro and in vivo features of human bladder cancer. The focus of this preliminary research was to successfully create OCM UC cell lines that mimic features of human UC. OCM urothelial cell lines (n=7) were isolated from whole bladders collected from male Oncopigs at euthanasia. OCM urothelial cell lines were exposed to Cre recombinase 48 hours post isolation to induce expression of KRASG12D and TP53R167H transgenes, resulting in development of OCM UC cell lines. Differences in migration rates were observed between OCM urothelial and UC cell lines, confirming cellular invasiveness. RT-PCR was performed after several passages to confirm KRASG12D and TP53R167H gene expression, which was observed in OCM UC but not urothelial cell lines. Uroplakin II and pan-Cytokeratin (PCK-26) are markers for UC and urothelial cells, respectively, and are used to diagnose UC clinically. OCM UC cells stained positively for Uroplakin II and PCK-26, confirming their identity as malignant UC cells. Future work is needed to confirm OCM and human UC display similar histological and molecular features, and to develop strategies for in vivo OCM UC tumor development. Citation Format: Aisha Qazi, Faith M. Thomas, Sulalita Chaki, Noah Robertson, Shovik Patel, Kyle M. Schachtschneider, Lawrence B. Schook. Development of oncopig urothelial carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3703.

Abstract 3711: Development, maintenance, and characterization of porcine hepatocellular carcinoma cell lines

Abstract Biomedical research of liver cancer requires effective model cell lines and animals in order to translate diagnostic and treatment strategies into clinical practice. An ideal translational model to study hepatocellular carcinoma (HCC) would be the use of a genetic pig model of HCC due to the many similarities between pigs and humans which include anatomy, physiology, metabolism and genetics. This study utilized the Oncopig Cancer Model (OCM), a transgenic pig model that develops site and cell specific tumors through Cre recombinase induced expression of KRASG12D and TP53R167H transgenes. Our objective was to develop an in vitro HCC model using primary hepatocytes isolated from Oncopig liver tissue. In order to develop Oncopig HCC cell lines, Oncopigs (n=36) underwent liver resection to remove a portion (5 to 20 grams) of their liver for hepatocyte isolation. Post isolation mean cell yield was 1.8 million cells per gram of tissue ranging from 2.2 million cells to 6.5 million cells. After incubation (24 hours post hepatocyte isolation), primary hepatocytes were transfected with adenoviral vector encoding Cre recombinase (AdCre), resulting in KRASG12D and TP53R167H expression and transformation of hepatocytes into HCC cell lines. Also, AdCre transfected cells will have GFP expression. Successful transfection rates (77% to 99% with a mean of 87.9%) were confirmed by fluorescent microscopy. Phenotypic characterization of OCM HCC cells was then performed. Cell migration assays were performed to characterize enhanced cell migration of transformed cells (mean t1/2 gap = 4 hours) and polymerase chain reaction (PCR) assays were done after several passages to confirm KRASG12D and TP53R167H gene expressions. HCC lines were injected subcutaneously in the abdomen of SCID or NSG mice (n=34). Each mouse was given 2 injections with each injection site receiving 5 million cells. Mouse injections resulted in 59 tumors (86.8% tumor growth success). Mean volume for the tumors was 285.3 mm3 ranging from 18.84 mm3 to 1766.3 mm3. Following confirmation of tumor development in SCID or NSG mice, HCC cell lines were autologously injected into Oncopigs (n=29). Each Oncopig was given 6 injections subcutaneously in the abdomen with each injection site having 10 million cells Autologous injections to Oncopigs were capable of growing tumors. These results indicate that a porcine HCC model can be created and used for further tumor biology research. Citation Format: Shovik S. Patel, Sulalita Chaki, Faith M. Thomas, Aisha Qazi, Kyle M. Schachtschneider, Matthew Stewart, Elizabeth Pollack, Ron C. Gaba, Lawrence B. Schook. Development, maintenance, and characterization of porcine hepatocellular carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3711.

Abstract 3105: Oncopig carcinoma cell lines: A foundation for co-clinical trials

Abstract The Oncopig cancer model (OCM) was developed to support a broad range of solid and liquid tumor malignancies while providing the appropriate anatomical and physiological models for preclinical and co-clinical trials to expedite human clinical trials. The OCM develops tumors following Cre recombinase induced expression of KRASG12D and TP53R167H transgenes in a temporally and spatially controlled manner. To date, in vitro transformation and in vivo tumor formation of soft tissue sarcomas (STS), hepatocellular carcinoma (HCC), and pancreatic ductal adenocarcinoma (PDAC) have been demonstrated. However, the OCM was designed to generate cancers of all tissue origins. To demonstrate the potential importance of the OCM, we have successfully isolated and transformed multiple OCM cell types, each a progenitor of clinically relevant human disease. Cell type specific cell isolations were performed, and cells cultured under appropriate conditions. Within the first 48 hours, isolated cells were treated with adenoviral vector encoding Cre recombinase (AdCre) at 200-400 MOI for 5 hours in low serum (5%) medium. Both control and transformed (AdCre treated) cells underwent basic histopathologic screening (H&E staining, immunohistochemical staining for vimentin and cytokeratin), and verification of transgene expression (RT-PCR) and tumorigenicity (xenograft into SCID mice). Cell types isolated include fibroblasts, hepatocytes, pancreatic ductal cells, dermal epithelial cells, splenocytes, ovarian surface epithelial cells, fallopian tube secretory epithelial cells, renal proximal tubule epithelial cells, urinary bladder epithelial cells, bone marrow (without specific cell isolation), and testis (no specific cell isolation). Each cell type was successfully transformed, as shown by in vitro phenotypic changes and tumor formation in vivo. Additional gene expression analysis (RNA-Seq) of the STS and HCC cell lines verified the OCM transformed cells recapitulate transcriptional hallmarks of human cancers. Consistent with human STS, OCM STS cell lines display altered TP53 signaling, Wnt signaling activation, and evidence of epigenetic reprogramming. OCM HCC cell lines show transcriptional evidence of TERT reactivation, apoptosis evasion, angiogenesis activation, and Wnt signaling activation, all similar to human HCC. While not an exhaustive list of Oncopig cell types, we have not encountered cells resistant to malignant transformation through Cre recombinase exposure. These results support the unprecedented potential for modeling of additional cancer types, including colorectal, ovarian, fallopian tube, uterine, renal, bladder, skin, and hematological cancers to support and expand preclinical, translational, and co-clinical investigations. Citation Format: Laurie A. Rund, Kyle M. Schachtschneider, Ron C. Gaba, Daniel R. Principe, Paul Grippo, Regina M. Schwind, Howard Ozer, Lawrence B. Schook. Oncopig carcinoma cell lines: A foundation for co-clinical trials [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 3105.

Abstract 4094: Oncopig and human hepatocellular carcinoma cell lines exhibit similar response to liver cancer chemotherapy agents

Abstract Preclinical animal models represent pivotal tools to develop novel therapeutics. While murine models are commonly used in preclinical drug research, many agents that show promise in mouse studies ultimately fail in human clinical trials. In contrast to mice, pigs share many genetic, physiological, and metabolic similarities with humans, which may facilitate relevant investigation of drug pharmacokinetic and therapeutic analyses. 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. Given similar drug metabolism between pigs and humans, we propose that the OCM can address unmet clinical needs by facilitating translation of results obtained in preclinical murine studies to human clinical practice. In order to investigate the feasibility of using the OCM for drug screening, we tested the hypothesis that OCM and human hepatocellular carcinoma (HCC) cell lines exhibit similar responses to chemotherapy agents commonly used in clinical settings. Responses to four cytostatic (sorafenib) and cytotoxic (doxorubicin, cisplatin, mitomycin C) HCC chemotherapies were compared between OCM derived porcine HCC cell lines and three representative human HCC cell lines (SNU-387, SNU-475, HepG2). Results of MTT time course assays (3x performance) revealed that OCM HCC and human HCC cell lines displayed similar sensitivities to all four chemotherapies when exposed to comparable, clinically relevant concentrations for 72 hours. Sorafenib had a cytostatic effect at 10-20 µM in all four cells lines tested, consistent with clinically relevant, 3-15 µM systemic therapeutic concentrations. Doxorubicin, usually administered at 1-5 µM for locoregional therapy in patients, had comparable half maximal inhibitory concentrations (IC50) for pHCC (0.19 µM), HepG2 (0.45 µM), and SNU-387 (0.94 µM), but was higher for SNU-475 (3.31 µM). Mitomycin C had similar IC50 for pHCC (1.77 µM), HepG2 (1.73 µM), and SNU-475 (2.93 µM), but was higher for SNU-387 (7.91 µM). Finally, IC50 for cisplatin was nearly identical for pHCC (7.54 µM) and HepG2 (8.34 µM), but higher for SNU-387 (25.89 µM) and SNU-475 (16.57 µM). These results show clinically relevant similarities in response to the tested chemotherapy agents between pHCC and HepG2 (the most widely used human HCC cell line for in vitro investigations). Notably, differential drug responses observed between human HCC cell lines are consistent with previous studies, and are likely reflective of differential pathway alterations driving oncogenesis in these lines. These results indicate the OCM represents a valuable tool for screening promising chemotherapy agents, providing a novel large animal bridge between preclinical investigation in small animal models and human clinical trials. Citation Format: Mario F. Neto, Eileena F. Giurini, Hanna H. Chen, Jordan L. Newson, Lawrence B. Schook, Kyle M. Schachtschneider, Ron C. Gaba. Oncopig and human hepatocellular carcinoma cell lines exhibit similar response to liver cancer chemotherapy agents [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 4094.

Abstract 4085: Development of porcine hepatocellular carcinoma cell lines: Comprehensive in vitro and in vivo characterization

Abstract 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.

Abstract 2088: Characterization of an inducible alcoholic liver fibrosis model for hepatocellular carcinoma investigation in a transgenic porcine platform

Abstract Hepatocellular carcinoma (HCC) is a deadly tumor that spans more than 780,000 new diagnoses and causes 750,000 annual deaths worldwide. While global cancer incidence and mortality are generally decreasing, HCC incidence is rising and projected to continually increase given the growing prevalence of chronic liver diseases. Alcoholic liver disease represents a common progressive chronic liver ailment that incites liver cirrhosis—a precancerous state of liver scarring—that increases the risk for HCC development. The health status of the liver can also affect HCC tumor biology and treatment responses. Therefore, a large animal model capable of exhibiting both HCC and alcohol induced liver cirrhosis would be a valuable resource for preclinical research investigating HCC in its native comorbid cirrhotic microenvironment. This study utilized the innovative Oncopig Cancer Model (OCM)—a transgenic swine model that recapitulates human cancer through development of site and cell specific tumors via induced expression of heterozygous KRASG12D and TP53R167H transgenes—to develop alcohol induced fibrosis in a porcine model capable of developing HCC tumors. Liver injury was induced in Oncopigs (n=5) via transcatheter infusion of absolute ethanol and ethiodized oil (1:3 v/v dosed at 0.75 mL/kg) into the hepatic arterial circulation. Histological assessment of resected liver specimens collected 8 weeks post-induction revealed METAVIR stage F2-F4 fibrosis, stage A2-A3 inflammation, and a median percent fibrosis of 15.3% (range 5.0-22.9%). All METAVIR and inflammation scores were elevated compared to age matched controls (F0-F1, A0-A1, median percent fibrosis 8.7%, range 5.8-12.1%). Liver injury was then induced via the same protocol in a second OCM cohort (n=5) for a time course study in which post-induction disease surveillance via general health assessment, lab analysis, and serial liver biopsies was performed every 2 weeks for 20 weeks post-induction. Histology revealed METAVIR stage F1-F3 fibrosis as early as 2 weeks post-induction. However, as clinically observed for patients presenting with pre-cirrhotic alcohol induced liver damage, a lack of persistent alcohol exposure resulted in liver recovery as evidenced by METAVIR F1-F2 fibrosis levels at 20 weeks post-induction. No behavioral or biochemical abnormalities were observed during the monitoring period to suggest liver decompensation. In conclusion, this study successfully validated a protocol to develop a pre-cirrhotic METAVIR F2-F3 model of alcoholic liver disease within 8 weeks using transarterial ethanol-ethiodized oil infusion. While these results support the OCM's capability to serve as a model for HCC in a cirrhotic liver background, the data indicate prolonged exposure to alcohol may be required to develop an irreversible METAVIR stage F4 porcine model of cirrhosis. Citation Format: Kyle M. Schachtschneider, Nasya Mendoza-Elias, Daniel P. Regan, Kelly D. Garcia, Lauretta A. Rund, Regina M. Schwind, Lawrence B. Schook, Ron C. Gaba. Characterization of an inducible alcoholic liver fibrosis model for hepatocellular carcinoma investigation in a transgenic porcine platform [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 2088.

Abstract 3047: Development and initial application of a porcine-specific MRI and MRE protocol for liver imaging in a large animal cancer model

Abstract While global cancer mortality is generally decreasing, the incidence of hepatocellular carcinoma (HCC) is projected to increase given the growing prevalence of chronic liver diseases that increase the risk for carcinogenesis. The current diagnosis of liver cirrhosis is invasive tissue biopsy, and those at-risk undergo an HCC surveillance program of ultrasound imaging to survey tumor development. Such screening programs center on static radiologic imaging snapshots prescribed at arbitrary intervals based on empiric histologic cirrhosis staging schemes and observational tumor developmental data that neither reflect the transition between normal and diseased states nor biologically relevant disease thresholds. In contrast, magnetic resonance imaging (MRI) and magnetic resonance elastography (MRE) may provide a more foundational portrayal of early stages of liver disease and liver oncogenesis that could result in early diagnosis when curative therapies could be employed. In addition, MRI/MRE-correlated systemic molecular biomarkers indicative of specific disease stages also have potential to serve as important non-invasive diagnostic tools. In this study, we developed a clinically translatable MRI/MRE liver imaging protocol in a clinically relevant large animal platform, the Oncopig Cancer Model (OCM). The OCM is a novel transgenic swine model that recapitulates human HCC through development of site and cell specific tumors after Cre recombinase induced expression of heterozygous KRASG12D and TP53R167H transgenes. A clinical imaging workflow consisting of respiratory gated acquisitions was developed with the Siemens 3 T Prisma MRI scanner to include the following: T1 Flash in-phase and out-of-phase, T2-HASTE/BLADE acquisitions to provide motion-robust imaging, VIBE imaging, diffusion-weighted imaging with IntraVoxel Incoherent Motion (IVIM) for estimating blood flow, and MRE for quantifying liver stiffness. Additional MRI imaging for tumor characterization was also performed, spanning multiphase (arterial, venous, delayed) contrast-enhanced T1-weighted imaging. This novel imaging protocol was successfully applied in normal control Oncopig subjects (n = 2), resulting in high quality, high resolution MRI/MRE images for clinical interpretation. In the future, integration of MRI/MRE, histology, and molecular measures in Oncopigs presenting with liver cirrhosis and HCC may be employed to investigate biomarkers that may define normal versus diseased liver, and may serve as a new approach to identify optimal time points for initiation of disease screening and treatment administration for clinical translation. Citation Format: Faith M. Thomas, Bradley P. Sutton, Tracey M. Wszalek, Megan J. Dailey, Kyle M. Schachtschneider, Lawrence B. Schook, Ron C. Gaba. Development and initial application of a porcine-specific MRI and MRE protocol for liver imaging in a large animal cancer model [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 3047.

Abstract 2148: Image-guided catheter-based ultrasound thermal ablation of intramuscular and retroperitoneal sarcomas in the transgenic Oncopig cancer model

Abstract Soft-tissue sarcomas (STS) are aggressive, often lethal mesenchymal tumors that represent roughly 1% of adult cancers. The 5-year survival rate of STS has remained unchanged for decades at 50%, highlighting the need for more effective therapies. We are developing protocols and assessing the treatment efficacy of 3D spatially-registered real-time image-guided needle/catheter based ultrasound (CBUS) ablative thermal therapy in STS induced in the transgenic Oncopig cancer model (OCM). The transgenic Oncopig carries a Cre recombinase inducible transgene encoding KRASG12D and TP53R167H; both mutations commonly in human cancers. Cre recombinase induces expression of the transgenes and transformation of cells in culture and rapid and reproducible development of leiomyosarcomas when introduced into the muscle of oncopigs. The flexibility of the model allowed induction of sarcomas in two clinically relevant intramuscular (IM) sites in both the hind limb and retroperitoneal (RP) regions, to closely simulate human disease. We have previously shown that these Oncopig tumors present the same transcriptional hallmarks as human STS including altered TP53 signaling, Wnt signaling activation and evidence of epigenetic reprogramming. Initial acute studies revealed that the tumors could be successfully grown in the muscle reaching mean tumor volume of 19.76cm3 (RP, n= 8) and 25.12cm3 (IM, n=14) cm3 within two weeks following AdCre (5.0x108 to 1.0x109 PFU) injections. Tumors were then treated utilizing ultrasound image guidance combined with 3D EM tracking to place the applicator in the target region. Sectored tubular transducers were introduced through a small skin incision and used to precisely deliver thermal energy to the treatment region. Tumors were treated for 6-9 minutes at 7 watts acoustic power (10.2W/cm2). Thermocouples were inserted into the tumors monitored temperature throughout the treatment. Post-treatment analysis revealed that the mean induced lesion volume exceeded the actual tumor volume for both IM (43.41 cm3) and RP (22.87 cm3) tumors with mean peak temperatures reaching 64.56 ° C (IM) and 58.75 ° C (RP). The mean peak thermal dose (thermal equivalent minutes at 43 ° C - TEM43) accumulated for IM tumors was 3.70E+08, and 1.46E+06 for RP tumors. Histopathology analysis verified that treatment areas reached past tumor margins using single applicator configuration. This study clearly demonstrates that the Oncopig is a suitable model in size and clinically relevant tumor type for evaluation of ultrasound ablation instrumentation. In addition, these STS treatment protocols can reliably and accurately target and treat tumor volumes plus surrounding margins. Future work will include monitoring of treatment lesions for up to 3 months post ablation. Funded by NIH 5R21CA195433 Citation Format: Laurie A. Rund, Goutam Ghoshal, Emery Williams, Paul Neubauer, Lance Frith, R Tamas Heffter, Patrick Roady, Lawrence B. Schook, E Clif Burdette. Image-guided catheter-based ultrasound thermal ablation of intramuscular and retroperitoneal sarcomas in the transgenic Oncopig cancer model [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 2148.

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

PurposeThis study used the Oncopig Cancer Model (OCM) to develop alcohol-induced fibrosis in a porcine model capable of developing hepatocellular carcinoma. Materials and MethodsLiver 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. ResultsIn 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. ConclusionsThis 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.

The Oncopig Cancer Model as a Complementary Tool for Phenotypic Drug Discovery.

The screening of potential therapeutic compounds using phenotypic drug discovery (PDD) is being embraced once again by researchers and pharmaceutical companies as an approach to enhance the development of new effective therapeutics. Before the genomics and molecular biology era and the consecutive emergence of targeted-drug discovery approaches, PDD was the most common platform used for drug discovery. PDD, also known as phenotypic screening, consists of screening potential compounds in either in vitro cellular or in vivo animal models to identify compounds resulting in a desirable phenotypic change. Using this approach, the biological targets of the compounds are not taken into consideration. Suitable animal models are crucial for the continued validation and discovery of new drugs, as compounds displaying promising results in phenotypic in vitro cell-based and in vivo small animal model screenings often fail in clinical trials. Indeed, this is mainly a result of differential anatomy, physiology, metabolism, immunology, and genetics between humans and currently used pre-clinical small animal models. In contrast, pigs are more predictive of therapeutic treatment outcomes in humans than rodents. In addition, pigs provide an ideal platform to study cancer due to their similarities with humans at the anatomical, physiological, metabolic, and genetic levels. Here we provide a mini-review on the reemergence of PDD in drug development, highlighting the potential of porcine cancer models for improving pre-clinical drug discovery and testing. We also present precision medicine based genetically defined swine cancer models developed to date and their potential as biomedical models.

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.