Hepatocellular Carcinoma Epigenetic Patterns Correspond to Differences in Ethnoracial Status and Treatment Response in a Single-Center Retrospective Study.

PURPOSE: To correlate epigenetic patterns with ethnoracial status and locoregional therapy (LRT) response in patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: DNA and RNA were extracted from 47 distinct formalin-fixed paraffin-embedded tumor samples from 42 patients with HCC (n = 14 Black, n = 19 White, n = 9 Hispanic). LRT response was determined using computed tomography (CT) or magnetic resonance (MR) imaging 3 months posttreatment of 35 tumors (n = 22 complete response, n = 13 retreatment candidates). RNA expression and DNA methylation were used to stratify patients by ethnoracial status and treatment response using partial least-squares discriminant analysis (PLS-DA). Results were validated using hierarchical clustering. Ingenuity pathway analysis was performed to identify upstream regulators and pathways. RESULTS: PLS-DA identified 100 genes and 12 methylated regions that differentiated tumors from Black from White/Hispanic patients. Hierarchical clustering clustered samples with the top 16 genes or the top 5 methylation regions. Dysregulated pathways included adrenomedullin pathway (P = .030), EIF2 signaling (P = .007), and several metabolic pathways. AGTR1 (log2fold = 1.59) and GSTM3 (log2fold = 2.53) represented potential differentially expressed therapeutic targets. PLS-DA identified 100 genes and 150 methylation regions that differentiated between complete responders and retreatment candidates. Hierarchical clustering clustered samples with the top 30 genes or the top 13 methylation regions. Dysregulated pathways included metabolic and DNA repair-related pathways. ASAP2 (log2fold = 0.29) and RAD50 (log2fold = 0.22) represented potential differentially expressed therapeutic targets. CONCLUSIONS: Variation in gene expression and DNA methylation patterns in patients with HCC corresponded to ethnoracial status and LRT response. These initial results suggest tumor profiling has the potential to close ethnoracial disparities and improve treatment stratification.

Pigs as Clinically Relevant Models for Synergizing Interventional Oncology and Immunotherapy.

Traditionally, rodent cancer models have driven preclinical oncology research. However, they do not fully recapitulate characteristics of human cancers, and their size poses challenges when evaluating tools in the interventional oncologists' armamentarium. Pig models, however, have been the gold standard for validating surgical procedures. Their size enables the study of image-guided interventions using human ultrasound (US), computed tomography (CT), and magnetic resonance (MR) imaging platforms. Furthermore, pigs have immunologic features that are similar to those of humans, which can potentially be leveraged for studying immunotherapy. Novel pig models of cancer are being developed, but additional research is required to better understand both the pig immune system and malignancy to enhance the potential for pig models in interventional oncology research. This review aims to address the main advantages and disadvantages of using a pig model for interventional oncology and outline the specific characteristics of pig models that make them more suitable for investigation of locoregional therapies.

Swine global genomic resources: insights into wild and domesticated populations.

Suids, both domesticated and wild, are found on all continents except for Antarctica and provide valuable food resources for humans in addition to serving as important models for biomedical research. Continuing advances in genome sequencing have allowed researchers to compare the genomes from diverse populations of suids helping to clarify their evolution and dispersal. Further analysis of these samples may provide clues to improve disease resistance/resilience and productivity in domestic suids as well as better ways of classifying and conserving genetic diversity within wild and captive suids. Collecting samples from diverse populations of suids is resource intensive and may negatively impact endangered populations. Here we catalog extensive tissue and DNA samples from suids in collections in both Europe and North America. We include samples that have previously been used for whole genome sequencing, targeted DNA sequencing, RNA sequencing, and reduced representation bisulfite sequencing (RRBS). This work provides an important centralized resource for researchers who wish to access published databases.

Swine models for translational oncological research: an evolving landscape and regulatory considerations.

Swine biomedical models have been gaining in popularity over the last decade, particularly for applications in oncology research. Swine models for cancer research include pigs that have severe combined immunodeficiency for xenotransplantation studies, genetically modified swine models which are capable of developing tumors in vivo, as well as normal immunocompetent pigs. In recent years, there has been a low success rate for the approval of new oncological therapeutics in clinical trials. The two leading reasons for these failures are either due to toxicity and safety issues or lack of efficacy. As all therapeutics must be tested within animal models prior to clinical testing, there are opportunities to expand the ability to assess efficacy and toxicity profiles within the preclinical testing phases of new therapeutics. Most preclinical in vivo testing is performed in mice, canines, and non-human primates. However, swine models are an alternative large animal model for cancer research with similarity to human size, genetics, and physiology. Additionally, tumorigenesis pathways are similar between human and pigs in that similar driver mutations are required for transformation. Due to their larger size, the development of orthotopic tumors is easier than in smaller rodent models; additionally, porcine models can be harnessed for testing of new interventional devices and radiological/surgical approaches as well. Taken together, swine are a feasible option for preclinical therapeutic and device testing. The goals of this resource are to provide a broad overview on regulatory processes required for new therapeutics and devices for use in the clinic, cross-species differences in oncological therapeutic responses, as well as to provide an overview of swine oncology models that have been developed that could be used for preclinical testing to fulfill regulatory requirements.

The molecular and cellular basis of copper dysregulation and its relationship with human pathologies.

Copper (Cu) is an essential micronutrient required for the activity of redox-active enzymes involved in critical metabolic reactions, signaling pathways, and biological functions. Transporters and chaperones control Cu ion levels and bioavailability to ensure proper subcellular and systemic Cu distribution. Intensive research has focused on understanding how mammalian cells maintain Cu homeostasis, and how molecular signals coordinate Cu acquisition and storage within organs. In humans, mutations of genes that regulate Cu homeostasis or facilitate interactions with Cu ions lead to numerous pathologic conditions. Malfunctions of the Cu+ -transporting ATPases ATP7A and ATP7B cause Menkes disease and Wilson disease, respectively. Additionally, defects in the mitochondrial and cellular distributions and homeostasis of Cu lead to severe neurodegenerative conditions, mitochondrial myopathies, and metabolic diseases. Cu has a dual nature in carcinogenesis as a promotor of tumor growth and an inducer of redox stress in cancer cells. Cu also plays role in cancer treatment as a component of drugs and a regulator of drug sensitivity and uptake. In this review, we provide an overview of the current knowledge of Cu metabolism and transport and its relation to various human pathologies.

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.

Transarterial Embolization of Liver Cancer in a Transgenic Pig Model.

PURPOSE: To develop and characterize a porcine model of liver cancer that could be used to test new locoregional therapies. MATERIALS AND METHODS: Liver tumors were induced in 18 Oncopigs (transgenic pigs with Cre-inducible TP53R167H and KRASG12D mutations) by using an adenoviral vector encoding the Cre-recombinase gene. The resulting 60 tumors were characterized on multiphase contrast-enhanced CT, angiography, perfusion, micro-CT, and necropsy. Transarterial embolization was performed using 40-120 µm (4 pigs) or 100-300 µm (4 pigs) Embosphere microspheres. Response to embolization was evaluated on imaging. Complications were determined based on daily clinical evaluation, laboratory results, imaging, and necropsy. RESULTS: Liver tumors developed at 60/70 (86%) inoculated sites. Mean tumor size was 2.1 cm (range, 0.3-4 cm) at 1 week. Microscopically, all animals developed poorly differentiated to undifferentiated carcinomas accompanied by a major inflammatory component, which resembled undifferentiated carcinomas of the human pancreatobiliary tract. Cytokeratin and vimentin expression confirmed epithelioid and mesenchymal differentiation, respectively. Lymph node, lung, and peritoneal metastases were seen in some cases. On multiphase CT, all tumors had a hypovascular center, and 17/60 (28%) had a hypervascular rim. After transarterial embolization, noncontrast CT showed retained contrast medium in the tumors. Follow-up contrast-enhanced scan showed reduced size of tumors after embolization using either 40-120 µm or 100-300 µm Embosphere microspheres, while untreated tumors showed continued growth. CONCLUSIONS: Liver tumors can be induced in a transgenic pig and can be successfully treated using bland embolization.

7-Chloroquinoline-1,2,3-triazoyl carboxamides induce cell cycle arrest and apoptosis in human bladder carcinoma cells.

In the present study, the antitumoral properties of a series of 7-chloroquinoline-1,2,3-triazoyl-carboxamides (QTCA) were investigated by analyzing their cytotoxic activities against human bladder cells (5637; grade II carcinoma). In addition, their effects on cell viability, cell cycle arrest mechanisms, apoptosis induction, in silico molecular docking, and detection of pro-apoptotic and anti-apoptotic proteins were evaluated. The cytotoxicity assay identified major dose- and time-dependent cytotoxic effects in 5637 cells after they were exposed to treatment with QTCA, only minimal effects were observed on normal cells. A live/dead assay confirmed that significant cell death, arrest in the G0/G1 phase and apoptosis were associated with treatment by 1-(7-Chloroquinolin-4-yl)-5-methyl-N-phenyl-1H-1,2,3-triazole-4-carboxamide (QTCA-1) and 1-(7-Chloroquinolin-4-yl)-N-(4-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxamide (QTCA-4). The in silico results indicated that these compounds acted through different mechanisms for the induction of cell cycle arrest and apoptosis. Western blotting confirmed the binding of the QTCAs to pro- and anti-apoptotic proteins. In conclusion, QTCA-1 and QTCA-4 are promising candidates for inducing cytotoxicity, cell cycle arrest, and apoptosis in human bladder cancer cells.

Synthesis and biological evaluation of new antioxidant and antiproliferative chalcogenobiotin derivatives for bladder carcinoma treatment.

Approximately 90% of bladder carcinomas are of the urothelial carcinoma type, which are characterized by high rates of recurrence and predisposition to progress to invasive tumors, representing one of the most costly neoplasms for health systems. Intravesical chemotherapy is a standard for the treatment of non-invasive bladder cancer. However, chemotherapy is usually aggressive and cytotoxic, which increases the death rates caused by cancer. Heterocyclic compounds which exhibit favorable pharmacokinetic and pharmacodynamic properties may enhance drug affinity for a target protein by targeting the treatment. Thus, this work presents the synthesis, characterization, and in vitro biological evaluation of new antioxidant (inhibition of lipid peroxidation, scavenging of free radical DPPH, and thiol peroxidase-like activity) and antiproliferative chalcogenobiotin derivatives and tests them against bladder carcinoma 5637 cells. A prominent response was obtained for the selected compounds, with tellurium biotin derivatives displaying effective antioxidant and antiproliferative activity. The effective compounds also demonstrated no toxicity in in vitro or in vivo studies.

Mycobacterium bovis BCG in metastatic melanoma therapy.

Melanoma is the most aggressive form of skin cancer, with a high mortality rate and with 96,480 new cases expected in 2019 in the USS. BRAFV600E, the most common driver mutation, is found in around 50% of melanomas, contributing to tumor growth, angiogenesis, and metastatic progression. Dacarbazine (DTIC), an alkylate agent, was the first chemotherapeutic agent approved by the US Food and Drug Administration (FDA) used as a standard treatment. Since then, immunotherapies have been approved for metastatic melanoma (MM) including ipilimumab and pembrolizumab checkpoint inhibitors that help decrease the risk of progression. Moreover, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) serves as an adjuvant therapy that induces the recruitment of natural killer NK, CD4+, and CD8+ T cells and contributes to antitumor immunity. BCG can be administered in combination with chemotherapeutic and immunotherapeutic agents and can be genetically manipulated to produce recombinant BCG (rBCG) strains that express heterologous proteins or overexpress immunogenic proteins, increasing the immune response and improving patient survival. In this review, we highlight several studies utilizing rBCG immunotherapy for MM in combination with other therapeutic agents.

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.

Gene and transposable element methylation in great tit (Parus major) brain and blood.

BACKGROUND: Studies on vertebrate DNA methylomes have revealed a regulatory role of tissue specific DNA methylation in relation to gene expression. However, it is not well known how tissue-specific methylation varies between different functional and structural components of genes and genomes. Using whole-genome bisulfite sequencing data we here describe both CpG and non-CpG methylation profiles of whole blood and brain tissue in relation to gene features, CpG-islands (CGIs), transposable elements (TE), and their functional roles in an ecological model species, the great tit (Parus major). RESULTS: We show that hypomethylation at the transcription start site (TSS) is enriched in genes with functional classes that relate directly to processes specific to each tissue type. We find that 6877 (~21 %) of the CGIs are differentially methylated between blood and brain, of which 1186 and 2055 are annotated to promoter and intragenic regions, respectively. We observe that CGI methylation in promoter regions is more conserved between tissues compared to CGI methylation in intra and inter-genic regions. Differentially methylated CGIs in promoter and intragenic regions are overrepresented in genomic loci linked to development, suggesting a distinct role for CGI methylation in regulating expression during development. Additionally, we find significant non-CpG methylation in brain but not in blood with a strong preference for methylation at CpA dinucleotide sites. Finally, CpG hypermethylation of TEs is significantly stronger in brain compared to blood, but does not correlate with TE activity. Surprisingly, TEs showed significant hypomethylation in non-CpG contexts which was negatively correlated with TE expression. CONCLUSION: The discovery that TSS methylation levels are directly linked to functional classes related to each tissue provides new insights in the regulatory role of DNA-methylation patterns. The dominant sequence motifs for brain non-CpG methylation, similar to those found in mammals, suggests that a conserved non-CpG regulatory mechanism was already present in the amniote ancestor. The negative correlation between brain non-CpG methylation and TE activity (not found for CpG methylation) suggests that non-CpG is the dominant regulatory form of methylation in TE silencing.

Impact of neonatal iron deficiency on hippocampal DNA methylation and gene transcription in a porcine biomedical model of cognitive development.

BACKGROUND: Iron deficiency is a common childhood micronutrient deficiency that results in altered hippocampal function and cognitive disorders. However, little is known about the mechanisms through which neonatal iron deficiency results in long lasting alterations in hippocampal gene expression and function. DNA methylation is an epigenetic mark involved in gene regulation and altered by environmental factors. In this study, hippocampal DNA methylation and gene expression were assessed via reduced representation bisulfite sequencing and RNA-seq on samples from a previous study reporting reduced hippocampal-based learning and memory in a porcine biomedical model of neonatal iron deficiency. RESULTS: In total 192 differentially expressed genes (DEGs) were identified between the iron deficient and control groups. GO term and pathway enrichment analysis identified DEGs associated with hypoxia, angiogenesis, increased blood brain barrier (BBB) permeability, and altered neurodevelopment and function. Of particular interest are genes previously implicated in cognitive deficits and behavioral disorders in humans and mice, including HTR2A, HTR2C, PAK3, PRSS12, and NETO1. Altered genome-wide DNA methylation was observed across 0.5 million CpG and 2.4 million non-CpG sites. In total 853 differentially methylated (DM) CpG and 99 DM non-CpG sites were identified between groups. Samples clustered by group when comparing DM non-CpG sites, suggesting high conservation of non-CpG methylation in response to neonatal environment. In total 12 DM sites were associated with 9 DEGs, including genes involved in angiogenesis, neurodevelopment, and neuronal function. CONCLUSIONS: Neonatal iron deficiency leads to altered hippocampal DNA methylation and gene regulation involved in hypoxia, angiogenesis, increased BBB permeability, and altered neurodevelopment and function. Together, these results provide new insights into the mechanisms through which neonatal iron deficiency results in long lasting reductions in cognitive development in humans.

Peripheral viral infection induced microglial sensome genes and enhanced microglial cell activity in the hippocampus of neonatal piglets.

Although poorly understood, early-life infection is predicted to affect brain microglial cells, making them hypersensitive to subsequent stimuli. To investigate this, we assessed gene expression in hippocampal tissue obtained from a previously published study reporting increased microglial cell activity and reduced hippocampal-dependent learning in neonatal piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus that induces interstitial pneumonia. Infection altered expression of 455 genes, of which 334 were up-regulated and 121 were down-regulated. Functional annotation revealed that immune function genes were enriched among the up-regulated differentially expressed genes (DEGs), whereas calcium binding and synaptic vesicle genes were enriched among the down-regulated DEGs. Twenty-six genes encoding part of the microglia sensory apparatus (i.e., the sensome) were up-regulated (e.g., IL1R1, TLR2, and TLR4), whereas 15 genes associated with the synaptosome and synaptic receptors (e.g., NPTX2, GABRA2, and SLC5A7) were down-regulated. As the sensome may foretell microglia reactivity, we next inoculated piglets with culture medium or PRRSV at PD 7 and assessed hippocampal microglia morphology and function at PD 28 when signs of infection were waning. Consistent with amplification of the sensome, microglia from PRRSV piglets had enhanced responsiveness to chemoattractants, increased phagocytic activity, and secreted more TNFα in response to lipopolysaccharide and Poly I:C. Immunohistochemical staining indicated PRRSV infection increased microglia soma length and length-to-width ratio. Bipolar rod-like microglia not evident in hippocampus of control piglets, were present in infected piglets. Collectively, this study suggests early-life infection alters the microglia sensome as well as microglial cell morphology and function.

Adult porcine genome-wide DNA methylation patterns support pigs as a biomedical model.

BACKGROUND: Pigs (Sus scrofa) provide relevant biomedical models to dissect complex diseases due to their anatomical, genetic, and physiological similarities with humans. Aberrant DNA methylation has been linked to many of these diseases and is associated with gene expression; however, the functional similarities and differences between porcine and human DNA methylation patterns are largely unknown. METHODS: DNA and RNA was isolated from eight tissue samples (fat, heart, kidney, liver, lung, lymph node, muscle, and spleen) from the adult female Duroc utilized for the pig genome sequencing project. Reduced representation bisulfite sequencing (RRBS) and RNA-seq were performed on an Illumina HiSeq2000. RRBS reads were aligned using BSseeker2, and only sites with a minimum depth of 10 reads were used for methylation analysis. RNA-seq reads were aligned using Tophat, and expression analysis was performed using Cufflinks. In addition, SNP calling was performed using GATK for targeted control and whole genome sequencing reads for CpG site validation and allelic expression analysis, respectively. RESULTS: Analysis on the influence of DNA variation in methylation calling revealed a reduced effectiveness of WGS datasets in covering CpG rich regions, as well as the usefulness of a targeted control library for SNP detection. Analysis of over 500,000 CpG sites demonstrated genome wide methylation patterns similar to those observed in humans, including reduced methylation within CpG islands and at transcription start sites (TSS), X chromosome inactivation, and anticorrelation of TSS CpG methylation with gene expression. In addition, a positive correlation between TSS CpG density and expression, and a negative correlation between TSS TpG density and expression were demonstrated. Low but non-random non-CpG methylation (<1%) was also detected in all non-neuronal somatic tissues, with differences in tissue clustering observed based on CpG and non-CpG methylation patterns. Finally, allele specific expression analysis revealed enrichment of genes involved in metabolic and regulatory processes. DISCUSSION: These results provide transcriptional and DNA methylation datasets for the biomedical community that are directly relatable to current genomic resources. In addition, the correlation between TSS CpG density and expression suggests increased mutation rates at CpG sites play a significant role in adaptive evolution by reducing CpG density at TSS over time, resulting in higher methylation levels in these regions and more permanent changes to lower gene expression. This is proposed to occur predominantly through deamination of 5-methylcytosine to thymidine, resulting in the replacement of CpG with TpG sites in these regions, as indicated by the increased TSS TpG density observed in non-expressed genes, resulting in a negative correlation between expression and TSS TpG density. CONCLUSIONS: This study provides baseline methylation and gene transcription profiles for a healthy adult pig, reports similar patterns to those observed in humans, and supports future porcine studies related to human disease and development. Additionally, the observed reduced CpG and increased TpG density at TSS of lowly expressed genes suggests DNA methylation plays a significant role in adaptive evolution through more permanent changes to lower gene expression.

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

Introduction: 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. Methods: 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: 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. Discussion: 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.

Induction and preliminary characterization of neoplastic pulmonary nodules in a transgenic pig model.

OBJECTIVES: Lung cancer models in large animals are lacking. Oncopigs are transgenic pigs that carry both KRASG12D and TP53R167H Cre-inducible mutations. This study aimed to develop and histologically characterize a swine model of lung cancer that could serve for preclinical studies evaluating locoregional therapies. MATERIALS AND METHODS: In two Oncopigs, an adenoviral vector encoding the Cre-recombinase gene (AdCre) was injected endovascularly through the pulmonary arteries or inferior vena cava. In two other Oncopigs, a lung biopsy was performed and incubated with AdCre, before reinjecting the mixture into the lungs percutaneously. Animals were clinically and biologically (complete blood count, liver enzymes and lipasemia) monitored. Obtained tumors were characterized on computed tomography (CT) and on pathology and immunohistochemistry (IHC). RESULTS: Neoplastic lung nodules developed following 1 (1/10, 10%) endovascular inoculation, and 2 (2/6, 33%) percutaneous inoculations. All lung tumors were visible at the 1-week CT, and appeared as well-circumscribed solid nodules, with a median longest diameter of 14 mm (range: 5-27 mm). Only one complication occurred: an extravasation of the mixture into the thoracic wall during a percutaneous injection that resulted in a thoracic wall tumor. Pigs remained clinically healthy during the entire follow-up (14-21 days). On histology, tumors consisted of inflammatory undifferentiated neoplasms composed of atypical spindle and epithelioid cells and/or a fibrovascular stroma and abundant mixed leukocytic infiltrate. On IHC, atypical cells diffusely displayed expression of vimentin and some showed expression of CK WSS and CK 8/18. The tumor microenvironment contained abundant IBA1 + macrophages and giant cells, CD3 + T cells, and CD31 + blood vessels. CONCLUSION: Tumors induced in the lungs of Oncopigs are fast growing poorly differentiated neoplasms associated with a marked inflammatory reaction that can be easily and safely induced at site specific locations. This large animal model might be suitable for interventional and surgical therapies of lung cancer.

Effect of CRISPR Knockout of AXIN1 or ARID1A on Proliferation and Migration of Porcine Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive disease lacking effective treatment. Animal models of HCC are necessary for preclinical evaluation of the safety and efficacy of novel therapeutics. Large animal models of HCC allow testing image-guided locoregional therapies, which are widely used in the management of HCC. Models with precise tumor mutations mimicking human HCC provide valuable tools for testing precision medicine. AXIN1 and ARID1A are two of the most frequently mutated genes in human HCC. Here, we investigated the effects of knockout of AXIN1 and/or ARID1A on proliferation, migration, and chemotherapeutic susceptibility of porcine HCC cells and we developed subcutaneous tumors harboring these mutations in pigs. Gene knockout was achieved by CRISPR/Cas9 and was validated by Next Generation Sequencing. AXIN1 knockout increased the migration of porcine HCC cells but did not alter the cell proliferation. Knockout of ARID1A increased both the proliferation and migration of porcine HCC cells. Simultaneous knockout of AXIN1 and ARID1A increased the migration, but did not alter the proliferation of porcine HCC cells. The effect of gene knockout on the response of porcine HCC cells to two of the most commonly used systemic and locoregional HCC treatments was investigated; sorafenib and doxorubicin, respectively. Knockout of AXIN1 and/or ARID1A did not alter the susceptibility of porcine HCC cells to sorafenib or doxorubicin. Autologous injection of CRISPR edited HCC cells resulted in development of subcutaneous tumors in pigs, which harbored the anticipated edits in AXIN1 and/or ARID1A. This study elucidates the effects of CRISPR-mediated knockout of HCC-associated genes in porcine HCC cells, and lays the foundation for development and utilization of genetically-tailored porcine HCC models for in vivo testing of novel therapeutic approaches in a clinically-relevant large animal model.