Benefits and Opportunities of the Transgenic Oncopig Cancer Model

Abstract

Cancer remains a leading cause of morbidity and mortality, and a paradigm shift is needed to fundamentally revisit drug development efforts. Pigs share close similarities to humans and may serve as an alternative model. Recently, a transgenic “Oncopig” line has been generated to induce solid tumors with organ-specificity, opening the potential of Oncopigs as a platform for developing novel therapeutic regimens.

Benefits of the Oncopig model over rodent and other pig cancer models

Rodents constitute a primary model for cancer research. However, most of the treatments developed by using mice as models have been failing at an alarming rate in the advanced stage of clinical trials, resulting in huge losses in capital, time, and patient lives. Findings in small animal models are rarely translated to the human cancer clinics [1]. Mice do not adequately mimic human cancer biology because of differences in body size, genetics/epigenetics, gene expression patterns, physiology and metabolism [2]. Therefore, a paradigm shift that uses alternative animal models like pigs in cancer research is long overdue. Pigs share many similarities with humans including a comparable body size, anatomy, physiology, diet, and genetics [2]. Even though there have been fewer studies involving pigs, research in pigs resulted in major contributions to our understanding of human disease and/or advancement of human patient care, including cystic fibrosis [3], cardiovascular [4], and other conditions [2].

Pigs have previously been used for cancer research. However, these models possess several limitations. For instance, in some studies human cancer cell lines were xenografted into pigs (some of them immunodeficient), while other pigs only carry a single and very specific (non-prevalent) mutations. These pig models have limited translatability as they do not recapitulate the local and systemic immune cell tumor microenvironment in vivo, or the mutations are not widely applicable and do not mimic the human tumor spectrum. Therefore, there exists an unmet need to develop an immunocompetent inducible pig model using human orthologous cancer mutations of high prevalence. To fill this gap, a transgenic pig (Oncopig) line carrying a key mutant tumor suppressor (TP53^R167H) [5] and a commonly found (20–50% depending upon the cancer type) oncogenic mutation (KRAS^G12D) [6] orthologous to humans was developed at the National Swine Research and Resource Center (NSRRC). The transgenic Oncopig utilizes a Cre-Lox system to control expression of the transgenes (CAG promoter-Lox-Stop-Lox-KRAS^G12D-IRES-TP53^R167H) [7]. The foundational Oncopig leveraged site-directed mutagenesis of KRAS and TP53 followed by somatic cell nuclear transfer and embryo transfer. While tumor suppressor gene TP53 is frequently mutated (and inactivated) in a majority of human cancers [5], the KRAS^G12D mutation is commonly detected in certain cancers (e.g., pancreatic, colorectal, bladder, ovarian, endometrial cancer [8]). Additionally, KRAS^G12D is a potential druggable target that can be readily tested in vivo in the Oncopig model before clinical translation to humans. The Oncopig model carries these two predominant and deterministic driver mutations (TP53^R167H, a dominant negative missense mutation, orthologous to humans TP53^R175H, and KRAS^G12D) of cancers and offers greater opportunities to study the pathophysiology of various devastating cancers through tissue-specific and targeted delivery of CRE via viral and non-viral vectors. Finally, the Oncopig model also allows the investigation of co-morbidities such as liver fibrosis or obesity [2]. Due to the Oncopigs’ high potential for the development of novel imaging modalities, surgical and radiological innovations, and theranostics, Oncopig offers great promise for clinical impact and precision oncology.

While there are many benefits of Oncopigs, there are some notable limitations of genetically engineered pig models compared to small animal models. These include higher cost of animals and husbandry, marginally longer time for the development of genetically engineered pigs, and availability of fewer models to-date. Some disadvantages include significant inflammatory reaction and spontaneous tumor regression in some Oncopigs models of cancer. However, the benefits outweigh the disadvantages, as Oncopigs hold much higher potential for understanding the complex pathophysiology of human cancer (Figure 1). Going forward, the Oncopig platform is expected to play a key role in developing novel cancer therapeutics.

Figure 1: Uses for the Oncopig cancer model.

Organ-specific Cre inducible tumors in Oncopigs. Translatability of the Oncopig with the use of diagnostic and therapeutic modalities that are widely used in the human clinic. PET, Positron Emission Tomography; ⁹⁰Y, Yttrium-90; SBRT, Stereotactic Body Radiation Therapy; TACE, Transarterial Chemoembolization. [Created with BioRender.com]

Applicability of Oncopigs for translational clinical research and precision oncology

Liver, pancreatic and lung cancer have been studied in Oncopigs by AdCre-mediated tumor induction in vivo. The same cancers have been extensively studied in mice using chemotoxic agents, implanted cancer cell lines, and genetically engineered mouse models such as KPC (LSL-Kras^G12D; LSL-Trp53^R172H; Pdx-Cre). Due to the sheer difference in anatomophysiology and tissue-specific epigenetics between mice and humans, the Oncopig model proves to be superior. Pancreatic cancer was successfully induced via laparoscopic AdCre injection into the pancreatic ducts of the Oncopig. The tumors presented similar molecular characteristics as human pancreatic cancer [9]. While there is a major inflammatory component with the tumors, analysis revealed undifferentiated carcinoma similar to those of human pancreaticobiliary system. In contrast to mice, pigs can undergo same advanced imaging performed in humans. For example, in pigs high-resolution CT can provide in-depth characterization on the vascularity of the tumor [10]. Pigs can also undergo multiphase contrast-enhanced CT and MRI angiography, making it optimal for longitudinal tracking of tumors and treatment response. Similar to humans, following transarterial embolization in Oncopigs non-contrast CT still showed retained contrast in liver tumors and follow-up scans revealed reduction in the size of tumors [11]. Therefore, due to anatomic similarities between human and pig liver, the Oncopig liver cancer model [12] is far superior to rodent models. While rabbits are currently used to test intra-arterial therapies, they have significantly different anatomy and physiology compared to pigs and humans. Because there are several limitations of intra-arterial therapies for human liver cancer (e.g., damage to the normal liver and high rate of recurrence), Oncopigs can serve as a platform to improve these types of minimally invasive therapies. While pigs can also undergo endoscopic interventions (e.g., via flexible bronchoscopy), a pilot Oncopig lung cancer model successfully induced tumors by endovascular and percutaneous inoculation with AdCre into the lungs [13]. Tumor histology showed epithelial and mesenchymal differentiations alongside significant peritumoral inflammation as previously observed in the pancreatic Oncopig cancer model [9]. However, the undifferentiated Oncopig lung tumors did not display any glandular or squamous epithelial differentiation. In contrast, human lung cancers mostly consist of classical histopathological characteristics of adenocarcinoma or squamous cell carcinoma. Developing a novel Oncopig with KRAS^G12C - the most prevalent KRAS mutation in lung cancer - may result in phenotypes found in human patients while allowing investigations of treatment responses to FDA-approved targeted drugs (e.g., sotorasib).

Immunocompetence of the Oncopig cancer model is a major advantage

All Oncopig-induced tumors showed intense immune reactions [9,12,13] underscoring a potential utility of Oncopigs to study immunotherapies. The porcine immune system is more robust than that of humans and may allow us to study immune mediated effects of antitumor immunity systemically and locally in the tumor microenvironment. Pigs have distinctly different T-cell lineages compared to humans that recognize foreign antigen in a non-MHC dependent fashion. Interestingly, the Oncopig immune system has been known to recognize and mount cytotoxic responses against tumor cells [14], which suggests adaptable recognition of induced tumors. Similarly, induction of lung cancer in Oncopig also displayed regression of the tumor secondary to intra-tumor immune cell infiltrations [13]. Oncopigs therefore are invaluable in studying and replicating cytotoxic T-cell-mediated antitumor response in humans [14]. This will likely open impactful new avenues for studying the effects of druggability of the immune system on cancer growth.

Concluding remarks

While the Oncopig model needs further development and characterization, it provides tremendous opportunities for translational cancer research. We have discussed the genetic make-up of the Oncopig, the importance of organ-specific large animal preclinical cancer models, and benefits of Oncopigs’ translatability to the human cancer patient. Given the abundance of TP53 and KRAS mutations in human cancers, we expect the Oncopig model to have a pivotal role in understanding the pathophysiology of a variety of cancers. Despite the limitations of cost and current lack of availability of established models and pig-specific reagents, the Oncopig model is anticipated to gain substantial popularity and be impactful for precision oncology research (Box 1).

Box 1: Advantages and Opportunities of the Oncopig Model.

• Superior to rodents due to body size, anatomophysiological characteristics, metabolism, genetics, epigenetics, and diet.

• Current Oncopig carries prevalent human orthologous mutations seen in many solid tumors.

• Option of targeted tumor induction in any organ of preference.

• Immunocompetent Oncopig model allows study of local and systemic immunity towards cancer.

• Large size allows for imaging, drug trialing and interventional testing modalities already used or applicable in humans.

• Transformative multiplex genetic modification in pigs by using CRISPR/Cas9 technology allows future customization of Oncopigs for specific cancer types.