Abstract 1659: Immunological characterization of the Oncopig model and detection of cell-mediated immune responses to cancer
Authors: Overgaard Nana H. , Principe Daniel R. , Jakobsen Jeanne T. , Rund Laurie A. , Grippo Paul J. , Jungersen Gregers , Schook Lawrence B. 
Published: July 2017

Abstract
In recent years, immunotherapy has shown great breakthroughs; however, the majority of preclinical studies has been based solely on rodent models and tends to experience a troublesome translation to human patients often related to the differences between mice and humans. Here, we utilized the Oncopig model that, due to its similarity in the immunome, metabolism, and size between humans and pigs, can serve as a supplementary large animal model for translational cancer research. With the establishment of the immunoscore as a new approach for staging cancer patients, it has become evident that the immune status of the tumor microenvironment has a crucial impact on the patient’s outcome and response to therapies. For this reason, we set out to perform an immunological characterization of Oncopig-derived tumors to elucidate the potential in using this model for testing cancer immune therapeutic approaches. Following injection of Cre recombinase, the transgenic Oncopigs express KRASG12D and TP53R167H, two mutations commonly found in human cancers, subsequently resulting in tumor formation at the site of injection. The expression of these two transgenes was confirmed using intracellular flow cytometry staining or immunohistochemistry (IHC). Subcutaneous, intramuscular, and testicular tumors all showed the presence of infiltrating CD3+ cells, which were found both in the periphery and in the core of the tumor as determined by IHC. With the establishment of a digestion protocol allowing isolation of the in vivo-generated tumor cells, we confirmed the CD3+ staining and found that the infiltrating T-cell pool was comprised of both cytotoxic and regulatory cells as indicated by positive flow cytometry staining for the CD8β chain and FoxP3, respectively. In order to investigate the immunogenicity of the tumor cells, we set up flow cytometry detection of porcine TNF-α, IFN-γ, and perforin in a PBMC-tumor cell co-culture. Despite the production of various cytokines, actual tumor cell killing is a desirable parameter to measure when evaluating both the immunogenicity and response to therapy. Therefore, we developed, to our knowledge, the first fluorescence-based in vitro porcine cytotoxicity assay and found a significant recognition and specific killing of autologous tumor cells in an effector-target titration dependent manner. Taken together, our results show that the established Oncopig tumors are infiltrated by T cells exhibiting an either cytotoxic or regulatory phenotype, thus indicative of a tumor microenvironment mimicking the complexity seen in human patients. Additionally, we were able to measure cell-mediated immune responses to cancer in this novel, large animal model, and both the cytokine production and tumor-specific killing hence underline the potential in using the Oncopig for future testing of immune therapies against human cancer.
Citation Format: Nana H. Overgaard, Daniel R. Principe, Jeanne T. Jakobsen, Laurie A. Rund, Paul J. Grippo, Gregers Jungersen, Lawrence B. Schook. Immunological characterization of the Oncopig model and detection of cell-mediated immune responses to cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1659. doi:10.1158/1538-7445.AM2017-1659