Immunological characterization of the Oncopig model and detection of cell-mediated immune responses to cancer

N. Overgaard, D.R. Principe, J.T. Jakobsen, L.A. Rund, P.J. Grippo, G. Jungersen, L.B. Schook
American Association for Cancer Research Annual Meeting, April 1-5 2017, Washington D.C.

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 confırmed using intracellular flow cytometry staining or immunohistochemistry (IHC). Subcutaneous, intramuscular, and testicular tumors all showed the presence of infıltrating 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 confırmed the CD3 staining and found that the infıltrating T-cell pool was comprised of both cytotoxic and regulatory cells as indicated by positive flow cytometry staining for the CD8 chain and FoxP3, respectively. In order to investigate the immunogenicity of the tumor cells, we set up flow cytometry detection of porcine TNF-, IFN-, 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 fırst fluorescence-based in vitro porcine cytotoxicity assay and found a signifıcant recognition and specifıc killing of autologous tumor cells in an effector-target titration dependent manner. Taken together, our results show that the established Oncopig tumors are infıltrated 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-specifıc killing hence underline the potential in using the Oncopig for future testing of immune therapies against human cancer.