Abstract
Abstract In order to take full advantage of the potential for new therapeutics and biotechnology applications, there is an urgent need for new animal models supporting cancer research. Pigs share many genetic and physiological similarities with humans. Our previous studies have shown that overexpression of several human oncogenes led to tumor development in pigs. Transgenic pigs were engineered to contain oncogenic KRASG12D and dominant-negative p53R167H, downstream of a LoxP-polyA (STOP)-LoxP sequence (LSL) and CAG promoter, such that exposure to Cre-recombinase induces their expression in desired tissues. Fibroblast cell strains generated from four such clones were infected with an adenovirus vector (Ad-Cre-GFP) encoding Cre recombinase and GFP protein or control vector (Ad-GFP) with GFP alone. Cells were analyzed for cell migration rates, cell proliferation, growth in soft agar, tumor development in immunodeficient mice, histopathological and characterization tumor and RT-PCR from tumor. All four cell strains expressed KRASG12D and p53R167H mRNA, transformed phenotypes such as increased cell migration rates, increase cell proliferation, and growth in soft agar. Migration rates in a wound assay were significantly different (184 vs 67 at 24 hr (p ≤ 0.01). CFSE assay determined that CRE cells divided twice as many times as control GFP cells in a 73 hr period (p ≤ 0.01). Additionally, GFP cells were unable to form colonies in soft agar, while each of the CRE cell lines formed over 100 colonies (p ≤ 0.01). CRE cell lines produced tumors in the mice (13/14) while no tumors developed from the GFP lines. Histopathological analyses revealed the tumors to be sarcomas, which were non-encapsulated, densely cellular and locally infiltrative with marked cellular and nuclear pleomorphism. Genetic analysis of tumor DNA showed the presence of CAG, KRASG12D and p53R167H transgene sequences. RT-PCR of tumor cDNA confirmed expression of the oncogenic KRASG12D and dominant-negative p53R167H mRNA from each tumor; therefore, demonstrating that induction of the transgenes in these porcine cells triggered a tumorigenic phenotype. Our next step will be to monitor tumor incidence following site-specific transgene induction in these Onco-pigs that can then be challenged with new anticancer therapies and interventional strategies. Citation Format: Tiago Collares, Fabiana K. Seixas, Laurie A. Rund, Wenping Hu, Fernanda M. Rodrigues, Ying Liang, Kuldeep Singh, Cristopher M. Counter, Lawrence B. Schook. Transgenic Onco-Pig cells mimic human cancer. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A21.
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