Abstract 6137: Development of ARID1A-deficient hepatocellular carcinoma in transgenic pigs
Authors: Elkhadragy Lobna , Totura William M. , Goli Kimia Dasteh , Schook Lawrence B. , Gaba Ron C. , Schachtschneider Kyle M. 
Published: September 2020

Abstract
Hepatocellular carcinoma (HCC) is an aggressive disease with poor response to treatment at advanced stages. Frameshift and nonsense mutations in ARID1A, a subunit of the SWI/SNF chromatin-remodeling complex, are among the most frequent genetic alterations in human HCC. Hence, it is necessary to develop animal models for preclinical assessment of targeted therapies for HCC with ARID1A deficiency. Due to the remarkable physiologic, genetic, and size similarities between pigs and humans, porcine models are excellent candidates for translational HCC studies. Here, we generated a porcine HCC model with ARID1A deficiency using the Oncopig, a transgenic pig that harbors heterozygous Cre recombinase-inducible TP53R167H and KRASG12D transgenes. Porcine HCC cells were developed by transduction of hepatocytes isolated from four Oncopigs with adenoviral vector encoding Cre recombinase. TP53R167H and KRASG12D expression in the developed HCC cell lines was confirmed by RT-PCR, and the hepatocyte origin of the cells was validated by positive arginase-1 staining. To disrupt ARID1A expression, Oncopig HCC cell lines were transfected with recombinant Cas9 nuclease complexed with a gRNA targeting ARID1A. This resulted in &gt;85% ARID1A editing in each of the four cell lines as determined by targeted Illumina sequencing. Single cell clones were isolated and screened for ARID1A mutations. The functional effect of ARID1A deficiency was evaluated in ARID1A-edited cell pools and ARID1A knockout single cell clones. Consistent with its reported tumor suppressor role, deficiency of ARID1A increased Oncopig HCC cell proliferation as measured by MTS assays. Following in vitro characterization, ARID1A-deficient Oncopig HCC cells were injected autologously into subcutaneous sites in Oncopig flanks, resulting in development of masses measuring 1-2 cm within two weeks. These subcutaneous masses were excised and fragmented, and 2 - 3 fragments were engrafted into the liver of the same animal by ultrasound-guided injection. Development of intrahepatic masses was detected by ultrasound imaging as well as CT scans. Following euthanasia of the Oncopigs, the intrahepatic masses were collected for histological and genomic analyses. Subcutaneous and intrahepatic masses were histologically characterized as HCC, and CRISPR-Cas9 mediated ARID1A deficiency was confirmed by targeted Illumina sequencing. To conclude, we have developed a novel large animal model of ARID1A-deficient HCC and demonstrated the feasibility of generating genetically-tailored HCC tumors in Oncopigs. These translational HCC models are promising tools for testing precision medicine approaches and investigating the contribution of clinically-relevant driver mutations on treatment susceptibility.
Citation Format: Lobna Elkhadragy, William M. Totura, Kimia Dasteh Goli, Lawrence B. Schook, Ron C. Gaba, Kyle M. Schachtschneider. Development of ARID1A-deficient hepatocellular carcinoma in transgenic pigs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6137.