Abstract
Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer. Transgenic (TG) minipigs were generated using somatic cell nuclear transfer by handmade cloning. The pigs encode two TG cassettes: (a) an Flp recombinase‐inducible oncogene cassette containing KRAS‐G12D, cMYC, SV40LT – which inhibits p53 – and pRB and (b) a 4‐hydroxytamoxifen (4‐OHT)‐inducible Flp recombinase activator cassette controlled by the intestinal epithelium‐specific villin promoter. Thirteen viable transgenic minipigs were born. The ability of 4‐OHT to activate the oncogene cassette was confirmed in vitro in TG colonic organoids and ex vivo in tissue biopsies obtained by colonoscopy. In order to provide proof of principle that the oncogene cassette could also successfully be activated in vivo, three pigs were perorally treated with 400 mg tamoxifen for 2 × 5 days. After two months, one pig developed a duodenal neuroendocrine carcinoma with a lymph node metastasis. Molecular analysis of the carcinoma and metastasis confirmed activation of the oncogene cassette. No tumor formation was observed in untreated TG pigs or in the remaining two treated pigs. The latter indicates that tamoxifen delivery can probably be improved. In summary, we have generated a novel inducible oncopig model of intestinal cancer, which has the ability to form metastatic disease already two months after induction. The model may be helpful in bridging the gap between basic research and clinical usage. It opens new venues for longitudinal studies of tumor development and evolution, for preclinical assessment of new anticancer regimens, for pharmacology and toxicology assessments, as well as for studies into biological mechanisms of tumor formation and metastasis.
Highlights
In cancer research, animal models play an important role for establishing a link between basic research and clinical application
The following plasmids were used in the construction: the pLVCT-rtTR-KRAB-2SM2 (Addgene plasmid #11779), and the pBABE-puroSV40LT (Addgene plasmid #13970); the yellow fluorescent protein (YFP)-F2AKRAS G12D-T2A-cMYC, cytomegalovirus early enhancer chicken b-actin rabbit b-globin (CAG)-red fluorescent protein (RFP), and the tet-responsive elements (TRE) element were ordered from GenScript (Piscataway, NJ, USA)
The first part consists of RFP and a puromycin selection gene followed by a polyA stop element, which prevents expression of the second part of the cassette
Summary
Animal models play an important role for establishing a link between basic research and clinical application. Numerous rodent models have been developed for studying intestinal cancer. They have provided insights into pathogenic mechanisms, served as tools for discovery and validation of novel therapeutic targets and for testing chemoprevention strategies (Le Magnen et al, 2016). The limitations of rodents as models of human cancer include large differences in lifespan, size, and physiology. The pig is a promising alternative model organism due to its high degree of similarity to humans, including longevity, size, organ anatomy, physiology, drug metabolism, genetics, and immunology (Flisikowska et al, 2013; Forster et al, 2010; Kuzmuk and Schook, 2011; Manno et al, 2016; Mote and Rothschild, 2006; Wernersson et al, 2005). The large size of pigs brings opportunities for longitudinal sampling and monitoring of tumor evolution during treatment, which is generally not possible in rodents
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