Engraftment of Allotransplanted Tumor Cells in Adult rag2 Mutant Xenopus tropicalis

SIMPLE SUMMARY: While the mouse is without doubt the most studied animal for experimental cancer research, aquatic vertebrates such as zebrafish have also contributed to the field. More recently, thanks to the Nobel-prize winning technology of CRISPR/Cas mediated genomic engineering, the frog Xenopus tropicalis has emerged as an additional powerful model for studying human cancer. Via CRISPR-mediated genome editing, several models for different human cancers have been obtained in this animal. However, what has been lacking in Xenopus is the possibility to transplant tumor cells between different frogs. This is important to allow better characterization of the tumor cells and exploration of therapeutic opportunities. In this paper, we describe the generation of a genetic mutant in Xenopus tropicalis that has a compromised immune system, thereby allowing the grafting and expansion of tumors obtained in this species. In addition, an optimized protocol is provided for the irradiation of wild-type Xenopus frogs that subsequently are temporarily immunocompromised and during that period allow tumor engraftment. This work will expand the toolbox for modeling human cancer in Xenopus tropicalis, thereby further establishing it as a powerful experimental cancer model. ABSTRACT: Modeling human genetic diseases and cancer in lab animals has been greatly aided by the emergence of genetic engineering tools such as TALENs and CRISPR/Cas9. We have previously demonstrated the ease with which genetically engineered Xenopus models (GEXM) can be generated via injection of early embryos with Cas9 recombinant protein loaded with sgRNAs targeting single or multiple tumor suppressor genes. What has been lacking so far is the possibility to propagate and characterize the induced cancers via transplantation. Here, we describe the generation of a rag2 knockout line in Xenopus tropicalis that is deficient in functional T and B cells. This line was validated by means of allografting experiments with primary tp53(−/−) and apc(+/−)/tp53(−/−) donor tumors. In addition, we optimized available protocols for the sub-lethal irradiation of wild-type X. tropicalis froglets. Irradiated animals also allowed the stable, albeit transient, engraftment of transplanted X. tropicalis tumor cells. The novel rag2(−/−) line and the irradiated wild-type froglets will further expand the experimental toolbox in the diploid amphibian X. tropicalis and help to establish it as a versatile and relevant model for exploring human cancer.