Tulkens D et al. (2022), Engraftment of Allotransplanted Tumor Cells in ...

XB-ART-59395

Cancers (Basel) 2022 Sep 20;1419:. doi: 10.3390/cancers14194560.

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Engraftment of Allotransplanted Tumor Cells in Adult rag2 Mutant Xenopus tropicalis.

Tulkens D , Dimitrakopoulou D , Boelens M , Van Nieuwenhuysen T , Demuynck S , Toussaint W , Creytens D , Van Vlierberghe P , Vleminckx K .

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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.

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Species referenced: Xenopus tropicalis
Genes referenced: rag2 tp53

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	Figure 1. Generation of the X. tropicalis rag2−/− knockout line. (A) Embryos were injected with an sgRNA targeting the rag2 gene along with Cas9 protein. When sexually mature, animals were outcrossed to wild-types to obtain heterozygous animals that were subsequently incrossed to obtain rag2 homozygous mutant animals in the F2 generation. (B) Scatter plot showing correlation between in vivo observed mutational CRISPR repair outcomes in injected embryos (x-axis) versus predicted outcomes using the inDelphi algorithm tool (y-axis). Dashed lines show the 95% confidence interval corresponding to the best-fit linear regression line (solid line). (C) Genotyping of sampled F2 animals. Images taken from DNA electrophoresis gels after performing a normal HMA (left) and mixed HMA (right). Normal HMA included heating of the sampled PCR amplicons followed by slowly cooling and loading on the gel, while, for mixed HMA, sampled PCR samples were first mixed with wild-type rag2 amplicons, after which the HMA was performed. Multiple bands present in both gels indicate heterozygous animals, while extra bands only appearing after performing the mixed HMA (right gel) relate to homozygous mutant animals. Absence of any extra bands is indicative of wild-type animals. Presence of a 4 bp deletion in homozygous mutant animals was confirmed by Sanger sequencing.
	Figure 2. Validation of allografting in X. tropicalis rag2−/− animals. (A) tp53−/− donor animal harboring a thymic tumor (black arrows). (B) Transplantation strategy including the generation of a cell suspension using a 40 µm strainer followed by IP injections in a rag2−/− adult and a wild-type adult control (both 5 × 106 live cells). (C) A rag2−/− transplanted animal with a subcutaneous outgrowth close to the injection site (white arrow, white dashed line) 10 weeks post-transplantation. (D) Microscopy images (ventral view) of rag2−/− transplanted animal showing the engrafted tumor at the injection site before and after removal of the skin (top panels) and internal (top right and bottom). The tumor is also visible upon opening of the abdominal cavity (white arrowheads, white dashed line), in addition to a large tumor mass associated with the intestinal mesenterium (yellow arrowhead). (E) H and E and IHC stained sections from the primary tumor in the tp53−/− donor animal and the tumor graft in the transplanted rag2−/− animal. Black arrowheads indicate regions with neovascularization. (F) Mixed HMA analysis for the tp53 gene on DNA from tp53−/− tumor sample (donor animal), liver (without grafts) and two tumor grafts obtained from the transplanted rag2−/− animal. (G) Transplantation experiment starting from frozen tp53−/− tumor cells. H and E images from the rag2−/− transplant kidney versus a stage matched wild-type are shown. (H) Transplantation experiment starting from tumor ascites cells from an apc+/−/tp53−/− liver tumor bearing animal. Dissection micrographs from kidney, liver and lungs of rag2−/− transplant. Yellow and white arrowheads show visible engraftment regions in liver and lungs, respectively (left). H and E images from the liver tumor in donor animal and liver grafts in the transplanted animal (middle). The presence of tumor cells in the liver of the transplanted rag2−/− animal is confirmed by amplicon deep sequencing at the apc and tp53 target site (right). All black scale bars are 50 µm. White scale bars are 2 mm.
	Figure 3. Allografting in irradiated wild-type X. tropicalis animals. (A) Plots showing hemocytometer cell counts as represented by white blood cell (WBC)/red blood cell (RBC) ratios of irradiated animals and non-irradiated controls. (B) H and E and anti-CD3 immunostained sections from spleens and livers of all 4 groups. Yellow arrows show CD3 positive zones in the spleen; black arrows show CD3 positive cells in the liver. (C) IHC quantified CD3 data of spleens and livers using the open source digital analysis tool QuPath [20]. * p < 0.05, p < 0.01, * p < 0.001, **** p < 0.0001. All scale bars are 50 µm. Bar charts shown represent means with SD as error bar.
	Figure 4. Engraftment of tp53−/− tumor cells in irradiated wild-type X. tropicalis froglet. (A) IP fluid from tumor cell transplanted irradiated and non-irradiated control animals, stained with Natt and Herrick reagent. Red and yellow arrows indicate an RBC and a lymphocyte, respectively. The blue arrow shows a tumor blast cell. (B) H and E- (left) and PCNA-stained (right) sections of engrafted regions in kidney (black arrows) from transplanted irradiated froglet compared with respective kidney sections in the transplanted non-irradiated control froglet. (C) H and E- (left) and PCNA-stained (right) sections of engrafted regions in liver (black arrows) from transplanted irradiated froglet compared with respective liver sections in the transplanted non-irradiated control froglet. All scale bars are 50 µm.

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Asfari, Mycobacterium-induced infectious granuloma in Xenopus: histopathology and transmissibility. 1988, Pubmed, Xenbase