Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ)

Abstract

Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAF^V600E in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.

Keywords: Cancer; Electroporation; Melanoma; Metastasis; Zebrafish.

Fig. 1.

TEAZ. (A) Schematic representation of the method applied for the introduction of ubb:tdTomato directly under the dorsal fin of adult zebrafish. The purified plasmid DNA (1 μl of a 1000 ng/μl solution of ubb:tdTomato) is injected into anesthetized zebrafish using a pulled glass micropipette. Electrical pulses are directed across the injected region (settings: LV mode, 45 V, 5 pulses, 60 ms pulse length and 1 s pulse interval). Reporter expression can be visualized by fluorescent microscopy (n=2/2). (B) Electroporation of a CMV:tdTomato plasmid was performed and the animal followed for a period of 8 months (n=2/2). The fluorescent signal can be visualized as early as 1 dpe, with intensity peaking at ∼1 week and maintaining for at least 8 months. (C) Multiple plasmids will co-integrate in TEAZ. casper zebrafish were electroporated with a total volume of 1.0 μl (0.5 μl of 1000 ng/μl ubb:GFP and 0.5 μl of 1000 ng/μl ubb:tdTomato) and imaged using BF, GFP and tdTomato (n=3/3), revealing co-expression of the plasmids. (D) Promoter specificity is maintained following TEAZ. AB fish were electroporated with 1.0 μl total volume (0.5 μl of 1000 ng/μl ubb:GFP and 0.5 μl of 1000 ng/μl mitfa:tdTomato) and displayed highly restricted expression of the mitfa reporter plasmid, but widespread expression of the ubb plasmid (n=9/9). High-resolution imaging of the tdTomato⁺ cells reveals a dendritic phenotype, consistent with the melanocytic lineage.

Fig. 2.

Generation of a novel melanoma model with TEAZ. (A) mitfa:BRAF^V600E;tp53^−/−;mitfa^−/− zebrafish (triple strain) were electroporated with the miniCoopR:GFP plasmid that both rescues melanocytes and expresses GFP under the mitfa promoter, with (n=10) or without (n=9) two additional plasmids to genetically knockout rb1 (ubb:Cas9 and zfU6:sgRNA against rb1). The electroporated zebrafish were then imaged over time by both fluorescence and brightfield to monitor tumor development. Overall, 17/20 electroporated zebrafish had GFP⁺ cells. Tumor development in a representative zebrafish from the melanoma model including rb1 knockout is shown. (B) Higher-magnification view of the tumor-bearing animal shown in A at 16 weeks postelectroporation. (C) At 9 weeks postelectroporation, 4/8 zebrafish had evidence of GFP⁺ distant micrometastases in the head. (D) The loss of rb1 is essential for tumor initiation as visualized by the Kaplan–Meier curve comparing zebrafish electroporated with miniCoopR:GFP with or without rb1 sgRNA. Log-rank (Mantel–Cox) test was used for statistical analysis (****P<0.0001).

Fig. 3.

Melanoma model using TEAZ show evidence of rapid progression. (A) Pathology of tumor-bearing zebrafish (n=1) (along with control zebrafish, n=2) at 16 weeks postelectroporation, stained with H&E or anti-GFP immunohistochemistry, demonstrates a large primary tumor that is uniformly GFP⁺. (B-D) Histology reveals evidence of extensive invasion into the muscle (arrows) (B) along with micrometastatic sites within the kidney (C) or along blood vessels (arrows) (D). Images are visualized at 4× and 40×. Scale bars: 500 μm (4×) and 50 μm (40×). Dashed line boxes indicate the area enlarged at 40×.

Fig. 4.

Histological comparison of the embryo injection melanoma model and TEAZ melanoma model. (A) The left images show a melanoma created by injection of an mitfa:BRAF^V600E-tdTomato (fusion) transgene into a tp53^−/− background (n=1). Right images show a TEAZ-based melanoma created by electroporation of miniCoopR:GFP plus ubb:Cas9 plus zfU6:sgRNA against Rb1 (n=1) (example shown is fish at 16 weeks also shown in Fig. 2A). (B) H&E staining of both tumors shows similar histology, although with increased melanin pigmentation in the TEAZ tumor (also shown in Fig. 3A). (C,D) Antibody staining against BRAF^V600E shows that both tumors are widely BRAF^V600E positive, which correlates with high levels of phospho-ERK staining. (E) Reflecting the neural crest origin of melanocytes, both tumors show strong nuclear expression of SOX10. Images are visualized at 4× and 40×. Scale bars: 500 μm (4×) and 50 μm (40×). Dashed line boxes indicate the area enlarged at 40×.

Fig. 5.

Cancer modeling with TEAZ enables sequential electroporation of transgenes. A tumor-bearing fish (created with rb1 sgRNA as in Fig. 2) was imaged using GFP and tdTomato. As expected, only GFP⁺ tumor cells were seen, with no expression in the tdTomato channel. This tumor was then electroporated with an mitfa:tdTomato plasmid and re-imaged 5 days later, showing areas that are now both GFP⁺ and tdTomato⁺ (n=2).