Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ)

Background

Zebrafish are proving themselves very useful as a model of complex pathologies, including melanoma. Researchers have generated numerous zebrafish transgenic melanoma model, which harbour mutations commonly found in human melanoma patients. These models are powerful tools that allow investigation into the molecular mechanisms behind melanoma, and crucially, into the discovery of drugs to target and kill cancerous cells.

These transgenic models are not perfect however, as often the timing and location of tumour formation can be quite variable^[1], making monitoring of carcinogenesis amongst cohorts tricky. Tumour transplantation studies give more spatial and temporal control, but there is often a requirement to use immunosuppressed animals which may differ from immunocompetent fish in terms of the tumour microenvironment^[2].

To address these problems, Callahan and colleagues have established a technique called TEAZ (Transgene Electroporation in Adult Zebrafish) in which different oncogenic transgenic constructs are electroporated directly into adult somatic tissue either simultaneously or sequentially to model melanoma.

TEAZ in a nutshell: plasmid DNA, in this case a ubb:tdTomato construct that will ubiquitously drive fluorescent protein expression, is injected into the adult zebrafish. Brief electrical pulses are applied to the anaesthetised fish to allow the plasmid to enter cells. The fluorescent protein is then observable using fluorescent microscopy.  From Callahan et al., 2018 Figure 1a (under CC-BY-NC-ND 4.0)

Key findings

1. The authors first electroporated multiple constructs to drive fluorescent protein expression in defined regions of the zebrafish. Not only was fluorescence still stable after eight months, but cell type-specific transgene expression was achieved by using a melanocyte-specific reporter construct (mitfa:tdTomato).
2. The team then took an existing melanoma model (mitfa:BRAF^V600E;p53^-/-;mitfa^-/-)^[3], and electroporated in CRISPR-Cas9 elements to disrupt the tumour suppressor rb1. Strikingly, the electroporated animals developed melanotic lesions in just 3-7 weeks, compared to the 3-6 months taken for non-electroporated fish.
3. These melanotic lesions were highly tumourigenic, and reminiscent of human high-grade melanoma. Histological analyses confirmed distant metastases in a range of locations – an event not documented with previous melanoma models.
4. As proof-of-principle, the team electroporated mitfa:tdTomato into an existing tumour, in order to see if established tumours could be sequentially modified. In one week, cell-specific fluorescence could be observed within the tumour, suggesting that TEAZ could be used to model secondary mutations after tumour onset.

Why I chose this preprint

As someone in a zebrafish disease model lab, I often get a ‘lightbulb moment’ where I think of a great experiment, followed by a sinking feeling as I realise that it would only be made possible by using my fish’s great-grandchildren after rounds of painstaking genotyping.  A way of introducing transgenes into an adult fish in any genetic background, and having spatial and temporal control of this could save labs so much time and reduce the numbers of fish required.

As our understanding of the molecular events behind cancer grows, it is important that our disease models reflect the nuances of human pathologies. Which genes drive tumour initiation vs tumour metastasis, and does the order of mutation acquisition matter? The thought of being able to reproducibly disrupt or repair genes in adult somatic cells using CRISPR, and to thereby screen for factors influencing tumour progression and metastasis is really exciting, and I look forward to seeing what this team and others do with TEAZ next. I also think TEAZ could be broadly useful as a quick way to introduce multiple fluorescent reporter constructs into a disease model, in order to image different subpopulations of cells in vivo within their microenvironment.

Open questions

• The analysis of a tumour from an rb1 sgRNA-electroporated fish revealed the formation of a mixed histology sarcoma and melanoma tumour. The authors attributed this to using a ubiquitous instead of tissue-specific Cas9. As only two electroporated fish were used for this study, it might be important to find out how often these unexpected tumours occur in a larger cohort relative to more typical melanomas. If using a tissue-specific Cas9, the sgRNA and Cas9 might modify a much smaller proportion of cells, so it would be interesting to see if melanomas form to the same extent as when using ubiquitously-expressed Cas9.
• Perhaps related to the above, Callahan and colleagues sequenced these tumour cells and found two different mutations in 2% of rb1 alleles each. This was evidently more than enough to drive aggressive melanomas, but it will be interesting to see whether this frequency might be too low to make a biological difference when investigating other genes, or whether this mosaicism is in fact a good thing as it is more representative of a malignant subpopulation of cells within a tumour.

References

1. Patton, E. E., et al. (2005). BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr. Biol. 15 , 249–254.
2. Tang, Q., et al. (2016). Imaging tumour cell heterogeneity following cell transplantation into optically clear immune-deficient zebrafish. Nat. Commun. 7 , 10358.
3. Ceol, C. J., et al. (2011). The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. Nature 471 , 513–517.

Tags: cancer models, skin cancer, transgenesis methods

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