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Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ)

Scott J Callahan, Stephanie Tepan, Yan M Zhang, Helen Lindsay, Alexa Burger, Nathaniel R Campbell, Isabella S Kim, Travis J Hollmann, Lorenz Studer, Christian Mosimann, Richard M White

Preprint posted on April 09, 2018 https://www.biorxiv.org/content/early/2018/04/09/297234

TEAZing fish melanoma models – Transgene Electroporation of Adult Zebrafish as a simple way of genetically modifying somatic tissues

Selected by Hannah Brunsdon

Categories: cancer biology, genetics

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:BRAFV600E;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

Read preprint (1 votes)




  • Author's response

    Scott Callahan shared

    Thank you so much for your interest and discussion of our work! We are very excited about the novel possibilities that open up with TEAZ and we are very hopeful that other labs will adopt the technique to answer novel questions that we haven’t even thought of. We agree that your open questions are exciting areas for further research.  We would like to add a little further insights into the questions with the hopes that this may spur further discussion and experiments!

    • 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.

    On your first point, we analyzed 9 fish in this initial pilot study and were excited to find melanomas develop in all 9 fish as identified by pigmentation, and typical morphology under brightfield and mitf expression as identified by GFP expression. The two fish that were chosen for further histology were chosen for distinct reasons. The fish highlighted in the body of the text represented an aggressive melanoma typical of the field and we elected to do histology to confirm our diagnosis and to look for metastatic events, which we found and highlighted in figure 3.  The second fish was chosen due to its uniqueness when compared to the field. This tumor was not distinct in that it was not uniformly GFP and therefore not uniform for mitf expression.  Without uniform mitf expression we were uncomfortable with diagnosing the fish further without histology. This histology work uncovered the sarcoma. We do not think this event is a common event as we have repeated the study without any sarcomas but we do not have enough experience yet to determine how infrequent this may be.

    • 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.

    We agree that the relatively low concentration of rb1 mutant alleles was very surprising. We are cautious to over interpret the result though, as the genomic DNA utilized for sequencing was isolated from the dorsal fin and was a mixed population of healthy tissue and tumor cells. We have not quantified the ratio of tumor cells to healthy cells in our MiSEQ experiment. It is possible that the low ratio of rb1 KO alleles is a result of low tumor gDNA representation in the population. In hindsight, we should have isolated out the tumor cells by FACS to more fully answer this question. This work was recently accepted to Disease Models and Mechanisms and as part of the review process we have stained the tumors for phospho-Rb1 and found that the TEAZ tumors have very low levels (albeit not zero staining) of pRB1 when compared to traditional F0 embryo injection tumors. This may suggest that Rb1 inactivation is happening in a greater percentage of the tumor than our sequencing results suggest.

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