UK Department of Molecular and Cellular Biochemistry

The field of cancer biology is entering a postgenomic era in which most types of human cancer will have been extensively sequenced through efforts such as The Cancer Genome Atlas and the International Cancer Genome Consortium.

This wealth of data has been truly eye-opening with regard to the extensive scope of genetic alterations that are present in most malignancies, and these data will be further complemented by the ongoing efforts to characterize the cancer epigenome and transcriptome. Now we must couple this genetic data with functional studies in order to take full advantage of our opportunity to translate these findings into new therapeutic advances.

No single model system can recapitulate the heterogeneity and evolving complexity of human cancer, but utilizing the combined strengths of various cancer models will help us link genomic data to functional effects in cancer cells. In recent years, zebrafish have emerged as a key model system for the in vivo study of cancer due to their unique capabilities.

Cancer can be induced in zebrafish through carcinogenic compounds, transgenesis, or xenograft. Zebrafish share many of the same genes as humans, so their tumors arise in much the same way as human malignancies, with much the same oncogenic driving lesions and altered signaling pathways as the human cancer they model. Cross-species comparisons between zebrafish and human tumors have been very useful in identifying important drivers of human malignancy.

Additionally, many types of human cancers, including breast, lung, and prostate cancers as well as leukemias, have been xenografted into zebrafish larvae, which do not have a developed immune system until 14 days of life.

Zebrafish larvae, as well as the Casper strain of adult zebrafish, are translucent, offering in vivo imaging capability of tumor progression, angiogenesis, and metastasis in real time and in a noninvasive manner.

Zebrafish are also incredibly useful in drug screening. A single mating pair will produce 200 to 300 embryos in a week, and the larvae can be arrayed in 96-well plates, enabling hundreds or thousands of animals to be used to test anticancer compounds in a very cost-effective manner. Compared with in vitro drug screens in cell culture, in vivo screening in zebrafish offers a better picture of the effect of drugs on both tumor cells and normal cells, providing a strong rationale for moving a drug hit to more expensive testing in a mouse.

My own basic science laboratory at the University of Kentucky takes advantage of all of these aspects of zebrafish to functionally dissect genomic data associated with pediatric cancers. We are working to sift through these large datasets to define the important drivers of pediatric cancer progression and relapse so that we can ultimately identify new anticancer targets.

The zebrafish pediatric T-cell acute lymphoblastic leukemia model developed at the University of Kentucky.

We use the zebrafish pediatric T-cell acute lymphoblastic leukemia (T-ALL) model and are working to develop B-cell acute lymphoblastic leukemia, neuroblastoma, and glioblastoma zebrafish models. Of particular relevance to our work is the ease with which transgenic animals can be developed to assess the functional effects of genes of interest. Using transient transgenic approaches, a large cohort of tumor-bearing transgenic animals can be developed in as little as 60 days, and these tumors can be maintained indefinitely through propagation in a syngeneic zebrafish line (REF).

We focus on genes that are recurrently mutated or mis-expressed in pediatric cancer datasets and work to functionally define the role that the gene has in cancer progression.

We examine everything from tumor onset and spread to proliferation and apoptosis, and to the development of drug resistance and relapse. We also use zebrafish to dissect intratumoral heterogeneity and tumor evolution, which is a major issue in driving relapse development. We have previously completed a screen in zebrafish in which 47 monoclonal T-ALL were derived from 16 heterogeneous T-ALL by 10-cell transplant, and then serially passaged into more than 6000 animals.

By carefully assessing T-ALL for functional changes at every transplant, were able to identify a subset of leukemia that evolved activation of the AKT pathway, leading to increased self-renewal of leukemia stem cells and spontaneous resistance to dexamethasone. The AKT pathway is frequently dysregulated in human T-ALL through PTEN inactivation, where is it associated with poor patient prognosis, although the reasons for this were previously unknown.

Importantly, we showed that using an AKT or PI3K inhibitor resensitized drug-resistant cells and killed leukemia stem cells in a zebrafish relapse model, providing a strong rationale for testing FDA-approved AKT pathway inhibitors in patients with T-ALL. Besides AKT, this zebrafish functional transplantation screen identified several other gene candidates that are recurrently mutated in human disease, and we are following these up to identify potentially new therapeutic targets in T-ALL.

There is little doubt that zebrafish cancer models will play an increasingly significant role in cancer research as the field works to translate our knowledge of recurrent genetic mutations and epigenetic alterations into new treatment options for patients. Beyond the investment of facility installation, zebrafish are low cost and easy for cancer centers to maintain for researchers to use as their needs arise. For example, at the UK Markey Cancer Center, our lab’s Aquarius Fish System automatically manages the water chemistry and sterility; we simply feed animals and perform routine health checks.

We also have the Danio Data server in place from Fulcrum that automatically pushes preset notifications about zebrafish stocks to researchers’ iPads, such as needs for tumor checks, genotyping, breeding, or other experimental timepoints. This server allows us to easily manage thousands of animals and for collaborators to keep apprised of their stocks remotely.

These types of technologies provide an ease of use to truly allow any cancer research group to take advantage of the unique capabilities of zebrafish to enhance their work.

Jessica Blackburn, PhD

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