Richard Mark White, MD, PhD

The evolution of metastasis in zebrafish

Metastatic capacity is the defining feature of malignancy. It is responsible for the vast majority of deaths from solid tumors. Cancers acquire this capacity as a result of an evolutionary game that capitalizes on four essential features of Darwinian evolution: 1) heritable mutations, 2) environmental selection of the fittest cells, 3) geographical distribution of individual cell types, and 4) clonal expansion. In this light, successful metastasis must always be considered as an interaction between the tumor cell and its host environment.

Cancer modeling in the zebrafish

Our laboratory utilizes zebrafish to study cancer evolution. The zebrafish has emerged as a unique vertebrate model organism that is especially useful for cancer because of its capacity for high-throughput, unbiased genetic and chemical screening approaches. Combined with its strengths in transgenic technology and in vivo imaging, our laboratory is addressing the genetic underpinnings of metastasis, considering both the tumor cell as well as the host genetic background. Our goal is to identify the molecular mechanisms that allow tumors to evolve and to determine whether such mechanisms could be exploited therapeutically.

The zebrafish is highly amenable to transgenic and carcinogenic tumor formation. We have developed a zebrafish model of melanoma in which the mutant human BRAF^V600E allele is expressed under the melanocyte specific mitf promoter. When crossed with p53 mutant fish, the resultant BRAF;p53 animals all develop melanoma within four to 12 months. These tumors strongly resemble human melanoma at both the histological and gene expression levels, and serve as a platform for unbiased genetic screening approaches. In addition to transgenic models of melanoma, fish are susceptible to transposon and retroviral induction of tumors in a wide variety of organs such as the pancreas, blood, and liver.

A transgenic zebrafish model of melanoma. The image on the left shows a wild-type zebrafish with alternating dark and light stripes. The dark stripes are composed of pigmented melanocytes. The image on the right shows a transgenic zebrafish expressing human BRAF^V600E under the melanocyte-specific mitfa promoter in the context of a p53 mutation. These transgenic animals develop severe aberrancy of the stripe pattern and clearly visible melanomas.

Studying metastasis in the zebrafish

Metastasis is a multistep process, and imaging and isolation of cells at each step is required to understand the genetic factors contributing to each stage. To enhance this ability, we developed a zebrafish strain named casper, which maintains its transparency throughout adulthood. The casper model provides exquisite single-cell analysis of metastatic cells at each step in the cascade. We have utilized this model to identify genes required for dissemination of T cell lymphoma and melanoma cells, and to determine how these cells interact with the host stroma and vasculature.

The zebrafish casper mutant as a model for metastasis. The casper strain (top) is translucent throughout its adult life cycle due to a lack of melanocytes and iridophores. The transplantation of GFP+ melanoma cells into the flank of a casper recipient yields both primary and metastatic growth (bottom).

Genomics of tumor evolution in zebrafish

We have now undertaken several large-scale efforts aimed at defining the genome of zebrafish melanoma. This investigation includes exome sequencing as well as whole-genome and RNA-seq analysis of matched DNA from primary and metastatic tumors, and normal germline DNA. Our research has revealed that zebrafish tumors undergo large-scale genomic alterations similar to those occurring in human tumors, with several copy number and mutation events conserved between the two species.  Furthermore, the development of these genomic platforms allows for an in-depth understanding of how tumor genomes evolve over time, and how these genomes evolve during metastatic progression.

Visualization of melanoma over time using the zebrafish, where colors represent fluorescent markers of cells within the tumor. These cells can be tracked longitudinally within the same animal, and the genomic lesion responsible for metastasis identified at its earliest stages.