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Visualizing the Birth of Cancer in Zebrafish

Fluorescent Zebrafish Model of Rhabdomyosacoma. Fluorescent proteins are expressed within distinct stages of muscle development, which is dynamically visualized by live cell imaging. GFP is expressed in the tumor propagating cells that drive continued tumor growth and relapse. Red fluorescent protein is localized to the nucleus and expressed in mid-differentiated myoblast-like cells. Blue fluorescent protein is membrane localized and confined to terminally differentiated cancer cells that express myosin. Ignatius et al, 2012 (Cancer Cell)

Cancer behaves differently in living animals than it does in a dish, so designing animal models is integral to both basic and clinical research in the DF/HCC Cancer Genetics Program. “The zebrafish augments what we can do with more traditional mouse models,” explains David Langenau, PhD (HMS). Zebrafish embryos and larvae are transparent, so by using live cell imaging and fluorescently labeling different cell types, Langenau can dynamically visualize the birth of a cancer and the functional consequences of tumor cell heterogeneity within established tumors.

Zebrafish are also ideal for screening and testing new drug compounds. “We sprinkle investigational drugs into the water. Remarkably, the fish swim around and absorb the drug through their skin and gills. This allows us to directly assess which drugs shrink the tumors overtime,” says Langenau, who applies this method to identify compounds that can both stop tumor growth and prevent relapse following drug therapy.

For a recent study, Langenau used a zebrafish model he had developed of rhabdomyosarcoma, a rare cancer of the muscle that affects about 350 children under the age of 10 each year, and that lends itself to exploring its early genesis. “We figured that if rhabdomyosarcoma happens so early in human life, it would happen early in fish.” Indeed, by day 10, when the fish measure just 3mm in length, they have prominent tumors protruding from their muscles. Langenau first color-coded cell populations in normal fish with fluorescent proteins that are expressed in distinct stages of tissue development. Green fluorescent protein (GFP) was expressed in the muscle stem cells (activated satellite cells) that can give rise to more differentiated muscle cells. Red fluorescent protein was expressed in the nuclei of mid-differentiated myoblast-like cells.  Blue was confined to the membrane of terminally differentiated cells that express myosin.

This color-coded developmental process was recapitulated in fish with rhabdomyosarcoma tumors, except that it was the tumor propagating cells (TPCs) rather than muscle stem cells that expressed GFP. TPCs have similar gene expression to the muscle stem cells, but instead of building a muscle they build a tumor, and they drive continued tumor growth and relapse.

Langenau could watch metastasis happen in these zebrafish as cancer cells escaped the primary tumor, settled elsewhere, and eventually took over the animal. Surprisingly, TPCs were not the cells responsible for initiating metastasis. The first cells that escaped the primary tumor were more differentiated cells, and over time they recruited the slower moving TPCs from the original tumor.

After identifying the distinctive TPC population based on gene expression, Langenau’s group has designed a chemical screen to find compounds that could cause TPCs to differentiate into terminally differentiated myosin-expressing cells. He hypothesized that without tumor propagating cells, the cancer cannot relapse, and that coaxing cancer to express myosin would stop metastasis. Working with Leonard Zon, MD (BCH), and others, Langenau screened an initial 40,000 compounds (which included about half of the FDA approved compounds) in the zebrafish model. They identified 12 drugs that altered rhabdomyosarcoma growth in both zebrafish and human cells, and a subset of those also caused the TPCs to differentiate and lose their stem cell function. The team is testing that subset of compounds on zebrafish to find ones that both slow the growth of tumors and cause TPCs to terminally differentiate, which they hope will identify new and existing candidates for preclinical studies. With the ability to conduct large and unbiased chemical screens in zebrafish models, finding radically new uses for approved compounds may become a common motif in cancer therapy.

Research detailed in this article was funded in part by NIH grants, including CA065164, AR055619, CA154923, and CA156056.

— Cathryn Delude