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Zebrafishing for a weapon against metastatic cancer

For many cancer patients and their doctors, the deadliest enemy is metastasis - the migration of cancer cells from the original tumor to other places in the body. In at least 90 percent of hard-tumor cancer deaths, the killer isn't the original cancer but rather its metastasized offspring.

Now, a team of National Institutes of Health (NIH) scientists has proposed a promising new approach for finding ways to impede or halt this deadly process. They have developed transgenic zebrafish as a live animal model of metastasis - offering cancer researchers a new, potentially more accurate way to screen for drugs and to identify new genetic targets against the disease.

Current screening of possible anti-metastasis compounds is based on in vitro procedures - basically, testing hundreds of thousands of substances against cancer cell lines in laboratory dishes. Such high-throughput screening has identified thousands of drug candidates over the past 10 years but has resulted in "very poor success rates when it came to therapeutic drugs," Viviana Gallardo, Ph.D., and Shawn Burgess, Ph.D., both of the National Human Genome Research Institute (NHGRI), and their colleagues wrote in Disease Models & Mechanisms, published online on March 25, 2015.

"Doing things in a live animal model, you can get different answers than you get in vitro," said Dr. Burgess. "In vivo studies can be more reliable because they more accurately reflect the complicated environment of the whole animal," he said.

Coauthors of the Disease Models & Mechanisms paper, in addition to Drs. Burgess and Gallardo, were: Gaurav Varshney, Ph.D., Minnkyong Lee, Ph.D., Sujata Bupp, Lisha Xu, M.S., and Nigel Crawford, M.B., Ch.B., Ph.D., all of NHGRI, and Paul Shinn and James Inglese, Ph.D., of NIH's National Center for Advancing Translational Sciences (NCATS). Dr. Inglese is also associated with NHGRI.

The zebrafish has become an important laboratory model for several reasons. As a vertebrate, it is much closer to humans than research models like fruit flies and roundworms. It reproduces hundreds of times more quickly than mammalian models such as mice (although not nearly so fast as flies). It can be made transgenic by injecting a small sequence of foreign DNA into a fertilized egg or a developing embryo. Importantly, it is transparent during embryonic development, so researchers can watch every developmental step, and its genome has close counterparts to about 70 percent of human genes.

Another feature makes the zebrafish especially appealing as a model for metastasis research. During zebrafish development, a cluster of neural cells called the "posterior lateral line primordium" (PLLp) mimics the behavior of metastasizing human cancer. The cluster of zebrafish neural cells - which will help the adult fish sense water pressure - travels the entire length of the embryo, driven by the same molecular pathways that drive human cancer cells to new sites in the body.  

"A large number of the genes that are involved in human cancer metastasis are being expressed here as the PLLp migrates down," said Dr. Burgess. "So, if we can find drugs that block it, we may find drugs that potentially block cancer metastasis as well."

To test their zebrafish screen, the researchers exposed genetically engineered embryos, in which the PLLp cells glowed brightly when exposed to blue light, to nearly 3,000 different chemical compounds. They found 165 compounds that seemed to inhibit migration of the zebrafish cells without severely damaging the embryo.  These compounds targeted "a wide variety" of pathways and genes with potential roles in metastasis. Those pathways and genes may be targets for further anti-metastasis research.

The researchers doublechecked to make sure their screen was doing what it was supposed to do - identifying substances that block cell migration - by performing cell culture assays with human melanoma cells. Some of the compounds showed "significant inhibition," while others didn't, proving once again that cancer is not a simple disease.

For one of the most promising compounds, the researchers implanted aggressively metastasizing human cancer cells in mice, and then treated the mice with an inhibitor that had seemed most effective in blocking cell migration in zebrafish. The mice treated with the inhibitor developed significantly fewer metastatic tumors than control mice that had not been treated. Some showed no metastasis events at all.

Despite the zebrafish screen's power, Dr. Inglese noted that "unfortunately, it still remains low-throughput relative to cell culture or molecular target-based methods." But he called it "ideal" for a physiological assessment of a smaller number of selected compounds. "In that sense it is highly complementary to generally practiced forms of high- throughput screening," he said.

In the current study, Dr. Gallardo and her colleagues weren't looking for new cancer therapies, at least not directly. Their goal was to demonstrate that their zebrafish model could spot promising anti-cancer compounds and identify genetic targets for therapy that current screening methods might miss. They believe they've accomplished that goal.

"The screen can successfully identify both compounds and new targets for targeting cancer metastasis and also starting points for future in-depth studies to develop new strategies," they wrote in their Disease Models & Mechanisms paper.

 "We are not the first to use zebrafish as a way to screen for new drugs," said Dr. Burgess, "and other people are starting to catch on to zebrafish being useful in this way."

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Posted: April 24, 2015

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Phenotype-driven chemical screening in zebrafish for collective cell migration inhibitors identifies multiple potential pathways for targeting metastasis
Disease Models & Mechanisms, March 25, 2015