By David Brill
The humble zebrafish has long been prized by aquarium owners for its hardy constitution and dramatic stripes. But this tropical freshwater species may become even more popular by helping point the way to cures for cancer and other deadly diseases.
Most of us might surmise that humans would have little in common with a tiny creature that hatches from an egg, breathes through gills, and swims around in a tank of water. However, important similarities lie deep in the genetics of both species.
“Zebrafish and humans share 70 percent of their genes and suffer 85 percent of the same diseases,” said Steven Ripp, research associate professor with UT’s Center for Environmental Biotechnology (CEB). “Those similarities make them ideal subjects for testing drug therapies that target human diseases.”
See-Through Subjects
Compared with mice and other rodents, which also share much of the human genome and disease states, zebrafish are inexpensive, easily housed, and capable of reproducing rapidly. They also mature quickly, reaching the larval stage in only five days. But perhaps the most distinctive and useful zebrafish characteristic is its transparent organs and skin during the early phases of life, allowing for direct observation of chemical and biological processes taking place inside the fish.
Unfortunately, the zebrafish’s window of transparency closes rather quickly—within three to four weeks—leaving researchers to wonder what’s happening beneath opaque skin.
That is why CEB has devised a new process to allow scientists to observe disease processes, including the spread and remission of cancers, occurring in the fish’s major organs and other body systems. Based on synthetic biology, the technique genetically induces glowing cells within the zebrafish to change their light signals as therapies attack specific diseases.
Innovative Illumination
Ever wonder what causes fireflies to create yellow-green flashes of light on a warm summer night? The answer is an enzyme called luciferase. Previous experiments have successfully transferred the firefly luciferase gene to a range of microorganisms. Unfortunately that process requires the addition of a second substance to trigger the glow, making it impractical for drug testing.
CEB’s latest innovation is a method that will cause target cells to illuminate throughout the fish’s lifetime without the need for an additional substance. According to Ripp, this genetically introduced trait may even be transferred to zebrafish offspring, creating a self-perpetuating population of glowing test subjects.
Although the new technique is still in the testing phase, Ripp reports that altered zebrafish are beginning to emit measurable light—which validates the process—and he expects to begin testing specific drug therapies within a year.
Shining Example
To use this novel platform to test a drug to treat liver cancer, for instance, zebrafish will be injected with CEB’s bacteria-based luciferase via a genetic sequence. Because the luciferase is programmed to follow the genetic pathway that promotes the growth of cancerous liver tumors, the diseased liver will begin to glow, or bioluminesce. The intensity of the light will increase as the cancer progresses.
Then the tiny fish will be placed into the individual wells of a standard test plate (about the size of an iPhone, with ninety-six individual wells). Various formulations of the candidate anticancer drug will be added to the water for the zebrafish to ingest.
The test plate will then be placed in a photomultiplier to continuously measure the intensity of the light emitted by the cancerous liver cells in each zebrafish. If a candidate drug is working, the light will steadily ebb as the cancer cells die. If the well goes dark, the promising new therapy could advance to the next stage in the FDA’s approval process.
Industrial Revolution
CEB’s pioneering approach comes at an opportune time and may help pharmaceutical companies reverse decades of industry stagnation.
“Since the 1990s, drug discovery has significantly slowed, with only about thirty new drug approvals per year despite R&D investments that have more than doubled during that period, from $48 billion to $106 billion,” Ripp said. The problem, in large part, results from an industry-wide reliance on test methods that are rapid but ineffective.
Two decades ago, in the interest of speed and economy, drug makers began to abandon early in vivo testing on whole living animals. Instead they opted to test their formulations in vitro, on isolated clusters of mammalian cells. With hundreds or even thousands of cell clusters—liver or breast cancer cells, for instance—loaded into the wells of a test plate, drug manufacturers could quickly assess the effectiveness of multiple drug formulations. But the gains in speed came at a cost.
“A person is much more complex than a collection of cells in a dish,” Ripp said. Indeed, tests on cell clusters often fail to evaluate the full long-term effects of the drug on a whole, living animal—whether it be a human, mouse, or fish.
“Once a drug enters the body, it gets metabolized in the liver and may come out as something else, and from there it migrates throughout the body,” Ripp explained. “In some cases, those metabolites might treat the disease but cause harm to other organs.”
Clearly, the pharmaceutical industry stands to benefit from CEB’s breakthrough technology, but other potential applications have emerged as well. Researchers at UT’s College of Veterinary Medicine are exploring the use of bioluminescent cancer cells to trace how the disease metastasizes and migrates to other organs.
And then there are the big national pet shop chains. Ripp has heard from them, too. It seems that there just might be a booming consumer market for glow-in-the-dark fish.
The Discovery Process
1. Fish eggs are collected (with a strainer) and put into a petri dish in a single column.
2. A microinjector with a fine-tipped needle point is used to inject embryos with the bioluminescent genes to produce glowing tumors or organs.
3. Within 4 days, embryos develop into tiny zebrafish in the petri dish and are then transferred into the wells of a 96-well microtiter plate.
4. An anticancer or other test drug is added to the water.
5. The tiny fish are observed with a sensitive camera that reveals if the drug is affecting tumor growth or producing side effects that might harm vital organs.
6. Drugs that prove to be effective and non-toxic are then tested more exhaustively, eventually moving to clinical trials where they can assist in treating or curing human disease.