Fluorescent probe may aid early cancer detection

Specially designed molecule lights up when it detects cellular activity that precedes tumors' spread

Bogyo Lab

The Stanford-designed tagging molecule (right, A) attaches to a protease (B). The complex stays dark until it is activated, when the quencher unit pops off (C).

Medical school researchers have developed a new way to spot subtle yet important chemical changes that take place early in the growth of tumors. The method could eventually help in the early detection of cancer and other diseases.

Matthew Bogyo, PhD, assistant professor of pathology, and his colleagues have created a molecule that can label proteases—protein-chewing enzymes that blast into overdrive in cancerous cells. Bogyo's new molecule contains a fluorescent tag that flashes brightly enough to be seen with conventional imaging equipment. The results appeared Aug. 14 in the online issue of Nature Chemical Biology.

While there are other enzyme tags, Bogyo's "activity-based probe" is unusual in that it only lights up when the proteases are active. Moreover, it works in living cells, so the probe could potentially be used for whole-body imaging in the not-too-distant future. Such techniques might be able to detect the warning signs of cancer long before tumors have a chance to spread.

Proteases are a general class of enzymes found in all healthy cells, where they clean up old proteins no longer needed by the cell. But proteases are often abnormally active in rapidly dividing cancer cells. They also work overtime in tumors that are getting ready to spread, or metastasize, and in cells that are recruiting new blood vessels in a process called angiogenesis.

"This is an important tool for understanding the biochemistry of proteases, and how they play a role in diseases like cancer," Bogyo said. "And it's non-invasive and fairly non-toxic, in that it doesn't involve radioisotopes."

So far, Bogyo's group has tested the probe only in cultured cells. But they soon plan to test it in mice to ensure that it is safe and bright enough to generate whole-body images of protease activity. If so, the probes may eventually be useful for imaging human bodies.

Bogyo's probe has several advantages over other enzyme imaging probes, and it's the combination of these characteristics that make it unique.

In one key innovation, Bogyo and his colleagues added a chemical subunit called a "quencher" that keeps the probe dark until the protease is activated. Much like a fluorescent light stick turns on when you crack it in half, the chemical changes that activate the enzyme also cause the quencher unit to pop off, switching the probe on "like a molecular beacon," Bogyo said.

The probe is small enough to pass easily across the cell membrane, allowing it to be used inside living cells. In contrast, some probes only report on enzyme activity outside the cell.

The probe also forms a permanent bond with its target protease, allowing it to track where the protease is active. This is an improvement over other non-bonding imaging molecules that can signal when—but not where—proteases do their job.

"It's a nifty trick, a real neat approach," said Ben Cravatt, a professor of cell biology at the Scripps Research Institute in La Jolla,

Calif., who was not involved in the research. He confirmed that the combination of permanent tagging and a quencher unit give the probe distinct advantages. "It's a great proof-of-principle," he added. "It will be interesting to see the method expanded to other proteases."

Cancer cells aren't the only ones with hyperactive proteases. They are also found in cells affected by arthritis, osteoporosis, atherosclerosis and neurodegenerative disorders such as Alzheimer's.

While Bogyo's probe specifically targets a small family of proteases that are activated in several different forms of cancer, he believes that the same method can be used to make new molecules to probe for many other proteases. For example, he plans to focus on making probes for caspases—a type of protease associated with conditions such as stroke and Huntington's disease.

Besides making new probes, Bogyo and his team are working on imaging applications. "We have made the tool, now we want to go and use it," said Galia Blum, a postdoctoral scholar in Bogyo's lab and the study's lead author. "Our next step is to generate whole-body images in mice." Such images may distinguish between areas of normal and abnormal protease activity, as well as track changes in activity over time.

If all goes well in the mouse models, the researchers plan to pursue tests in human subjects. Bogyo believes fluorescent probes hold a lot of promise for studying cancer progression without relying on invasive procedures such as biopsy or surgery. If the probe is safe and useful for clinical applications, it might help to advance the timetable on the course of cancer therapies.

For example, many researchers are looking into anti-protease drugs as treatments for cancer. Instead of waiting to see if these drugs actually shrink tumors, activity-based enzyme probes could provide a way to verify immediately whether the drug does what it was designed to do.

"These probes have direct applications to human health, but they are also important research tools," Bogyo added. "People can apply them to their own experiments, and move the field forward in a lot of different areas."