Targeted fluorescent probes for high-contrast cancer detection

Cancer is a universal issue that affects uncountably many people around the world. Many will turn to surgery in the hope that a surgeon will be able to completely remove a tumor, leaving healthy tissues unaffected. Various tools and techniques have been developed over the years to improve the way these surgeries are performed, and visual imaging methods such as glowing dyes have proven to be very useful.

But one drawback is that some probes can also be activated in healthy tissues by endogenous enzymes, creating background fluorescence and making it harder to judge what should be removed. The opposite is also possible, where cancer cells are left unmarked and are missed during surgery, increasing the chance of recurrence.

Our group acknowledged this current shortcoming and improved upon this way to make cancer cells light up inside the body. In tests on mice, we delivered a special enzyme to tumors and used a fluorescence probe that only turns on when that enzyme is present.

Ryosuke Kojima, Associate Professor, Laboratory of Chemical Biology and Molecular Imaging, University of Tokyo

Kojima added, "Older probes often light up healthy tissue by mistake, creating background noise, but our highly selective, or bioorthogonal, dye probe is designed to stay completely off unless it meets its matching engineered enzyme. We essentially trained the enzyme through repeated mutation and selection, a form of directed evolution, so it could activate the probe strongly enough to work inside living animals."

Kojima, with Professor Yasuteru Urano and their team, created a special fluorescent probe that is not easily activated by natural enzymes in the body, which helps prevent unwanted background glow. This probe was paired with a matching reporter enzyme specially tailored to switch it on, so fluorescence appears mainly where the enzyme is delivered. When tested in mice bearing peritoneal cancer, the engineered enzyme reached the tumors in the abdominal wall lining and was followed up by the probe which lit up as expected.

"This allowed us to see tiny, millimeter-sized tumor lesions with extremely low background noise, a level of contrast that could be very useful during surgery," said Kojima. "In the near term, this system could become a powerful research tool, and in the longer term, it may help surgeons remove tumors more completely by clearly highlighting cancer cells. A major hurdle for clinical use will be ensuring that the engineered enzyme does not trigger an unwanted immune response in patients."

The system could be adapted to other kinds of cancers too, beyond the peritoneal cancer used in these trials, as many cancers present corresponding antigens, telltale markers of tumor tissue. By swapping the tumor-targeting component (for example, an antibody or nanobody against a chosen antigen), the same enzyme-probe pair could in principle be redirected to other cancer types.

Looking even further ahead, this research could even be helpful in highly targeted drug delivery, where instead of glowing dyes, cancer-fighting drugs would be sent to the sites they're needed, and nowhere else. But as Kojima stresses, it's still early days, the trials have only been done in mice, and much work is needed before it's deemed safe enough for human trials.