New DNA Label Improves Visualization of Clinical Cancer Samples

Researchers at the University of Pittsburgh have developed a new fluorescent label for use in superresolution fluorescence microscopy that gives a clearer picture of how DNA architecture is disrupted in cancer cells. The findings, published recently in Science Advances, could improve cancer diagnoses for patients and classification of future cancer risk.

Pathologists routinely use traditional light microscopes to visualize disruption to the DNA-protein complex, or chromatin, as a marker of cancer or precancerous lesions. “Although we know that chromatin is changed at the molecular scale during cancer development, we haven’t been able to clearly see what those changes are. This has bothered me for more than 10 years,” says senior author Yang Liu, Ph.D., associate professor of medicine and bioengineering at the University of Pittsburgh and a member of the University of Pittsburgh Medical Center (UPMC) Hillman Cancer Center. “To improve cancer diagnosis, we need tools to visualize nuclear structure at much greater resolution.”

In superresolution fluorescence microscopy, a molecule of interest is labelled with a special fluorescent dye that flashes on and off like a blinking star. Only a subset of the labels are switched on at a given moment, and, when overlayed, the collection of images provide a higher resolution picture. 

Unfortunately, these fluorescent dyes didn’t work well on DNA or in processed clinical cancer samples. So, Liu and her team formulated a new label called Hoechst-Cy5 by combining the DNA-binding molecule Cy5 and a fluorescent dye called Hoechst with ideal blinking properties for superresolution microscopy.

After showing that the new label produced higher resolution images than other dyes, the researchers compared colorectal tissue from normal, precancerous and cancerous lesions. In normal cells, chromatin is densely packed, especially at the edges of the nucleus. Condensed DNA glows brightly because a higher density of labels emits a stronger signal, while loosely packed chromatin produces a dimmer signal. The images show that as cancer progresses, chromatin becomes less densely packed, and the compact structure at the nuclear border is severely disrupted.

While these findings indicate that the new label can distinguish normal tissue from precancerous and cancerous lesions, Liu said that superresolution microscopy is unlikely to replace traditional microscopes for routine clinical diagnoses, but could be useful in risk stratification. “Early-stage lesions can have very different clinical outcomes,” said Liu. “Some people develop cancer very quickly, and others stay at the precursor stage for a long time. Stratifying cancer risk is a major challenge in cancer prevention.”

To see if chromatin structure could hold clues about future cancer risk, Liu and her team evaluated patients with Lynch syndrome, a heritable condition that increases the risk of several cancer types, including colon cancer. They looked at non-cancerous colorectal tissue from healthy people without Lynch syndrome and Lynch patients with or without a personal history of cancer.  The differences were striking; in Lynch patients who previously had colon cancer, chromatin was much less condensed than in healthy samples, suggesting that chromatin disruption could be an early sign of cancer development—even in tissue that looks completely normal to pathologists.

For Lynch patients without a personal history of cancer, some will go on to develop cancer, while others will not. “We see a much larger spread in this group, which is very interesting,” said Liu. “Some patients resemble healthy controls, and some are closer to Lynch patients who previously had cancer. We think that patients with more open chromatin are those who are more likely to develop cancer. We need to follow these patients over time to measure outcomes, but we’re pretty excited that chromatin disruption in normal cells could potentially predict cancer risk.”

In future work, Liu and her team are interested in examining chromatin structure in endometrial tissue from Lynch patients, who also have elevated risk of endometrial cancer. The researchers also received funding recently to look at sputum samples from smokers for early detection of lung cancer.