Cancer can often be detected by looking for signs of chromatin disruption under a microscope, as the disease can cause the DNA-containing material to become less densely packed within the nucleus. However, conventional light microscopy methods for examining clinical samples are limited when it comes to viewing the molecular-scale changes that chromatin undergoes as cancer develops. Superresolution microscopy, which could allow such structural features to be viewed at nanoscale, has also been limited in clinical settings due to a lack of fluorescent dyes that can bind well to DNA in highly processed clinical tissue. Now, a team at the University of Pittsburgh has overcome this hurdle and developed a fluorescent DNA label that can allow pathologists to visualize previously “invisible” cancer markers.
Superresolution fluorescence microscopy creates clearer biological images through the blinking on and off of specific labels at different times, allowing a composite image to be reconstructed showing all of the labeled components in high resolution. The researchers’ new dye combines the small-molecule DNA binding dye Hoechst with cyanine 5 (Cy5), which has optimal photoswitching properties for superresolution microscopy, to create Hoechst-Cy5. The team was able to validate that Hoechst-Cy5 produced high-quality superresolution images of DNA in clinically prepared formalin-fixed, paraffin-embedded (FFPE) tissue, with high signal, low background and good separation of molecules within the nucleus.
The researchers then tested the potential of their new DNA dye for helping to gauge cancer risk based on nanoscopic changes in chromatin structure. The team compared images of normal colorectal tissue to that taken from precancerous or cancerous lesions and found that in the normal cells, the chromatin was densely packed, especially at the edges of the nucleus, while the chromatin became less densely packed at different stages of cancer progression. The superresolution images showed characteristic DNA nanodomains from three distinct stages in cancer progression, the authors wrote, and disruption at the edges of the nucleus in cancerous cells was more visible in the superresolution images than in those taken by a conventional widefield fluorescence microscope.
The team also examined colorectal tissue from patients with Lynch syndrome, a heritable condition that increases the risk of several cancer types. The Lynch patients who had previously had colon cancer had much less condensed chromatin than healthy controls, while the chromatin structure of Lynch patients who had never had cancer varied widely, with some resembling healthy controls and others resembling the previously affected patients. The results suggest that superresolution fluorescence imaging, even of otherwise healthy-appearing tissue, could help clinicians to better stratify cancer risk and work with patients over time to manage and monitor this risk. This research was published in Science Advances.
“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,” said senior author Yang Liu.
In the future, the team plans to examine chromatin structure in endometrial tissue from Lynch patients, who also have an elevated risk of endometrial cancer, as well as sputum samples from smokers in order to study lung cancer risk.
Video Credit: University of Pittsburgh Medical Center