Pitfalls on sample preparation for ex vivo imaging of resected cancer tissue using enzyme-activatable fluorescent probes

In vivo and ex vivo fluorescence imaging-assisted surgery can aid in determining the margins of tumors during surgical resection. While a variety of fluorescent probes have been proposed for this task, small molecule enzyme-activatable fluorescent probes are ideal for this application. They are quickly activated at tumor sites and result in bright signal with little background, resulting in high sensitivity. Testing in resected specimens, however, can be difficult. Enzymes are usually stable after freezing and thawing but catalytic reactions are generally temperature-dependent. Therefore, tissue sample temperature should be carefully considered. In this study two enzyme activatable probes, γ-glutamylhydroxymethyl rhodamine green (gGlu-HMRG) that reacted with γ-glutamyltransferase and SPiDER-βGal that reacted with β-galactosidase, were employed to determine the effects of temperature on fluorescence signal kinetics in both fresh and frozen and then thawed ex vivo experimental ovarian cancer tissue samples. The results suggest γ-glutamyltransferase was less sensitive to temperature than β-galactosidase. Fresh samples showed higher fluorescence signals of gGlu-HMRG compared with thawed samples likely because the freeze-thaw cycle decreased the rate of internalization of the activated probe into the lysosome. In contrast, no significant difference of SPiDER-βGal fluorescence signal was observed between fresh and frozen tissues. In conclusion, although imaging of fresh samples at 37°C is the best condition for both probes, successful imaging with gGlu-HMRG could be achieved even at room temperature with thawed samples. We demonstrate that temperature regulation and tissue handling of resected tissue are two pitfalls that may influence ex vivo imaging signals with enzyme-activatable fluorescent probes.