Abstract

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.

Highlights

  • Intraoperative optical fluorescence molecular imaging provides real-time image guidance to surgeons to identify precise tumor margins and detect tiny tumor foci both of which result in improved resections with less residual disease, decreasing the risk of recurrence [1]

  • In this study two enzyme activatable probes, γ-glutamylhydroxymethyl rhodamine green 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 thawed ex vivo experimental ovarian cancer tissue samples

  • Conventional imaging probes, called “always-on” probes, continuously emit signals similar to conventional contrast agents used in computed tomography (CT), magnetic resonance imaging (MRI), and angiography and the images depend on the biodistribution of the probe, resulting in relatively high background signals and requiring time to wash out the background signal from the body

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Summary

Introduction

Intraoperative optical fluorescence molecular imaging provides real-time image guidance to surgeons to identify precise tumor margins and detect tiny tumor foci both of which result in improved resections with less residual disease, decreasing the risk of recurrence [1]. In order to overcome this limitation, “activatable” probes have been introduced They are turned on only after binding to a specific target found on tumors and, as a result, maximize the target www.oncotarget.com signal while minimizing the background signal, resulting in a high target to background ratio (TBR) [6,7,8]. Small molecule enzyme-activatable fluorescence probes are rapidly cleaved and activated by a single enzymatic reaction using targeted endopeptidases. Often this process occurs in less than an hour making it practical for clinical translation [9, 10]. In some cases small molecule enzyme-activatable probes can be applied topically during surgery and can be rapidly activated, causing minimal disruption to workflow

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