Surgical tissue handling methods to optimize ex vivo fluorescence with the activatable optical probe γ-glutamyl hydroxymethyl rhodamine green

Optical fluorescence imaging has been developed as an aid to intraoperative diagnosis to improve surgical and endoscopic procedures. Compared with other intraoperative imaging methods, it is lower in cost, has a high safety margin, is portable and easy to use. γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) is a recently developed activatable fluorescence probe that emits strong fluorescence in the presence of the enzyme γ-glutamyl transpeptidase (GGT), which is overexpressed in many cancers, including ovarian cancer. Ex vivo testing is important for clinical approval of such probes. The diagnostic performance of gGlu-HMRG in fresh excised surgical specimens has been reported; however, details of tissue handling have not been optimized. In this study, we investigated four different tissue handling procedures to optimize imaging in excised tumor specimens. The fluorescence intensity time courses after the different tissue handling methods were compared. Additionally, the fluorescence positive areas were correlated with the presence of red fluorescent protein (RFP) in an RFP positive cell line as the standard of reference for cancer location. In the 'intact' groups, tumors yielded quick and homogeneous activation of gGlu-HMRG. In the 'rinse' and 'cut' groups, the fluorescence intensity of the tumor was a little lower than that in the intact group. In the 'pressed' groups, however, fluorescence intensity from gGlu-HMRG was lower over the entire time course, suggesting a decrease or relocation of excreted GGT. In conclusion, we demonstrate that the method of tissue handling prior to ex vivo imaging with the activatable probe gGlu-HMRG has a strong influence on the signal derived from the specimen. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.