Enzyme-activatable Fluorescence Probe and Reagents. gGlu-HMRG, an activatable fluorescence probe targeting GGT, and HMRG were prepared as previously described [7]. They were stored as 10 mM solutions in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, Missouri) at − 80°C. Before ex vivo application to clinical specimens, the DMSO stock solution was thawed at room temperature and diluted to a final concentration of 50 µM (gGlu-HMRG) or 1 µM (HMRG) in phosphate-buffered saline (PBS, Life Technologies, Carlsbad, California). All organic solvents and reagents were commercial products of guaranteed grade and were used without further purification. Water was doubly distilled and deionized by a MilliQ water system before use.
Clinical Samples. 22 patients with thymoma or thymic carcinoma who underwent surgery between February 2013 and January 2021 at the University of Tokyo were included in the study. Twenty patients were enrolled for the ex vivo fluorescence imaging study (Figs. 1 − 4 and Supplementary Table S1), one patient was enrolled for the experiment involving application to serial sections of tumor and normal tissue (Fig. 5) and one patient was enrolled for the chemical inhibition experiment (Fig. 6). Some patients were included in plural analyses. Written informed consent was obtained from all patients, and this study was approved by the ethics committee of The University of Tokyo and the local ethics committees. All experiments were performed in accordance with guidelines and regulations approved by the ethics committees. All specimens were taken intraoperatively. Fluorescence images, except for the inhibition experiment, were collected within a day after resection. For the inhibition experiment, frozen specimens were thawed at room temperature and used within six months.
Ex Vivo Fluorescence Imaging Study of Patients’ Specimens. Images were captured with a Maestro in vivo imaging system (PerkinElmer) before and at 1, 3, 5, 10, 20, and 30 minutes after applying 50 µM gGlu-HMRG solution in PBS containing 0.5% v/v DMSO to tumor and normal specimens at room temperature. A sufficient amount of solution was used to ensure that the specimen was fully immersed. The excitation and emission wavelengths were 445–490 nm and 515 nm long pass, respectively. The Maestro’s tunable filter was automatically switched in 10 nm increments from 500 to 720 nm, while the camera sequentially captured images at each wavelength interval. Fluorescence at 540 nm was extracted, and fluorescence intensities were quantified by drawing regions of interest (ROIs) with the Maestro software. Exposure time was set at 50–100 ms depending on the fluorescence intensity. From 2013 to 2016 (8 cases in total), 50 ml of probe was applied to a relatively large specimen (about 1 cm) and three ROIs were set for both tumor and normal tissues, then the mean fluorescence intensity was calculated using Maestro software. From 2017 to 2021 (12 cases in total), specimens were cut into pieces a few millimeters in size, which were placed individually in wells of an 8-well chamber (µ- Slide 8 well; Ibidi), and 200 µl of probe solution was added to each well. ROIs were set for entire specimens. In three of the latter cases HMRG only (1 µM) was applied in order to examine the effect of the difference in color between tumor and non-tumor tissues, as the fluorescence intensity of reddish non-tumor tissues tends to be under-recorded. In one experiment, we performed fluorescence imaging of serial specimens of tumor and normal tissue after topically applying 2 ml of probe to the entire specimen. Increase in fluorescence intensity was calculated by subtracting the initial fluorescence intensity from that measured after incubation for 30 minutes with the probe. Fluorescence images were also obtained after treating paired samples with gGlu-HMRG (50 µM) with or without GGsTop (50 µM; Wako Pure Chemical Industries), which is a specific irreversible inhibitor of GGT, under the same conditions [17]. This inhibition experiment was performed using frozen specimens, which were allowed to thaw naturally at room temperature.
Histological Analysis. Resected specimens were immediately fixed with formalin for at least 24 h and embedded in paraffin. Paraffin-embedded tissues were sectioned at 4 µm thickness and stained with HE for histopathological evaluation. Certified pathologists made diagnosis according to the 5th edition of the World Health Organization (WHO) classification of thymic tumors [18]. Tumor stage was determined according to the 8th edition of the TNM staging system of the Union for International Cancer Control (UICC) and the Masaoka-Koga classification system [8], [19].
Immunohistochemical Analysis of GGT Expression. For IHC, sections were deparaffinized in Histo-Clear, sequentially washed in 100%, 90%, 80%, and 70% ethanol, and then washed in PBS. After heat-induced antigen retrieval (Tris-EDTA buffer, pH 9) using a microwave oven, each slide was pre-incubated in 3% H2O2 for 20 min, reacted with primary antibodies (mouse polyclonal antibody, H00002678-M01; Abnova, Taipei, Taiwan) in 5% skim milk for 90 min, and reacted with secondary antibodies (Takara POD conjugate anti Mouse for GGT, Japan) for 30 min at room temperature. Each slide was visualized with a 3,3’-diaminobenzidine tetrahydrochloride (DAB) detection kit (Product number: MK210, TaKaRa), and counter-stained with hematoxylin. GGT antibody was diluted to 1/3000 and the DAB reaction time was 5 min.
Statistical Analysis. Statistical analyses were carried out using software R 3.3.2 (R Foundation for Statistical Computing, Vienna, Austria). Sensitivity, specificity, PPV, NPV, and accuracy were evaluated from the ROC curves, and data were expressed as means ± SD.