Abstract 5113: Rapid Cancer Imaging By GGT-targeted Fluorescence Probe For Primary Lung Cancer
Authors: Hino Haruaki , Kawashima Mitsuaki , Murayama Tomonori , Ichinose Junji , Kitano Kentaro , Nagayama Kazuhiro , Nitadori Jun-ichi , Anraku Masaki , Murakawa Tomohiro , Mizuno Kasue , Tanaka Sayaka , Kamiya Mako , Nishiyama Nobuhiro , Kataoka Kazunori , Miyazono Kohei , Urano Yasuteru , Nakajima Jun 
Published: August 2015

Abstract
Introduction
Lung cancer is the leading cause of cancer death in Japan, however, it has been difficult to detect and diagnose precisely lung cancer with a diameter less than 1cm to date. The purpose of this study is to investigate clinical application of novel GGT-targeted fluorescence probe for detecting the primary lung cancer in an intraoperative manner.
Methods
As a fluorescence probe for γ-glutamyltranspeptidase (GGT), γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) was used. gGlu-HMRG is non-fluorescent, but is converted to a highly fluorescent hydroxymethyl rhodamine green (HMRG) upon reaction with the enzyme, which tends to accumulate in GGT-overexpressing cancer cells. First, we examined GGT activity of lung cancer cell lines, A549, H460, H441, H82 and H226, by applying gGlu-HMRG and evaluating fluorescence intensities by confocal fluorescence microscopy. We also compared mRNA expression level of GGT1 (one of the subtypes of GGT) by qRT-PCR. Further, by transfecting siRNA targeted to GGT1, we investigated the target of gGlu-HMRG. Next we performed in vivo imaging of orthotopic A549 lung cancer xenograft model in nude mouse to confirm the validity of fluorescence imaging. Finally, we carried out ex vivo fluorescence imaging of 73 human lung cancers and normal lung tissues which were surgically resected, and the fluorescence intensities were analyzed by Receiver Operating Characteristics curve.
Results
A549, H460 and H441 cells with high GGT1 expression could be visualized with high fluorescence intensity after application of gGlu-HMRG within several minutes, whereas H82 and H226 cells with relatively low GGT1 expression could not. We ascertained that the target of gGlu-HMRG was GGT1 by fluorescence imaging and qRT-PCR with lung cancer cell lines transfected with siRNA for GGT1. In lung cancer xenograft model, pleural dissemination, hilar and mediastinal lymph node metastasis and the surface of lung cancer were clearly detected within 15 minutes after topical drip of gGlu-HMRG. We confirm that every fluorescent lesion was adenocarcinoma pathologically. In ex vivo human lung cancer fluorescence imaging, the sensitivity, specificity and accuracy were calculated to be 43.8% (32/73cases), 84.9% (62/73cases) and 64.4% (94/146), respectively. The adenocarcinomas, cancer of female or never smoker were more significantly detected by fluorescence imaging (p&lt;0.05-0.001). Limited to 19 cases of adenocarcinomas, cancer of female and never smoker, sensitivity, specificity, and accuracy were 78.9% (15/19), 73.7% (14/19) and 76.3% (29/38), respectively.
Conclusions
We suggest that intraoperative application of gGlu-HMRG to detect pleural dissemination, small mediastinal lymph nodal metastasis, or other small foci of the lung cancer cells on surgical margin might be feasible when the cancer cells overexpress GGT. Intraoperative application of fluorescence probe is highly expected in near future.
Citation Format: Haruaki Hino, Mitsuaki Kawashima, Tomonori Murayama, Junji Ichinose, Kentaro Kitano, Kazuhiro Nagayama, Jun-ichi Nitadori, Masaki Anraku, Tomohiro Murakawa, Kasue Mizuno, Sayaka Tanaka, Mako Kamiya, Nobuhiro Nishiyama, Kazunori Kataoka, Kohei Miyazono, Yasuteru Urano, Jun Nakajima. Rapid Cancer Imaging By GGT-targeted Fluorescence Probe For Primary Lung Cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5113. doi:10.1158/1538-7445.AM2015-5113