Hydroxymethyl Rhodamine Green Research Articles

Fluorescence Imaging Using Enzyme-Activatable Probes for Detecting Diabetic Kidney Disease and Glomerular Diseases.

A clear identification of the etiology of glomerular disease is essential in patients with diabetes. Renal biopsy is the gold standard for assessing the underlying nephrotic pathology; however, it has the risk for potential complications. Here, we aimed to investigate the feasibility of urinary fluorescence imaging using an enzyme-activatable probe for differentiating diabetic kidney disease and the other glomerular diseases. Hydroxymethyl rhodamine green (HMRG)-based fluorescent probes targeting gamma-glutamyl transpeptidase (GGT) and dipeptidyl-peptidase (DPP) were used. Urinary fluorescence was compared between groups which were classified by their histopathological diagnoses (diabetic kidney disease, glomerulonephritis, and nephrosclerosis) as obtained by ultrasound-guided renal biopsy. Urinary fluorescence was significantly stronger in patients with diabetic kidney disease compared to those with glomerulonephritis/nephrosclerosis after DPP-HMRG, whereas it was stronger in patients with nephrosclerosis than in patients with glomerulonephritis after GGT-HMRG. Subgroup analyses of the fluorescence performed for patients with diabetes showed consistent results. Urinary fluorescence imaging using enzyme-activatable fluorescence probes thus represents a potential noninvasive assessment technique for kidney diseases in patients with diabetes.

Rapid visualization of mammary gland tumor lesions of dogs using the enzyme-activated fluorogenic probe; γ-glutamyl hydroxymethyl rhodamine green.

Since gamma-glutamyl transpeptidase (GGT) is highly and locally expressed in human breast cancer, a GGT-enzymatically activatable fluorescent probe, gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), has been developed to detect the human breast cancer area with high performance. In this study, GGT expression and the efficacy of gGlu-HMRG on visualization were investigated in canine mammary gland tumors (MGT). Seventeen non-fixed fresh-frozen MGT specimens and each peritumoral control tissue were utilized. The GGT mRNA levels were highly observed in the tumor specimens compared with the control. GGT immunostaining was mostly observed on the cell membrane and cytosol of the alveolar and duct mammary epithelium of MGT tissues. These signals were strongly positive in several cases while they were mild to not observed in other cases. When gGlu-HMRG solution was dropped to the non-fixed tissue pieces of MGT or control tissues, the fluorescence intensities (FIs) were measured using Maestro in-vivo imaging device. FIs in MGT tissues were significantly higher than each control tissue 20 min after treatment. Based on Youden index method said that the maximum sensitivity and specificity of FI was 82.4% and 82.4%. These findings suggest that GGT is highly expressed in several MGTs in dogs and gGlu-HMRG could visualize at least a part of MGT tissues in dogs. Nevertheless, it should be needed to assess the false-negative areas more carefully in canine than human cases.

FLGS-01. Development of novel topical fluorescent probe for intraoperative rapid detection of glioma

Abstract PURPOSE Fluorescence imaging is an important surgical adjunct in malignant glioma surgery. 5-aminolevulinic acid (5-ALA) has been proven effective for radical tumor resection and extended progression-free survival in a phase III randomized trial and therefore integrated into surgery for malignant glioma. Importantly, however, some limitations still exist in its use, which include false positivity and false negativity as well as inability of re-administration. In this study, we aimed to develop a novel, spray-type fluorescent probe using hydroxymethyl rhodamine green (HMRG) as a fluorescent scaffold. METHODS We have previously established a fluorescent probe library comprised of more than 320 kinds of HMRG probes. They have HMRG as a fluorescent scaffold with various types of dipeptides attached to it. Primary probe screening was performed using the homogenized tumor samples from patients with glioblastoma operated at our institution. Secondary screening followed using the selected probes and fresh tumor samples obtained from patients with glioblastoma operated from 2016 until 2018. Diced electrophoresis gel (DEG) assay, two-dimensional gel electrophoresis followed by a multi-well plate-based fluorometric assay, was performed to identify responsible enzymes for the selected probe. Further experiments with inhibitors, real-time PCR, immunohistochemistry, and western blotting were performed for confirmation. RESULTS Proline-arginine-HMRG (PR-HMRG) was selected as a candidate probe based upon the above two-step screenings. It achieved 79.4% accuracy in receiver operating characteristic curve analysis. Calpain-1 was found to be responsible to cleave PR-HMRG probe by DEG-proteome analysis. Calpain-1 protein was highly expressed in tumor tissues which reacted to PR-HMRG probe. CONCLUSIONS Our innovative screening method was able to find PR-HMRG as a novel fluorescent probe effective for rapid detection of glioblastoma. A preclinical study is planned to assess the efficacy and safety of the selected probe.

SURG-10. DEVELOPMENT OF NOVEL TOPICAL FLUORESCENT PROBE FOR INTRAOPERATIVE RAPID DETECTION OF GLIOMA

Abstract PURPOSE Fluorescence imaging is an important surgical adjunct in malignant glioma surgery. 5-aminolevulinic acid (5-ALA) has been proven effective for radical tumor resection and extended progression-free survival in a phase III randomized trial and therefore integrated into surgery for malignant glioma. Importantly, however, some limitations still exist in its use, which include false positivity and false negativity as well as inability of re-administration. In this study, we aimed to develop a novel, spray-type fluorescent probe using hydroxymethyl rhodamine green (HMRG) as a fluorescent scaffold. METHODS We have previously established a fluorescent probe library comprised of more than 320 kinds of HMRG probes. They have HMRG as a fluorescent scaffold with various types of dipeptides attached to it. Primary probe screening was performed using the homogenized tumor samples from patients with glioblastoma operated at our institution. Secondary screening followed using the selected probes and fresh tumor samples obtained from patients with glioblastoma operated from 2016 until 2018. Diced electrophoresis gel (DEG) assay, two-dimensional gel electrophoresis followed by a multi-well plate-based fluorometric assay, was performed to identify responsible enzymes for the selected probe. Further experiments with inhibitors, real-time PCR, immunohistochemistry, and western blotting were performed for confirmation. RESULTS Proline-arginine-HMRG (PR-HMRG) was selected as a candidate probe based upon the above two-step screenings. It achieved 79.4% accuracy in receiver operating characteristic curve analysis. Calpain-1 was found to be responsible to cleave PR-HMRG probe by DEG-proteome analysis. Calpain-1 protein was expressed at significantly higher level in tumors that were fluoresced by PR-HMRG than in those that were not. CONCLUSIONS Our innovative screening method was able to find PR-HMRG as a novel fluorescent probe effective for rapid detection of glioblastoma. A preclinical study is planned to assess the efficacy and safety of the selected probe.

A Novel Topical Fluorescent Probe for Detection of Glioblastoma.

Five-aminolevulinic acid (5-ALA) is widely used as an intraoperative fluorescent probe for radical resection of high-grade glioma, and thus aids in extending progression-free survival of patients. However, there exist some cases where 5-ALA fails to fluoresce. In some other cases, it may undergo fluorescence quenching but cannot be orally readministered during surgery. This study aimed to develop a novel hydroxymethyl rhodamine green (HMRG)-based fluorescence labeling system that can be repeatedly administered as a topical spray during surgery for the detection of glioblastoma. We performed a three-stage probe screening using tumor lysates and fresh tumor tissues with our probe library consisting of a variety of HMRG probes with different dipeptides. We then performed proteome and transcript expression analyses to detect candidate enzymes responsible for cleaving the probe. Moreover, in vitro and ex vivo studies using U87 glioblastoma cell line were conducted to validate the findings. The probe screening identified proline-arginine-HMRG (PR-HMRG) as the optimal probe that distinguished tumors from peritumoral tissues. Proteome analysis identified calpain-1 (CAPN1) to be responsible for cleaving the probe. CAPN1 was highly expressed in tumor tissues which reacted to the PR-HMRG probe. Knockdown of this enzyme suppressed fluorescence intensity in U87 glioblastoma cells. In situ assay using a mouse U87 xenograft model demonstrated marked contrast of fluorescence with the probe between the tumor and peritumoral tissues. The novel fluorescent probe PR-HMRG is effective in detecting glioblastoma when applied topically. Further investigations are warranted to assess the efficacy and safety of its clinical use.

Serum Exosomal Gamma-Glutamyltransferase Activity Increased in Patients with Renal Cell Carcinoma with Advanced Clinicopathological Features

Background: There has been no clinically useful diagnostic or prognostic biomarker for renal cell carcinoma (RCC). Serum γ-glutamyltransferase (GGT) activity has been reported to be a prognostic marker for several types of cancer including RCC. Exosomes or small extracellular vesicles present in body fluids have potential as a biomarker. We have recently demonstrated that GGT activity on exosomes isolated from serum is useful for the differential diagnosis of prostate cancer and benign prostate hyperplasia. In this study, we aimed to examine if serum exosomal GGT activity could be a marker for RCC. Methods: We examined GGT1 expression and GGT activity in cell lysates and exosomes from culture medium of HK-2 proximal tubule epithelial and RCC cell lines. GGT activity was measured using a fluorescent probe for GGT, γ-glutamyl hydroxymethyl rhodamine green. Serum and serum exosomal GGT activities were measured in patients with RCC. GGT1 expression in RCC tissues was evaluated by immunohistochemical staining. Results: GGT1 levels in exosomes from KMRC-1, OS-RC-2 and 786-O cells were elevated compared with those from HK-2 cells. In exosomes, GGT1 expression correlated with GGT activity determined using a fluorescent probe for GGT. In RCC patients, serum exosomal GGT activity was elevated in those with advanced stages (III/IV vs. I/II, p = 0.037) and those with microvascular invasion (with vs. without, p = 0.034). Immunohistochemical analysis showed that membranous GGT1 expression was increased in RCC with microvascular invasion. Notably, preoperative serum exosomal GGT activity could predict the likelihood of having microvascular invasion diagnosed by pathological examination of surgically resected specimens. Conclusions: Our results suggest that serum exosomal GGT activity could be a clinically useful marker for advanced clinicopathological features of RCC patients, and its combined use with conventional diagnostic modalities may improve the diagnosis and treatment of patients.

ɤ-glutamyl hydroxymethyl rhodamine green fluorescence as a prognostic indicator for lung cancer.

ɤ-glutamyltranspeptidase is an enzyme expressed in various malignancies including lung cancer. It rapidly activates non-fluorescent ɤ-glutamyl hydroxymethyl rhodamine green to highly fluorescent hydroxymethyl rhodamine green. The resultant tumor fluorescence is therefore an indicator of cellular ɤ-glutamyltranspeptidase activity. We have explored the use of ɤ-glutamyl hydroxymethyl rhodamine green as an intraoperative imaging tool for visualizing cancers. Herein, we evaluated the potential of the tumor fluorescence as a postoperative prognostic indicator. We included patients with non-small cell lung cancer who had undergone radical resection from 2012 to 2014 in the study. We assessed the fluorescence intensity of the resected tumor and normal lung tissue by ex vivo imaging using ɤ-glutamyl hydroxymethyl rhodamine green. Sixty-seven patients were eligible for the study (adenocarcinomas, n = 44; squamous cell carcinoma, n = 14; other histologies, n = 8). The pathological stages were I, II, III, and IV in 39, 15, 12, and 1 patient, respectively. Based on the fluorescence of the tumor tissue, the patients were divided into high fluorescence (n = 33) and low fluorescence (n = 34) groups. The 5-year overall survival rate was significantly higher in the high fluorescence group (72.7%) compared to the low fluorescence group (47.1%, P = 0.025). Similarly, pathological stage I patients of the high fluorescence group had higher 5-year overall survival (85.7% vs. 44.4%, P = 0.009) and recurrence-free survival (76.2% vs. 44.4% P = 0.044) rates compared to those of the low fluorescence group. ɤ-glutamyl hydroxymethyl rhodamine green fluorescence is a good postoperative prognostic indicator in patients with non-small cell lung cancer.

BOT-03 INVESTIGATION OF NOVEL SPRAY TYPE FLUORESCENT PROBE FOR GLIOBLASTOMA DETECTION

PURPOSE5-ALA is commonly used as an intraoperative tool in malignant glioma surgery, which has been proven effective for radical tumor resection and extended progression-free survival. However, there are some limitations in its use, such as false positivity, false negativity, and inability of re-administration. We aim to develop a novel fluorescent labeling system which can be repeatedly administered by spray during surgery, using hydroxymethyl rhodamine green (HMRG) as fluorescent scaffold originally designed at our university for cancer detection.METHODSPrimary probe screening was performed using the homogenized glioblastoma (GBM) samples with the fluorescent probe library comprised of more than 320 kinds of HMRG fluorescent scaffold combined with various types of dipeptides. Second probe screening was performed using fresh GBM specimens and the selected probes in primary screening. To identify the responsible enzymes, diced electrophoresis gel (DEG) assay was performed. This method utilizes the combination of two dimensional electrophoresis (isoelectric point and molecular weight) and a multiwell-plate-based fluorometric assay to find protein spots with the specified activities.RESULTSThe prominent probes were selected based upon the above two-step screenings. We identified two enzymes by proteome analysis and experiments using inhibitors, which was further confirmed with real-time PCR and western blotting.DISCUSSIONThis screening methodology is innovative in that it is based on selecting probes from the probe library that respond to clinical samples rather than creating probes from the responsible enzymes. Practical fluorescent probes can be established even for low-grade gliomas, which would be a breakthrough for rapid intraoperative diagnosis in glioma surgery.CONCLUSIONHMRG-based aminopeptidase fluorescent probes may be effective for GBM detection.

DDIS-02. DEVELOPMENT OF NOVEL SPRAY-TYPE FLUORESCENT PROBES FOR GLIOBLASTOMA DETECTION

Abstract PURPOSE: ALA is commonly used as an intraoperative tool in malignant glioma surgery, which has been proven effective for radical tumor resection and extended progression-free survival. However, there are some limitations in its use, such as false positivity, false negativity, and inability of re-administration. We aim to develop a novel fluorescent labeling system which can be repeatedly administered by spray during surgery, using hydroxymethyl rhodamine green (HMRG) as fluorescent scaffold originally designed at our university for cancer detection. [Methods]Primary probe screening was performed using the homogenized glioblastoma (GBM) samples with the fluorescent probe library comprised of more than 320 kinds of HMRG fluorescent scaffold combined with various types of dipeptides. Second probe screening was performed using fresh GBM specimens and the selected probes in primary screening. To identify the responsible enzymes, diced electrophoresis gel (DEG) assay was performed. This method utilizes the combination of two dimensional electrophoresis (isoelectric point and molecular weight) and a multiwell-plate-based fluorometric assay to find protein spots with the specified activities. [Results] The prominent probes were selected based upon the above two-step screenings. We identified two enzymes by proteome analysis and experiments using inhibitors, which was further confirmed with real-time PCR and western blotting. [Discussion] This screening methodology is innovative in that it is based on selecting probes from the probe library that respond to clinical samples rather than creating probes from the responsible enzymes. Practical fluorescent probes can be established even for low-grade gliomas, which would be a breakthrough for rapid intraoperative diagnosis in glioma surgery. [Conclusion] HMRG-based aminopeptidase fluorescent probes may be effective for GBM detection.

SAT-190 COMPREHENSIVE ANALYSIS OF URINARY AMINOPEPTIDASE ACTIVITIES FOR INVESTIGATING PROGNOSTIC MARKERS TO PREDICT THE PROGRESSION OF CHRONIC KIDNEY DISEASE

Renal prognosis is better correlated with the degree of tubulointerstitial damage than with that of glomerular injury (J Am Soc Nephrol. 2006: 17: 17–25), therefore a biomarker for tubular damage is important to predict chronic kidney disease (CKD) progression although a gold standard of tubular injury marker is yet to be established. Past reports showed that urinary GGT (γ-glutamyl transpeptidase) was one of renal prognostic factors (Transplant Proc. 2014; 46: 1812-8, Ren Fail. 2014; 36: 390-2). Urinary GGT activity is a tubular damage marker, considering that GGT is expressed in apical side of proximal tubule. In this viewpoint, other aminopeptidases are also expressed abundantly in proximal tubules, therefore it is possible that a certain aminopeptidase activity better reflects tubular damage than GGT. In addition, urinary aminopeptidase activity measurement has an advantage in quickness and cost-effectiveness once established. In this report, we examined comprehensive analysis of aminopeptidase activities in the urine of patients with CKD in order to search novel biomarkers to predict CKD progression, with a comparison to GGT activity. This study was approved by the ethical committee of University of Tokyo Graduate School of Medicine (11783, 11799). We collected the urine of adult patients with CKD G3b or G4. We measured urinary aminopeptidase activities by using our HMRG (hydroxymethyl rhodamine green)-based probe library, 384 kinds of fluorogenic probes for aminopeptidases. Urine was incubated with each probe for one hour and measured a fluorescent level, which was regarded as a target aminopeptidase activity. Each fluorescent probe activity was standardized by urinary creatinine concentration. We examined the association between urinary aminopeptidase activities and declines in eGFR for the past three years. We defined those whose eGFR decline rates per year, which was calculated by dividing eGFR, were over 5% as a ‘rapid’ group and the others as a ‘stable’ group. We also studied their clinical parameters obtained from medical records. A two-tailed paired Students’ t-test for continuous valuables and Fisher’s exact test for categorical valuables were used for the comparison of two groups. All statistical analysis was done with JMP Pro ver13.2.1. A p value < 0.5 was considered statistically significant. Forty patients were included in this study (70.2±13.2 years old: 19 women: estimated glomerular filtration rate (eGFR) 31.6±7.9 ml/min/1.73m2). Overall eGFR decline per year was 2.2±2.7 ml/min/1.73m2 and the decline rate was 8.3±10.7%. Twenty patients (50.0%) were in a rapid group. We analyzed 120 probes (31.3%), since the other 264 probes showed no fluorescence signals in almost all of the patients. We found several probes including ɤ-glutamyl-HMRG, a fluorogenic probe for detecting GGT, were significantly associated with CKD progression (Figure). Comparison of baseline characteristics between rapid and stable group was shown in Table.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We confirmed it was possible to measure urinary aminopeptidase activities by using our HMRG-based probes. Urinary GGT was significantly associated with eGFR decline, which was compatible with past reports. Other urinary aminopeptidase activity of cutting Lysine (Lys)-, LysLys- or Leucine-HMRG which are associated with eGFR decline may also become a novel biomarker for CKD progression.

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.

Rapid diagnosis of lymph node metastasis in breast cancer using a new fluorescent method with γ-glutamyl hydroxymethyl rhodamine green.

Sentinel lymph node biopsy is performed as a standard procedure in breast cancer surgery, and the development of quick and simple methods to detect metastatic lesions is in high demand. Here, we validated a new fluorescent method using γ-glutamyl hydroxymethyl rhodamine green to diagnose metastatic lymph nodes in breast cancer. One hundred and forty-nine lymph nodes from 38 breast cancer patients were evaluated in this study. Comparison of fluorescent and pathological images showed that this fluorescent method was successful for visualizing breast cancer cells in lymph nodes. This method had a sufficiently high sensitivity (97%), specificity (79%) and negative predictive value (99%) to render it useful for an intraoperative diagnosis of cancer. These preliminary findings suggest that this novel method is useful for distinguishing non-cancerous specimens from those in need of careful examination and could help save time and cost for surgeons and pathologists.

Asymmetric Rhodamine-Based Fluorescent Probe for Multicolour In Vivo Imaging.

To achieve rapid and sensitive detection of cancer, activatable fluorescent probes targeting proteases that are overexpressed in various types of cancer have been developed, based on the hydroxymethyl rhodamine green (HMRG) scaffold. However, to visualize altered activities of multiple enzymes in cancer sites, other scaffolds with distinct fluorescence properties from those of HMRG are needed. A novel asymmetrically modified rhodamine with suitable absorption/emission, brightness and equilibrium constant of intramolecular spirocyclization, working in the yellow/orange region, is introduced. As a proof of concept, a probe targeting γ-glutamyl transpeptidase (gGlu-HMJCR) was developed on the basis of the new scaffold. Simultaneous visualization and discrimination of tumours expressing γ-glutamyl transpeptidase (with gGlu-HMJCR) and cathepsins (with Z-Phe-Arg-HMRG) by colour were achieved in a mouse model in vivo.

Abstract 5113: Rapid Cancer Imaging By GGT-targeted Fluorescence Probe For Primary Lung Cancer

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&amp;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

Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green.

We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

In vivo imaging of intraperitoneally disseminated tumors in model mice by using activatable fluorescent small-molecular probes for activity of cathepsins.

It is difficult to completely remove carcinomas in unguided ablative surgery because they cannot be distinguished with the unaided human eye. Therefore, in order to precisely visualize tiny tumors and the borders between cancerous lesions and normal tissues, we have been developing fluorescence probes activatable only in cancer cells. We previously reported the hydroxymethylrhodamine green (HMRG)-based fluorescence probe gGlu-HMRG for γ-glutamyltransferase (GGT), which is overexpressed in a variety of cancer cells, and we showed that it enables in vivo rapid detection of human ovarian cancer SHIN-3 nodules with a high tumor-to-background (T/B) fluorescence ratio in model mice. However, cancer cell lines with low GGT expression could hardly be detected with gGlu-HMRG. Here we developed two new HMRG-based fluorescence probes for the cathepsin family of cysteine proteases, including cathepsin B (CatB) and cathepsin L (CatL), which show increased expression and/or activity, secretion, and altered localization in many kinds of cancer cells. The developed probes, Z-Phe-Arg-HMRG and Z-Arg-Arg-HMRG, are colorless and nonfluorescent at the physiological pH of 7.4, but are hydrolyzed to HMRG upon reaction with purified cathepsins, resulting in a more than 200-fold fluorescence increase. These probes could visualize human ovarian cancer cell lines SHIN-3, SK-OV-3, and OVCAR-3, of which the latter two were hardly detectable with gGlu-HMRG. Z-Phe-Arg-HMRG showed higher applicability than Z-Arg-Arg-HMRG for in vivo imaging, and we confirmed that 0.5-mm-sized SK-OV-3 tumor nodules disseminated on the mesentery in a mouse model could be rapidly visualized by Z-Phe-Arg-HMRG, with a T/B fluorescence ratio of 4.2. Further, intraperitoneally disseminated tumor could be visualized in real time in vivo by fluorescence endoscopy after spraying Z-Phe-Arg-HMRG, with a T/B ratio of 3. In conclusion, our HMRG-based activatable probes targeted to cathepsins have expanded the detectable range of cancers, and appear to be suitable for clinical application.

JS1-3 - Rapid in vivo Cancer Imaging by Topically Spraying a Newly Designed Activatable Fluorescence Probe

ABSTRACT Fluorescence imaging is one of the most powerful techniques currently available for continuous observation of dynamic responses in living cells and animals. For highly efficient development of novel fluorescence probes, we have established several rational strategies for controlling the properties of visible light to NIR-excitable fluorophores based on the intramolecular photoinduced electron transfer. These strategies are quite powerful and versatile and, indeed, based on these strategies, we have succeeded to develop a series of fluorescence probes for reactive oxygen species, ions, and various enzymatic activities. Recently, we have established another strategy to control the fluorescent properties of various fluorophores based on intramolecular spirocyclization. For example, if one of the amino groups of hydroxymethyl rhodaminegreen (HMRG), a new rhodaminegreen derivative bearing a hydroxymethyl group instead of the original carboxy group, is amidated with an amino acid, the resultant product is colorless in neutral pH buffer due to the preferred spirocyclization. This compound can be a highly sensitive fluorescence probe for the target aminopeptidase, because it is selectively reactive towards the target enzyme to yield highly fluorescent HMRG. Based on these HMRG-based fluorescence probes, we have succeeded to establish a novel strategy for in vivo tumor imaging by utilizing upregulated enzymatic activities in cancer cells. Gamma-glutamyltranspeptidase (GGT) is well known to be significantly overexpressed in several human tumors, and we have developed a novel fluorescence probe (gGlu-HMRG) for GGT based on HMRG scaffold, which is quickly activated by GGT and yields an over 350-fold increase in fluorescence signal compared with the quenched state. gGlu-HMRG showed a large fluorescence increase in several cancer cell lines, but not in a normal cell line. Indeed, tumor cells in a mouse model of peritoneal metastases were successfully visualized with high signal contrast between the tumor and background. The activation occurred within 1 min of spraying the probe, and the signal was strong enough to be detected even with our naked eyes. This probe is believed to be practical for clinical application during surgical or endoscopic procedures.