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Small-molecule based activatable fluorescence probes for detecting specific enzyme activity with high sensitivity can visualize the expression site of marker genes and cancers where the enzyme is highly expressed. However, the enzyme-catalyzed fluorescent hydrolysis product easily leaks out and diffuses from the reaction site, making it difficult to perform long-term tracking and immunohistochemical analysis which needs washing/fixation procedure. Our group have focused on quinone methide chemistry and developed series of activatable fluorescence probes with excellent intracellular retention that are converted to quinone-methide or aza-quinone-methide intermediates upon reaction with enzymes, which are then react with intracellular nucleophiles such as proteins and glutathione to be retained in cells and to exhibit significant increase in fluorescence. Based on this molecular design, we have developed fluorescence probes targeting β-galactosidase and γ-glutamyltranspeptidase with different colors. We also developed photo-functional probes such as activatable photosensitizers and caged fluorophores. These probes can visualize or kill target enzyme-expressing cells with high selectivity by suppressing the leakage of hydrolysis products from target cells, and fluorescence imaging in combination with immunostaining was possible due to the high tolerance of the obtained fluorescence signal even after washing and fixation.

In cancer surgery, complete surgical resection of tumor lesions is critical to optimize the outcome. However, it is sometimes difficult to distinguish the boundary between tumor and normal tissues, and residual tumor tissue can result in cancer recurrence. Intraoperative imaging with fluorescent molecular probes can assist surgeons to visualize tumor lesions and their boundaries during surgery. Here, we review molecular probes for fluorescence image-guided cancer surgery, focusing especially on recent developments in high-performance tumor-imaging probes and the strategies used for their design., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We previously showed that spraying the fluorescent probe gGlu-HMRG (γ-glutamyl hydroxymethyl rhodamine green) can visualize even tiny tumors on the mesentery and peritoneal wall of tumor-bearing mice. However, during surgery, repeated spraying is necessary to detect tumors located deep within organs. Here, we examine whether deeply located tumors can be stained by intravenous administration of this probe. In mice bearing subcutaneous tumors, intravenous administration of gGlu-HMRG resulted in a rapid and specific increase of fluorescence in the tumor, which was visible to the naked eye within 5 min, and the maximum fluorescence intensity ratio of tumor to normal tissue (T/N = 4.3) was reached at 30 min. In mice bearing lung tumors, the T/N ratio reached approximately 20 at 30 min after administration, and deeply located tumors were clearly visualized. These results suggest that intravenous administration of gGlu-HMRG may be a useful technique in fluorescence-guided surgery of tumors.

γ-Glutamyltranspeptidase (GGT) is overexpressed in several types of cancer. Existing GGT-targeting fluorescence probes can image these cancers, but the fluorescent hydrolysis product leaks from the target cancer cells during prolonged incubation or fixation. Here, we present a functionalized fluorescence probe for GGT, 4-CH 2 F-HMDiEtR-gGlu, which is designed to generate an azaquinone methide intermediate during activation by GGT; this intermediate reacts with intracellular nucleophiles to generate a fluorescent adduct that is trapped inside the cells, without loss of the target enzyme activity. Application of the probe to patient-derived xenograft (PDX) mice enabled in vivo cancer imaging for a prolonged period and was also compatible with fixation and immunostaining of the cancer tissue., (© 2020 Wiley-VCH GmbH.)

Fluorescence (FL)-guided detection of cancer is one of the most promising approaches to achieve intraoperative assessment of surgical margins. Enzymes, such as aminopeptidase, carboxypeptidase, and glycosidase, whose activities are increased in cancer, have attracted great interest as imaging targets for rapid and sensitive visualization of cancerous tissues with fluorescent probes. Activatable probes, which are initially nonfluorescent but become strongly fluorescent upon rapid one-step cleavage of their substrate moiety by the target enzyme, are especially promising for practical clinical application during surgical or endoscopic procedures due to the highly amplified FL change generated by enzyme-catalyzed turnover at lesion sites. Here, we describe robust protocols for using activatable fluorescent probes targeting cancer-associated enzyme activities to visualize cultured cancer cells, metastatic cancer in a mouse model, and cancerous lesions in surgical specimens from patients.

It is a long-term goal of cancer diagnosis to develop tumor-imaging techniques that have sufficient specificity and sensitivity to detect small tumor nodules during surgery or endoscopic surgery. Here, we introduce an avidin-conjugated fluorescence probe, Avidin-Leu-HMRG, which consists of a cancer-targeting macromolecule (avidin) and a protease-activatable probe. The conjugate has a high affinity for lectin on cancer cells and undergoes endocytosis, followed by irreversible fluorescence activation due to cleavage by lysosomal leucine aminopeptidase. In a mouse model of peritoneal ovarian metastases, the probe could detect submillimeter-sized tumor nodules with a high S/N ratio at 1 h after intraperitoneal injection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We designed and synthesized an activatable near-infrared (NIR) fluorescent probe for γ-glutamyltransferase, based on an asymmetric silicon rhodamine scaffold with an optimized equilibrium of intramolecular spirocyclization. The synthesized probe exhibits dramatic NIR fluorescence activation and, in combination with previously reported probes, enables discrimination of tumors with different enzymatic profiles.

Fluorescence-guided diagnostics is one of the most powerful techniques for real-time in situ tumor detection. Here, we introduce two categories of fluorescence probes used for tumor imaging (always-on probes and activatable probes) and briefly summarize recent advances in tumor-targeted fluorescence imaging probes and their clinical/preclinical applications, including our recent work on rational design of activatable fluorescence probes for tumors expressing aminopeptidases and glycosidases. These probes enable rapid and sensitive detection of tiny tumors as small as <1mm in diameter, both in vivo and in clinical specimens., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ability of the unaided human eye to detect small cancer foci or accurate borders between cancer and normal tissue during surgery or endoscopy is limited. Fluorescent probes are useful for enhancing visualization of small tumors but are typically limited by either high background signal or the requirement for administration hours to days before use. We synthesized a rapidly activatable, cancer-selective fluorescence imaging probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), with intramolecular spirocyclic caging for complete quenching. Activation occurs by rapid one-step cleavage of glutamate with γ-glutamyltranspeptidase (GGT), which is not expressed in normal tissue, but is overexpressed on the cell membrane of various cancer cells, thus leading to complete uncaging and dequenching of the fluorescence probe. In vitro activation of gGlu-HMRG was evident in 11 human ovarian cancer cell lines tested. In vivo in mouse models of disseminated human peritoneal ovarian cancer, activation of gGlu-HMRG occurred within 1 min of topically spraying the tumor, creating high signal contrast between the tumor and the background. The gGlu-HMRG probe is practical for clinical application during surgical or endoscopic procedures because of its rapid and strong activation upon contact with GGT on the surface of cancer cells.

A long-term goal of cancer diagnosis is to develop tumor-imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from nontarget tissues is crucial. Here we achieve highly specific in vivo cancer visualization by using a newly designed targeted 'activatable' fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH-activatable probes based on the boron-dipyrromethene fluorophore were synthesized and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex vivo and in vivo imaging of human epidermal growth factor receptor type 2-positive lung cancer cells in mice was performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific, cell surface-targeting molecules that result in cellular internalization.

Beta-galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TG-betaGal (J. Am. Chem. Soc. 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-betaGal, provides a dramatic fluorescence enhancement upon reaction with beta-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-betaGal, after confirming that tumors in the model could be labeled with an avidin-beta-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-betaGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 microm. This strategy has potential clinical application, for example, in video-assisted laparoscopic tumor resection.