In Vivo Imaging of Intraperitoneally Disseminated Tumors in Model Mice by Using Activatable Fluorescent Small-Molecular Probes for Activity of Cathepsins

Abstract

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.

Cathepsin B‐Specific Metabolic Precursor for In Vivo Tumor‐Specific Fluorescence Imaging

“…Therefore, cathepsin B-specific imaging probes and pro-drugs have received much attention for the development of new theranostic platforms for cancer therapy. [14] Thec athepsin Bspecific peptide caged metabolic precursor consists of three major components,acathepsin B-specific cleavable peptide moiety (Lys-Gly-Arg-Arg,K GRR), as pacer linker of paminobenzyloxycarbonyl (S), and the metabolic precursor of triacetylated N-azidoacetyl-d-mannosamine (Ac 3 ManNAz), resulting in RR-S-Ac 3 ManNAz (Scheme 1a). We have confirmed that the cathepsin Bs pecifically cleavable peptide moiety of Lys-Gly-Arg-Arg (KGRR) was successfully cleaved by the targeting cathepsin Be nzyme using in vitro enzyme tests and in vivo tumor-bearing mice.…”

Novel theranostic agents for next-generation personalized medicine: small molecules, nanoparticles, and engineered mammalian cells

“…In this respect, a new method reported by Urano et al which allowed rational development of intramolecularly caged fluorescence probes that were designed to be activated only when specific peptidases expressed on cancer cells were detected [50], could advance usefulness of theranostic agents. If more cancer-specific enzymes are discovered [51], their strategy may serve as an attractive foundation for increasing the variety of small-molecule-based theranostic agents, owning to its flexibility against different enzymes, multiple potential caging sites for forming an AND gate, and easy convertibility to a photosensitizer [52]. Additionally, the discovery of enzymes that can cleave specific molecular bonds that are specific biomarkers of other diseases would also expand the availability of small-molecule-based theranostic agents.…”

A Bright Future for Precision Medicine: Advances in Fluorescent Chemical Probe Design and Their Clinical Application

“…Urano et al have functionalized this fluorophore with various substrate moieties to produce a fluorescent signal when removed by a target enzyme. Targets include leucine aminopeptidase (Leu-HMRG) (Sakabe, et al, 2013), fibroblast activation protein (Ac-GlyPro-HMRG) (Sakabe, et al, 2013), β-galactosidase (βGal-HMRG) (Kamiya, et al, 2011), γ-glutamyltransferase (gGlu-HMRG) (Urano, et al, 2011), and most recently cathepsins (ZFR-HMRG) (Figure 4E) (Fujii, et al, 2014). This type of turn-on dye has great promise, as it is relatively small and does not require additional large quencher groups.…”