Development of Novel Fluorogenic Probes for Realizing Rapid Intraoperative Multi-color Imaging of Tiny Tumors
We found that hydroxymethyl rhodamine green (HMRG) strongly fluoresces, while mono-amidated HMRG derivatives are colorless and non-fluorescent due to the preferred spirocyclized structure in aqueous solution at pH 7.4. Based on these findings, we have established a rational design strategy for novel fluorogenic probes, and developed various aminopeptidase-sensitive probes which were applicable for living cell system, including gGlu-HMRG, a novel HMRG-based “activatable” fluorescence probe for γ-glutamyltranspeptidase (GGT). Tiny tumors could be detected in vivo by spraying gGlu-HMRG onto tissue surfaces that are suspected of harboring tumors, creating high signal contrast between the tumor and the background within 1 min. Furthermore, we succeeded to develop HMJCR as another-color fluorogenic scaffold for protease activity by optimizing intramolecular spirocyclization property of longer wavelength light-excitable asymmetric hydroxymethyl rhodamines. Based on this scaffold, gGlu-HMJCR could be developed, and was proved to be capable of detecting GGT activity in living cancer cells. Finally, simultaneous and rapid detection of different two peptidase activities in vivo was successfully achieved by making a cocktail of GGT-sensitive HMJCR probe and cathepsin-sensitive HMRG probe. This would enable the surgeons to detect wider types of cancers, and also to characterize and diagnose the tumors according to the difference in protease activity profiles.
KeywordsFluorescence Cancer Probe Multi-color imaging Peptidase
24.1 Rational Design of Organic Fluorogenic Probes Based on Unique Spirocyclization of Rhodamines by the Intramolecular Hydroxymethyl Group
Fluorescence imaging is one of the most powerful techniques currently available for continuous observation of dynamic intracellular processes in living cells. In recent years, this technique is not restricted to biological experiments but applicable to clinical fields. Among them, intraoperative fluorescence imaging of lesions or structures that are difficult to identify with the naked eye enable real-time characterization of tumors, thereby aiding clinical decisions on both intra- and postoperative treatments (Stewart and Wild 2014; Hanahan and Weinberg 2000).
Suitable fluorogenic (activatable fluorescence) probes that are initially non-fluorescent and turn to be highly fluorescent upon binding/reaction with target biological molecules of interest, are naturally of critical importance for fluorescence imaging. We have been working for developing novel fluorogenic probes, and succeeded to construct several versatile rational design strategies based on the concept of photoinduced electron transfer (Miura et al. 2003; Urano et al. 2005) and intramolecular spirocyclization (Kamiya et al. 2011; Sakabe et al. 2013). As for the latter design strategy, we have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open non-spirocyclic structure. More precisely, the pKcycl value, the pH at which the absorbance or fluorescence of the compound decreases to a half of the maximum value as a result of spirocyclization, of HMRG was calculated to be 8.1, but that of AcHMRG where one of the amines on the xanthene moiety was amidated, decreased to 5.3. Therefore at the physiological pH of 7.4, HMRG predominantly takes the ring-open form with strong fluorescence at λfl of 524 nm, but its acetylated derivative AcHMRG would predominantly be the in spirocyclized closed form with reduced absorbance or fluorescence in the visible region.
24.2 Development of Novel Fluorogenic Green Probes for Biological and Medical Purposes, Especially for Intraoperative Rapid Tumor Imaging
Furthermore, not only with tumor mice models, but with freshly resected real human tumor samples, we started to examine the efficacy of gGlu-HMRG as an intraoperative tumor detecting agent by collaborating with many surgeons. Indeed, gGlu-HMRG was proved to be effective for rapid intraoperative imaging of breast (Ueo et al. 2015; Shinden et al. 2016), oral (Shimane et al. 2016), and hepatic (Miyata et al. 2017) cancers. For example, breast cancer could be visualized regardless of its types like invasive/non-invasive or ER and HER2(+)/(−) with high specificity and sensitivity, both exceeded 90% (Ueo et al. 2015), and metastases in sentinel lymph nodes could be examined within a few minutes with high negative predictive values (>99%) (Shinden et al. 2016).
24.3 Development of Novel Fluorogenic Scaffold for Detecting Protease Activity in Longer Wavelength by Optimizing the Spirocyclization Properties: Novel Strategy for Fluorescence-Assisted Surgery with Multicolor Protease Imaging (Iwatate et al. 2016)
However, as can be easily imagined, because cancer cells are known to have big heterogeneity, gGlu-HMRG can only image cancer with elevated GGT activity, which accounts for only a small portion of all cancer. Two of the realistic clinical applications of fluorescent probes are preoperative diagnosis by fluorescent endoscopy and intraoperative diagnosis. Yet for fluorescence to be used for these purposes, the fluorescent probe must be able to detect, in principle, all types of cancer. Although this is difficult to realize with a single probe targeting one protease, by spraying or administering a cocktail of fluorescent probes targeting various proteases that can cover for each other, it would become possible to simultaneously detect multiple proteases. If the cocktail is composed of probes with different fluorescence wavelengths, this would also enable the surgeons to distinguish and characterize the tumors according to the difference in protease activity profiles.
If protease probes that function in a similar manner to gGlu-HMRG but with different target and wavelength were developed, such imaging can be realized. For a protease probe to be used for multicolour imaging with HMRG, the scaffold dye must have a spectroscopic property that can be differentiated from HMRG. If the scaffold was a spirocyclizing rhodamine that bears an amine on xanthene moiety available for peptide conjugation to be the switch for fluorescence activation and retains the hydroxymethyl group on the benzene ring moiety of HMRG for intramolecular spirocyclization to occur, some of the favourable properties of HMRG may also be applicable for the new probes. Thus, modifying parts of HMRG was deemed to be an appropriate strategy to develop a series of protease probes for such purposes.
Has longer λabs and λfl than HMRG for better tissue penetration
Emits fluorescence in the visible region for detection by the naked human eye
Single reaction point for more sensitive and quantitative detection
Structures of synthesised asymmetrical rhodamines bearing a hydroxymethyl group and their acetylated derivatives
Acetylated derivative of a rhodamine has been shown to function as a model compound for protease probes on HMRG. The changes to the absorption and fluorescence spectra matched well and most importantly, the change to pKcycl can be modelled with an easy-to-synthesise compound with a single amidation reaction. Thus, acetylated derivatives of HMDiMeR, HMDiEtR and HMJR were also synthesised (Table 24.1: 1d, 2d, 3d).
Spectroscopic properties of asymmetric N-alkyl rhodamines bearing a hydroxymethyl group
As intramolecular spirocyclization occurs through intramolecular nucleophilic attack of the hydroxymethyl group on the 9 position of the xanthene moiety, a hypothesis that making the 9 position less electron-rich would reduce the pKcycl value to the acidic side was contrived. One easy and synthetically-feasible solution was to introduce electron-withdrawing groups such as halogens to the xanthene moiety. Therefore, we started to synthesize three asymmetrical rhodamines with fluorination or chlorination to the 2 position of the xanthene moieties (Table 24.1: 1b, 1c, 2b, 2c, 3b, 3c), together with their acetylated derivatives. (Table 24.1: 1e, 1f, 2e, 2f, 3e, 3f).
The spectroscopic properties of the synthesised compounds supported the hypothesis that halogenation would have no negative effect on the spectroscopic properties such as λabs, λfl and Φfl, but would reduce pKcycl values (Table 24.2). The reduction in pKcycl values was larger for chlorinated compounds.
HMDiMeR derivatives and HMDiEtR derivatives were very dark compared to HMRG with Φfl of around 0.05, and their λabs were relatively short at around 530 nm: HMJR derivatives were brighter with Φfl of around 0.4 and had longer λabs at around 550 nm. The pKcycl value of AcHMJCR was 7.3, which suggests a half of the molecules would take open form under physiological pH, but this increases to 90% after the reaction. Even though these sets of values were not quite optimal in themselves, taking account the changes in Φfl and ε, fluorescence increase of about 40-fold could have been expected. Therefore HMJCR was chosen as the novel scaffold for a red-shifted protease probe.
The pKcycl value of gGlu-HMJCR was calculated to be 7.7, suggesting that approximately 33% of the probe exists in non-fluorescent spirocyclized form at the physiological pH of 7.4, whereas the putative hydrolysis product HMJCR with pKcycl of 8.9 exists predominantly in the fluorescent open form. In addition, the open form of gGlu-HMJCR was found to have remarkably reduced Φfl (0.06) and ε (20,000) compared to those of HMJCR (0.42 and 50,000, respectively). The combination of the diminutions in pKcycl, Φfl and ε resulted in efficient quenching of the fluorescence of gGlu-HMJCR.
Z-Phe-Arg-HMRG reacts with cysteine proteases in the cathepsins family such as cathepsin B and L, which are lysosomal endopeptidases with the increase in fluorescence of over 800-fold. As both cathepsins play a variety of roles in maintaining normal cellular metabolism, they are also naturally upregulated, secreted and show changes in localisation in various diseases, including breast, colorectal, gastric, lung, prostate, and ovarian cancers. These cathepsins are also known to contribute to the invasion of tumor cells to basal membrane, by degrading extracellular matrix components such as collagen I, collagen IV, laminin, fibronectin and elastin.
Mouse models of dual tumour implant were developed using two ovarian cancer cell lines with differing protease activities: SKOV-3/RFP and SHIN3. SHIN3 cells exhibit both cathepsins and GGT activities, while SKOV-3/RFP cells only show cathepsins activity. Thus, whereas Z-Phe-Arg-HMRG should label both SHIN3 and SKOV-3/RFP derived tumors, gGlu-HMJCR should only label SHIN3 derived tumors.
This was the first example known to date of such discrimination, and would serve as a guide for real-time and accurate characterization of tumor. The relationship between protease activity and therapeutic effect or prognosis are largely yet to be discovered, but such knowledge could be beneficial for aiding clinical decisions on intra- and postoperative treatments, leading to personalised medicine.
We have succeeded to construct a new fluorogenic scaffold HMJCR by optimizing its color, brightness, and spirocyclization property. HMJCR-based probes are capable of detecting various aminopeptidase activities in different color from so far-developed HMRG-based green probes. Thus, simultaneous live imaging of different protease activities in living cells, clinical specimen, and preferably in human patients in the future, are doable by the cocktail of HMRG- and HMJCR-probes.
Wide range of tumors that cannot be detected and characterised with a single protease activity can be captured simultaneously.
Tumors with a difference in protease activity profiles can be distinguished intraoperatively.
Some of the probes emit fluorescence in the visible region and are detectable and distinguishable from each other by the naked human eye, suggesting good translatability to clinical purposes such as intraoperative imaging.
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- Stewart BW, Wild CP (2014) World cancer report 2014. IARC Press, LyonGoogle Scholar
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