Urano Y, Sakabe M, Kosaka N, Ogawa M, Mitsunaga M, Asanuma D, et al. Rapid cancer detection by topically spraying a γ-glutamyltranspeptidase–activated fluorescent probe. Sci Transl Med 2011;3:110ra119. (Reprinted with permission.)
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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.
Translation of fundamental biomedical optics principles and methods from bench to bedside has become an intriguing venture in medical research. A widely known success story is pulse oximetry, a noninvasive technique that reports the oxygen levels in arterial blood, and arguably a mainstay of vital sign assessments in clinical medicine.1, 2 More sophisticated near-infrared (NIR) optical spectroscopy techniques, which rely on the intrinsic absorption properties of tissue in the NIR wavelengths, have been used successfully to interrogate tissue compositions and functional status, with the goal of detecting human diseases or monitoring physiologic events.3–5 However, the application of optical imaging in clinical settings has generally lagged behind its spectroscopy counterpart. This is partly due to the difficulties in reporting physiological or molecular processes with high quantitative accuracy, especially in deep tissue such as human liver. Some of these challenges have been addressed by advances in quantitative image reconstruction algorithms, improved laser technology, and use of highly sensitive optical detectors. Consequently, optical imaging is now applied to diverse organs such as the breast, skin, joints, gastrointestinal, bladder, and the oral cavity. A niche that has recently emerged for optical imaging in the clinical arena is real-time image guidance in the surgical resection of tumors.
Surgery remains the primary treatment paradigm for most solid tumors. Today's surgeons are exceptionally skilled in the art of open and minimally invasive surgeries with good patient outcomes. However, real-time image guidance can facilitate intraoperative assessment of surgical margins and the detection of small positive nodules that are not visible to the unaided human eye. These needs have inspired the development of optical imaging instruments for use in the operating room. The simplicity, use of nonionizing radiation, and capability of real-time image guidance without disrupting normal surgical procedures in the operating room favor optical imaging methods.6 In addition to commercially available intraoperative optical instruments,6 a recent study reported the development of a simple wearable goggle system that enables the surgeon to navigate the surgical bed and identify positive tumor nodules in real time.7 To enhance tumor-to-normal tissue contrast for intraoperative assessment of the surgical bed and margins, these systems rely on contrast agents that stain tumors selectively. Thus, a combination of optical imaging device and tumor-selective fluorescent molecular probes can facilitate the identification of micron-sized tumors and the assessment of surgical margins with high sensitivity and specificity, and improve the extent of resection.
Researchers are actively pursuing two major reporting strategies in developing molecular imaging probes for intraoperative image-guided oncologic surgery. Affinity molecular probes are contrast agents that selectively accumulate in tumor tissue relative to the surrounding normal tissue by binding to overexpressed proteins in malignant tumors or through other uptake mechanisms. In its simplest form, a dye such as heptamethine cyanine was recently shown to have an intrinsic tumor-targeting capability without conjugation to a biological carrier,8 although this approach is subject to further scrutiny. With this exception, affinity probes typically involve the conjugation of a fluorescent dye to tumor-targeting biomolecules such as monoclonal antibodies or high affinity peptide ligands. This approach has been successfully used in nuclear imaging, where radiolabeled biomolecules have been shown to detect human cancer noninvasively.9 Replacement of the radionuclide with a fluorescent dye has become a viable approach in optical imaging. In fact, the first NIR fluorescent dye-labeled peptide (octreotate) used to demonstrate molecular optical imaging of tumors10 was modeled after the first US Food and Drug Administration-approved radiolabeled peptide (111In-DTPA-octreotide) (OctreoScan; Covidien, Hazelwood, MO), which is used clinically to image neuroendocrine tumors in humans.9 Using the tumor affinity targeting approach, a recent pilot human study showed that fluorescein-labeled folate, which targets the folate receptor, significantly improved detection of ovarian cancer metastases intraoperatively.11 However, a lingering concern with many affinity probes is the lengthy time lag between administration of the imaging agent and the onset of surgery required to minimize background fluorescence through removal of the circulating or nonreceptor bound molecular probes.
To address this problem, another research group had earlier used an endoscopic spray catheter to topically administer a fluorescein-labeled tumor-targeted heptapeptide to detect colonic dysplasia in human patients.12 In this study, the background fluorescence was minimized by rinsing off the excess fluorescein labeled peptide with water, followed by imaging within 5 minutes of administering the molecular probe. These studies demonstrate the feasibility of tumor-targeted optical molecular probes in humans, but also reveal the need for rapid contrast enhancement in tumors through suppression of background signal.
A solution to the problem of high background fluorescence is within the purview of activatable molecular probes. These molecular probes are designed to have low fluorescence yield until they encounter a molecular target (e.g., enzyme activatable probes)13 or localize in favorable physiological medium (e.g., pH activatable probes).14, 15 The enzyme activatable molecular probes were designed to report the presence and functional status of diagnostic enzymes such as cathepsins and matrix metalloproteinases, which are highly active in many tumors.13 Although these probes have low background fluorescence, the polymeric materials used for their development results in very slow rate of fluorescence enhancement, requiring several hours for optimal signal enhancement in tumors. Moreover, systemic administration of such macromolecules can elicit side reactions in different patients, a risk that may limit their use in humans.
To overcome the above limitations, Urano et al.16 recently reported a new class of activatable molecular probes that combine rapid fluorescence enhancement in tumors with high specificity. Taking advantage of the overexpression of γ-glutamyltranspeptidase (GGT) on the cell surface of diverse tumors, the authors developed fluorogenic substrates for GGT. They synthesized a spirocyclic γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) and explored its use as a fluorogenic substrate for GGT. Upon interaction with GGT, cleavage of the glutamyl group results in the spontaneous conversion of the structurally constrained spirocyclic molecules to the highly fluorescent hydroxymethyl rhodamine green (HMRG) derivative (Fig. 1). Because GGT is a cell surface enzyme, and the substrate is a small molecule, the conversion of spirocyclic gGlu-HMRG to HMRG would be expected to result in rapid clearance of the fluorophore from the tumor site, a condition that would lead to nonspecific or low fluorescence in the region of interest. Instead, the authors observed highly localized fluorescence signal in tumors.
This fortuitous observation was attributed to the generation of a more hydrophobic HMRG that rapidly internalizes in cells than the relatively hydrophilic gGlu-HMRG that is less permeable to cell membranes. Although this is a reasonable explanation, additional studies are needed to delineate the exact mechanism of HMRG's cellular uptake. A closer examination of the cellular and in vivo images shows that the fluorescence emanates almost exclusively from the intracellular compartment. These data suggest the possibility of a multistep activation pathway, where the spirocyclic structure remains intact after rapid cleavage of the γ-glutamate, followed by internalization of the more hydrophobic non-fluorescent spirocyclic HMRG, which subsequently isomerized to the highly fluorescent “open form” HMRG in the acidic lysosomes. Regardless of the mechanism of internalization, the specificity and rapid activation of gGlu-HMRG by tumors represents a major advance in the fields of molecular imaging and image-guided surgery.
An important outcome of the rapid and specific fluorescence activation is the potential to develop aerosolized activatable molecular probes for topical application in the surgical field. Urano et al.16 showed that small tumor nodules are identifiable within 10 seconds of spraying the activatable molecular probe gGlu-HMRG. For surgical guidance, the topical application of the molecular probe has several advantages, including the elimination of systemic toxicity and the use of small amounts of the molecular probes. These features favor human translation of the method. However, extensive preclinical studies are needed before human studies become feasible. Unlike affinity molecular probes that are now in pilot human studies at different medical centers, translation of this fast-acting activatable molecular probes to humans will tread an uncharted course in regulatory approval territory.
Clearly, Urano et al.16 have uncovered an exciting molecular contrast generation pathway with direct clinical translation potential. Their findings represent a major shift in the use of activatable molecular probes for real-time surgical guidance. The authors effectively demonstrated, for the first time, the use of γ-glutamyltranspeptidase as a molecular target for synergistic and selective real-time fluorescence image-guided surgery. The fast and specific transformation of the quenched spirocyclic to the highly fluorescent derivative created a product with distinct physical and biochemical properties from the substrate. This interesting strategy could be used to design activatable probes for detecting other molecular signatures of disease in vivo. A clear understanding of the internalization process will be useful for optimizing the nature of the anticipated fluorescent product from an enzyme substrate to further improve the specificity and sensitivity of the method. Opportunities to use the spray-and-image paradigm for identifying tumor boundaries, guide resection, and ensure complete removal of microscopic positive tumor nodules using the strategy reported by this group are enormous.
Although the authors used an ovarian cancer model to demonstrate the rapid tumor detection in vivo, the approach is applicable to various forms of hepatic tumors as well. The incidence of hepatocellular carcinoma (HCC) worldwide continues to rise, with more than 500,000 deaths per year.17, 18 In late stages, organ transplant is currently the only curative option for patients with HCC, but viable organs are in short supply. This leaves HCC resection as the alternative treatment regimen for some patients, because traditional chemotherapy or external beam radiation is generally ineffective.19, 20 These limitations have resulted in the use of open HCC resection, which will benefit from the fast activatable molecular probes approach (Fig. 1) and the development of minimally invasive image-guided HCC ablation methods, including the use of radiofrequency or cryoablation techniques. Other options for the treatment of HCC that are too large for direct ablation are the use of endovascular techniques such as hepatic arterial chemoembolization or small particle embolization. Regardless of the treatment method, real-time image guidance is crucial to the success of these techniques. Contingent on the overexpression of diagnostic aminopeptidases such as GGT, the method described by Urano et al.16 will bring all the advantages described above to improve the treatment of HCC patients. The ease of spraying the activatable probes in open surgery or applying them through catheters will provide alternative image guidance during treatment. Of course, an important limitation of optical imaging methods is the inability to detect deeply embedded tumors in the liver, particularly when using visible light wavelengths, because of the high attenuation of light by this organ. In collaboration with liver surgeons, the Achilefu group at Washington University has conducted a pilot human study using an NIR fluorescence imaging goggle system7 to guide HCC resection (unpublished work). In this scenario, an NIR molecular probe enabled visualization below the surface of the liver. The use of real-time optical imaging techniques for intraoperative procedures will only continue to increase in the future, positioning the rapid activatable probe paradigm as a viable option to improve patient outcomes.