FocusCancer Surgery

Glowing Tumors Make for Better Detection and Resection

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Science Translational Medicine  23 Nov 2011:
Vol. 3, Issue 110, pp. 110fs10
DOI: 10.1126/scitranslmed.3003375


Tumor-specific fluorescent probes that can be administered topically make tumors glow selectively and thus have great potential for improving cancer detection and removal.

The ability of the surgeon to accurately visualize primary tumor margins and identify metastatic nodules at the time of surgery is an important factor in determining the success of any cancer operation. In this issue of Science Translational Medicine, Urano et al. (1) describe a rapid cancer-labeling method that involves topically spraying tumors with a fluorescence-imaging probe, gamma-glutamyl hydroxymethyl rhodamine green (γGlu-HMRG); the probe is activated in a tumor-specific manner by cleavage of the γGlu moiety from HMRG by an enzyme commonly found in cancer cells, gamma-glutamyltranspeptidase (GGT). Fluorescence imaging is appropriate for cancer navigation and offers higher resolution and sensitivity compared to radiological imaging and to visual inspection and palpation during surgery (2).


Clinical studies. A variety of labeling compounds have been used for fluorescence-guided surgery in human subjects. For example, sentinel lymph nodes—the first reached by metastatic cells—in breast cancer patients were detected and similarily labeled by the near-infrared (NIR) fluorescing dye indocyanine as well as by 99mTc-lymphoscintigraphy, a radioactive tracer and state-of-the-art imaging method (3). However, indocyanine does not specifically label tumor cells. In contrast, the metabolite 5-aminolevulinic acid, a precursor of hemoglobin, can drive the accumulation of porphyrins within malignant glioma. Porphyrin fluorescence in the brain can then be visualized with the use of a modified neurosurgical microscope (4). In one study, patients with malignant gliomas were given 5-aminolevulinic acid orally three hours before undergoing either bright light– or fluorescence-guided neurosurgery. In the latter group, 65% of 139 patients displayed complete removal of their tumors, while in the former group only 36% of 131 patients showed complete tumor resection. Furthermore, patients who underwent fluorescence-guided surgery had higher 6-month progression-free survival rates (41%) than did those who had surgery under white light (21%).

Van Dam et al. (2) conjugated folate to fluorescein isothiocyanate (FITC) for targeting folate receptor–α (FR-α)—which is often overexpressed in ovarian cancers—in 10 ovarian cancer patients who were undergoing abdominal surgery. The surgeons used a real-time multispectral intra­operative fluorescence imaging system for tumor detection and achieved fluorescence-guided resection of tumor deposits less than 1 mm in size. However, overexpression of FR-α varies greatly among different tumors types, which reduces the general applicability of this approach.

Fluorescent switches. Activatable cell-penetrating peptides (ACPPs) are composed of a fluorescently labeled polycationic cell-penetrating peptide (CPP) that is coupled via a cleavable linker to a neutralizing peptide. If tumor tissue contains the appropriate proteases, the linker is cleaved, thereby allowing the CPP to enter tumor cells and make them fluorescent (5). ACPPs have been used to label human and mouse breast tumors as well as a melanoma transplanted in mice. The peptide-labeled tumors were able to be resected under fluorescence guidance, and the surgery resulted in better long-term tumor-free and overall survival relative to animals from which tumors were resected with the use of brightfield illumination only (5).

In another study, a monoclonal antibody directed against one of two cancer-specific proteins—cancer antigen 19-9 (CA19-9) or carcino-embryonic antigen (CEA)—was conjugated to a green fluorophore and delivered intravenously into nude mice with orthotopic human pancreatic or colon tumors (6). Once the fluorescent antibodies adhere to the CEA or CA19-9 antigens of CEA- or CA19-9–expressing cancer cells, respectively, the tumors become fluorescent. Tumors that were invisible with standard bright-field imaging demonstrated clear fluorescence and were resected under fluorescence guidance (6).

Researchers have also performed genetic labeling of tumors in situ with green fluorescent protein (GFP). For example, Fong et al. (7) used a herpes simplex virus (NV1066) vector that expresses the gfp gene to label, in a mouse model, metastatic lung tumor foci of less than 1 mm located in the pleural cavity. The tumor foci became fluorescent because NV1066 selectively replicates in cancer cells and expresses GFP. The fluorescently labeled tumors were detected by using a thoracoscopic endoscope system (which can be inserted into the thoracic cavity) equipped with fluorescent filters. Kishimoto et al. selectively and accurately labeled tumors with GFP using a telomerase-dependent adenovirus (OBP-401) that expresses the gfp gene only in cancer cells, which, in contrast to normal cells, express the telomerase enzyme (8). The labeled tumors could then be resected under fluorescence guidance (Fig. 1). The authors also demonstrated that tumors that recurred after fluorescence-guided surgery maintained GFP expression (9). Because the recurrent cancer cells stably express GFP, detection of cancer recurrence and metastasis is also possible with OBP-401 GFP labeling. Maintenance of label in recurrent tumors is not possible with nongenetic probes.

Fig. 1. Guiding lights.

Fluorescence-guided surgical removal of peritoneal-disseminated human colon tumors after in situ GFP labeling with the gfp-expressing OBP-401 adenovirus vector. Noncolored human colon cancer cells were injected into the abdominal space of nude mice. Ten days later, OBP-401 was injected intraperitoneally. (A) Disseminated nodules were efficiently labeled by OBP-401 and noninvasively visualized by GFP expression. (B) Laparotomy was performed to remove intra-abdominal disease under GFP-guided navigation. (C) Disseminated nodules visualized by GFP-guided navigation were removed [Scale bars: (A)–(C), 10 mm]. [Adapted from H. Kishimoto et al. (8), with permission.]


Laparoscopic, or minimally invasive, surgical systems that contain a standard bright-lighting function were recently enhanced with a fluorescence excitation light that enables imaging of fluorescently labeled tumors along with the surrounding anatomy in orthotopic mouse models of cancer (10). Fluorescence-guided laparoscopy is sufficiently sensitive under fluorescence-light excitation to detect fluorescent metastatic lesions smaller than 1 mm, which are not visible under bright light when unlabeled.

In the current study, Urano et al. (1) used fluorescence-guided laparoscopy to visualize and remove tumors illuminated by their newly developed probe, γGlu-HMRG. The probe effectively and selectively labeled, in a mouse model, invasive human ovarian cancer, which expresses the probe-activating GTT enzyme. Small tumor nodules were sharply defined in the peritoneum as early as 10 seconds after spraying the abdominal cavity with the γGlu-HMRG probe, and the tumors remained fluorescent for at least 1 hour. In the living mouse, implants as small as 1 mm were then removed by laparoscopy under fluorescence guidance. The rapidity of activation of γGlu-HMRG and simplicity of the topical application are the main advantages of this new probe.

The means to make tumors glow offers great potential advantages for tumor detection during fluoresence-guided laparoscopy and surgery of all types. But a major question remains: What is the best means of labeling tumors? The method developed by Urano et al. (1) to label tumors by topically spraying with a tumor-specific probe is rapid (on the order of seconds to minutes) and facile. However, this new method is limited to those tumors that overexpress GTT. The use of FR-α for targeted fluorescence labeling also may be limited to a few tumor types such as ovarian cancer, which overexpresses FR-α (4). And 5-aminolevulinic acid may also have limited labeling applicability beyond glioma (2). Also promising are the tumor-labeling methods that use fluorescent tumor-specific antibodies (6), which may have wider applicability. However, antibodies require intravenous injection, and labeling takes hours or days. Furthermore, this method is limited to only those cancers for which tumor-specific antigens have been characterized. The ACPP labeling method (5) is useful only in those tumors that express an appropriate protease with which to cleave and thus activate the probe.

Because tumors of all types express telomerase, the genetic labeling method that uses a telomerase-dependent adenovirus to deliver GFP specifically to tumors offers the potential of widespread application. This genetically stable labeling system also allows detection of cancer recurrence, which is potentially a great advantage, especially when coupled with fluorescence laparoscopy. However, the virus vector must be further tested for safety in humans before this method can be translated.

References and Notes

  1. Acknowledgments: : The authors’ laboratories are supported by NIH grants CA132971 and CA142669.
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