PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Clin Cancer Res. Author manuscript; available in PMC Aug 5, 2013.
Published in final edited form as:
PMCID: PMC3733255
NIHMSID: NIHMS495409
Integrin αvβ3-Targeted IRDye 800CW Near-Infrared Imaging of Glioblastoma
Ruimin Huang,1,2,3 Jelena Vider,1,8 Joy L. Kovar,7 D. Michael Olive,7 Ingo K. Mellinghoff,1,4 Philipp Mayer-Kuckuk,6 Moritz F. Kircher,2,3,5 and Ronald G. Blasberg1,2,3,5,9
1Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
2Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
3Brain Tumor Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
4Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
5Nanotechnology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065
6Bone Cell Biology and Imaging Laboratory, Hospital for Special Surgery, New York, NY 10021
7Translational Research, LI-COR Biosciences, Lincoln, NE 68504
8Current address: Host Response to Cancer Laboratory, School of Medical Science, Griffith University, Parklands Dr, Gold Coast, QLD 4222, Australia.
9Corresponding author: Departments of Neurology and Radiology, Box 52, Molecular Pharmacology & Chemistry Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065. Tel: 646-888-2211; Fax: 646-422-0408; blasberg/at/neuro1.mskcc.org.
Purpose
Integrin αvβ3 plays an important role in tumor angiogenesis, growth and metastasis. We have tested a targeted probe to visualize integrin receptor expression in glioblastomas using near-infrared fluorescent (NIRF) imaging.
Experimental design
A transgenic glioblastoma mouse model (RCAS-PDGF-driven/tv-a glioblastoma, which mimics the infiltrative growth pattern of human glioblastomas), and two human orthotopic glioblastoma models, (U-87 MG with high integrin β3 expression and TS543 with low integrin β3 expression), were studied. An integrin-targeting NIRF probe, IRDye 800CW-cyclic-RGD peptide (IRDye 800CW-RGD), was tested by in vivo and ex vivo NIRF imaging.
Results
We demonstrate that the IRDye 800CW-RGD peptide: 1) specifically binds to integrin receptors, 2) is selectively localized to glioblastoma tissue with overexpressed integrin receptors and is retained over prolonged periods of time, 3) is associated with minimal autofluorescence and photobleaching due to imaging at 800 nm, 4) provides delineation of tumor tissue with high precision due to a high tumor-to-normal brain fluorescence ratio (79.7±6.9, 31.2±2.8, and 16.3±1.3) in the U-87 MG, RCAS-PDGF, and TS543 models, respectively; p<0.01) and 5) enables fluorescence-guided glioblastoma resection. Importantly, small foci of residual fluorescence were observed after resection was completed using white light imaging alone, and these fluorescent foci were shown to represent residual tumor tissue by histology.
Conclusions
NIRF imaging with the IRDye 800CW-RGD probe provides a simple, rapid, low-cost, non-radioactive and highly translatable approach for improved intraoperative glioblastoma visualization and resection. It also has the potential to serve as an imaging platform for noninvasive cancer detection and drug efficacy evaluation studies.
Keywords: Near-Infrared Fluorescence, RGD, IRDye 800CW, Glioblastoma, Integrin
Integrin-targeting agents, based on the cyclic RGD peptide, provide the opportunity to visualize and quantify integrin receptor expression levels noninvasively. They have been extensively used in the study of tumor angiogenesis, growth and metastasis (1). A decade ago, a series of RGD peptides attached to different radionuclides were developed for positron emission tomography (PET) (2) and single-photon emission computed tomography (SPECT) (3) imaging. Although nuclear imaging modalities have high sensitivity and allow accurate quantification of tracer distribution in the body, they are associated with ionizing radiation, high-cost, and comparatively poor spatial and temporal resolution.
In contrast, fluorescence imaging is low-cost and associated with high spatial resolution, but has limited depth of penetration (due to photon scatter and absorption) and can be complicated by autofluorescence (high background) and photobleaching (4). Recently, the concurrent development of near-infrared (NIR) fluorophores and equipment has led to a revolution in optical imaging. NIR dyes are excited and emit photons in the so-called NIR window (650-900 nm), allowing deeper light penetration through tissues as well as decreased light scattering and autofluorescence. Various NIR agents have been developed and are currently available for research and clinical use, including fluorescein, indocyanine green (ICG), and IRDye 800CW (5). ICG has been used for decades as an angiographic contrast agent and has been translated to the clinic for noninvasive mapping of the lymphatic system (6). Unfortunately, the clinically approved form of ICG lacks a reactive functional group to enable the attachment of targeting moieties (7).
More recently, interest has moved to the NIR dyes at ~800 nm. IRDye 800CW is a fluorophore with a peak excitation at 774 nm and emission at 805 nm. It exhibits deeper tissue penetration and a higher signal-to-background ratio than Cy 5.5, due to the higher excitation frequency (8). Using a dual-labeled cyclic RGD motif for NIR fluorescence (NIRF) imaging (IRDye 800CW) and gamma scintigraphy (111In), the NIRF from IRDye 800CW provided greater signal to noise than the radiotracer 111In (9). Furthermore, IRDye 800CW can be functionalized with either an NHS or maleimide reactive group, allowing it to be attached to a number of biomolecules for targeting. Examples include Trastuzumab (10) and 2-deoxy-D-glucose (11).
Integrins are heterodimeric transmembrane cell surface receptors coupling the extracellular environment to the cytoskeleton, as well as receptors for transmitting signals important for cell migration, invasion, proliferation and survival. A subset of integrins, including αvβ3, recognizes a common motif in their ligands, the RGD sequence (argine-glycine-aspartic acid) (12). The integrin αvβ3 is highly expressed not only on the tumor vasculature and angiogenic endothelial cells, but also on tumor cells, including human gliomas (13). In this report, we focus on demonstrating the utility of NIRF-800 imaging for the tumor detection in the well-established RCAS-PDGF-driven/tv-a mouse glioblastoma model (RCAS-PDGF) (14), U-87 MG (15) and TS543 (16) human orthotopic glioblastoma models. The RCAS-PDGF, U-87 MG and TS543 models exhibit different levels of integrin receptor up-regulation. We show that an IRDye 800CW-conjugated RGD peptide can specifically target the overexpressed integrin receptors in these glioblastomas, and visualization of the tumor and tumor margins can be achieved by NIRF imaging. This has significant implications for the use of optical molecular imaging in both the preclinical and potentially clinical setting to facilitate more sensitive cancer detection and more complete surgical resection of glioblastomas.
Reagents
Cyclic Arg-Gly-Asp-D-Phe-Lys peptide (cRGD) and IRDye 800CW-conjugated RGD peptide (IRDye 800CW-RGD) were provided by LI-COR Bioscience (Lincoln, NB). IRDye 800CW-conjugated cyclic Arg-Ala-Asp-D-Phe-Lys peptide (IRDye 800CW-RAD) (LI-COR) was used as the nonspecific control.
Quantitative near-infrared imaging in vivo
All animal experiments were approved by the Institutional Animal Care and Use Committees of Memorial Sloan-Kettering Cancer Center (MSKCC) (protocol number: 06-07-11).
Mice were anesthetized with 2.5% isoflurane throughout the imaging procedure. Before image acquisition, mouse heads were shaved. The Pearl Impulse Imager (LI-COR) was used for quantitative NIRF imaging in vivo. The excitation/emission settings for the 700- and 800-nm channels were 685/720 and 785/820 nm, respectively.
Animals were imaged prior to injection to establish a background level of fluorescence. IRDye 800CW-RGD peptide alone (1 nmol), IRDye 800CW-RAD peptide alone (1 nmol) and blocking peptide mixture (1 nmol IRDye 800CW-RGD peptide plus 20 nmol cRGD peptide), respectively, were injected via tail vein and imaged at 0.5, 24 and 48 hours. The images were analyzed using Pearl Impulse Software (version 2.0). Regions of interest (ROIs) for both head and background were selected from equivalent-sized areas containing the same number of pixels. ROIs were quantified for total pixel values.
Quantitative near-Infrared Imaging ex vivo
Quantitative analysis of IRDye 800CW-fluorescence in the sliced brain tissues and the frozen sections were measured ex vivo in the 800 nm channel by the Odyssey Infrared Imaging System (LI-COR). Odyssey Application Software (version 3.0) was used to acquire fluorescence (sensitivity=5, resolution=21 μm) and to quantify the absolute fluorescence intensities. The background fluorescence was subtracted from the signal intensities. The area-weighted fluorescence signal was used to compare targeting agent specificity.
Statistical Analysis
Statistical comparisons were performed using the Sigma Plot 8.0 (SPPS Inc., Chicago, IL). The presented data are shown as mean±SEM. Comparisons between mean values were performed using the Student’s paired t-test. Statistical significance was set at p<0.01.
Differential expression of integrins in the RCAS-PDGF, U-87 MG and TS543 glioblastoma models
Three different glioblastoma models were chosen to test different expression levels of integrin receptor. The RCAS-PDGF transgenic mouse glioblastoma model recapitulates many of the histological and MRI features of human glioblastomas (17, 18). It is a very aggressive and infiltrating tumor, with the formation of oligodendrogliomas in ~60% of mice by 12 wk of age (19). The U-87 MG human glioblastoma model was selected as a positive control because it has been shown previously to express high levels of integrin αvβ3 (15). The TS543 glioblastoma model was developed from a human patient with the PDGFRA gene amplification (20), and has been used to investigate the function of glioblastoma-associated oncogenes/tumor suppressor genes (21, 22) and microRNA (23, 24), as well as response to receptor tyrosine kinase inhibitors (16, 20). The TS543 orthotopic xenografts are very invasive, with isolated microscopic tumor foci (25). To assess the expression level of integrin β3 in tumor cells and tumor endothelial cells, co-immunofluorescence staining of integrin β3 with tumor cell markers (HA-tag for the RCAS-PDGF model, EGFP for the U-87 MG model and human-specific vimentin for the TS543 model) and an endothelial cell marker (CD31) was performed. In the RCAS-PDGF model (Fig. 1A), integrin β3 was significantly overexpressed in tumor endothelial cells (right panel) and in some, but not all glioblastoma cells (left panel). Overexpression of integrin β3 was shown in the U-87 MG tumor cells and its expression was barely detected in tumor endothelial cells (Fig. 1B). In the TS543 glioblastoma, very low expression of integrin β3 was detected in both tumor cells and tumor endothelial cells (Fig. 1C), and this model was used as an integrin β3 “low model” in further studies. Different expression levels of integrin β3 in the cultured U-87 MG and TS543 tumor cells was confirmed by Western blotting assay (Fig.1D).
Fig. 1
Fig. 1
Expression of integrin β3 (red) in the RCAS-PDGF (A), U-87 MG (B) and TS543 (C) glioblastomas by immunofluorescence co-staining. CD31 was used as a marker of endothelial cells. HA-tag (A), EGFP (B) and human-specific vimentin (C) were used as (more ...)
Validation of the IRDye 800CW-RGD probe in vitro and in vivo
To examine the specificity of the IRDye 800CW-labeled RGD probe, tumor sections from the RCAS-PDGF glioblastomas were stained with the IRDye 800CW-RGD peptide (Fig. 2A). Non-fluorescent cyclic RGD peptide (cRGD) was used as a negative control, as well as a competitive binding reagent. As shown in Fig. 2A, fluorescence emitted at 800 nm (fluorescence-800) was detected in the presence of IRDye 800CW-RGD probe, but not cRGD peptide. Fluorescence-800 decreased significantly when the blocking reagent (cRGD) competed with the IRDye-800CW RGD probe for binding to integrin receptors. Fluorescence-800 from the IRDye 800CW-RAD probe, a nonspecific binding control (26), was detected at very low levels and did not co-localize with the integrin β3 protein. These data demonstrated specific binding of the IRDye 800CW-RGD probe to integrin receptors in vitro.
Fig. 2
Fig. 2
A. Specific binding to integrin receptors by IRDye 800CW-labeled RGD and RAD peptides (green). Immunofluorescence staining using the integrin β3 antibody is shown in red, and DAPI staining of nuclei in blue. Four panels are shown: IRDye 800CW-RGD (more ...)
The IRDye 800CW-labeled RGD probe (1 nmol) was injected intravenously (via tail vein) into normal nude mice to determine its biodistribution, spectrum-specificity and clearance in vivo. At different time points after probe injection (2, 24, 48 and 72 hour), nude mice were monitored by noninvasive dynamic NIRF imaging from both ventral and dorsal views using the Pearl Impulse Imager (Fig. 2B). Fluorescence emitted at 800 nm (from the probe) and 700 nm (fluorescence-700, autofluorescence from the animal) were concomitantly acquired from the same mouse and position. NIRF-800 images from ventral view showed that IRDye 800CW-RGD probe accumulated in the bladder, indicating renal clearance of the agent. NIRF-700 images from both ventral and dorsal views revealed that significant fluorescence-700 was detected from several areas, including the abdomen reflecting gut/food autofluorescence.
The fluorescence signal intensity (fluorescence-800 and fluorescence-700) was quantified using a rectangular ROI, covering the entire body except the tail (indicated in Fig. 2B). Fluorescence-800 from both ventral and dorsal views shared a similar, rapid clearance pattern (Fig. 2B and 2C). Less than 50% of fluorescence-800 remained 24 hours after injection; at 72 hours, less than 20% of fluorescence-800 was detected. However, fluorescence-700 intensity remained constant from 2 to 72 hours. These results demonstrate that fluorescence-800 emissions did not interfere with fluorescence-700 emissions, and that the acquisition of the IRDye 800CW spectrum was channel-specific. The IRDye 800CW-RGD peptide NIRF imaging results demonstrate low background and high signal-to-background ratio in vivo.
NIRF Imaging of IRDye 800CW-RGD probe in glioblastomas – in vivo
To assess whether we could noninvasively image the binding of the IRDye 800CW-RGD peptide to integrin receptors expressed in the RCAS-PDGF, U-87 MG and TS543 glioblastomas, tumor-bearing and non-tumor-bearing control mice were imaged. Both groups of animals were intravenously injected with the IRDye 800CW-RGD probe (1 nmol). Animals were monitored by dynamic NIRF imaging at multiple time points (0, 0.5, 24 and 48 hours). NIRF-800 images from the head area are shown in Fig. 3A. At 0.5 hour post-injection, fluorescence-800 reached a peak in both tumor-bearing and normal animal groups; signal intensity at 0.5 hour was defined as 100% in Fig. 3B. At 24 hours, tumor-targeting of IRDye 800CW-RGD peptide was observed, as the fluorescence signal intensity from the tumor areas was clearly greater than the signal intensity from corresponding areas in normal mice. At 48 hour post-injection, the head fluorescence tumor-to-normal brain ratio (T/N) was maximal; 2.98±0.22 in the RCAS-PDGF model (p<0.01), 3.26±0.18 in the U-87 MG model (p<0.01) and 1.42±0.11 in the TS543 model.
Fig. 3
Fig. 3
A. Non-invasive dynamic NIRF in vivo imaging of the RCAS-PDGF (upper panel), U-87 MG (middle panel) and TS543 (lower panel) glioblastoma-bearing mice. IRDye 800CW-RGD alone, IRDye 800CW-RAD alone (nonspecific peptide control) and IRDye 800CW-RGD plus (more ...)
Binding of the IRDye 800CW-RGD peptide specifically to tumor integrin receptors was established in two ways. First, fluorescence-800 signal intensity was compared to that of a control nonspecific IRDye 800CW-RAD peptide. In all three glioblastoma models, there was a highly significant difference in signal intensity between the RGD and RAD peptides in tumor-bearing animals (p<0.01). Further, there was only a small, insignificant difference in signal intensity of the IRDye 800CW-RAD control peptide between tumor-bearing and normal animals (Fig. 3B). Second, a blocking assay, involving the co-injection of the IRDye 800CW-RGD probe with 20x the amount of unlabelled cRGD probe, was performed. The unlabelled cRGD peptide significantly reduced tumor uptake of the IRDye 800CW-RGD probe in the integrin β3-high glioblastomas (Fig. 3B, left and middle panels). For example, at 48 hour post-injection in the RCAS-PDGF model, the normalized NIRF-800 intensity from the IRDye 800CW-RGD peptide alone and in the RGD blocking study was 24.8±3.1% and 11.1±3.0%, respectively (p<0.01).
NIRF Imaging of IRDye 800CW-RGD probe in glioblastomas – ex vivo
In order to confirm the presence of tumor and further characterize the distribution pattern of IRDye 800CW-RGD fluorescence in the glioblastoma-bearing brains, animals were sacrificed 48 hours after probe injection for ex vivo assays. Whole brains from both glioblastoma-bearing mice and normal mice were harvested and ex vivo NIRF imaging was performed. In the integrin β3-high glioblastoma-bearing brains (Fig. 4A and 4B), fluorescence-800 intensity was very high in the tumor area of the IRDye 800CW-RGD-injected animals. Fluorescence-800 from the IRDye 800CW-RAD peptide (non-specific probe) was barely above background in the tumor area. In the cRGD+IRDye 800CW-RGD blocking experiment, a substantial reduction in signal intensity was observed which was consistent with a partial blockade of IRDye 800CW-RGD binding to integrin receptors. In the integrin β3-“low” glioblastoma-bearing brains (Fig. 4C), even the Fluorescence-800 from the IRDye 800CW-RGD peptide in the tumor area was very low. In all normal non-tumor-bearing brains, fluorescence-800 intensity was undetectable.
Fig. 4
Fig. 4
NIRF imaging of the RCAS-PDGF (A&D), U-87 MG (B&E) and TS543 (C&F) glioblastomas ex vivo. Mice brains were dissected 48 hours after injection of IRDye 800CW-RGD alone, IRDye 800CW-RAD alone (nonspecific peptide control) and IRDye (more ...)
To confirm the presence or absence of tumor, whole brains were sliced coronally to expose the tumor. H&E staining showed the precise tumor localization. Brain slices were further analyzed by an Odyssey imager (Fig. 4D-4F). The images from this high-resolution laser scanner confirmed that the IRDye 800CW-RGD probe had accumulated in the integrin β3-high glioblastoma tissue (Fig. 4D and 4E, left). Fluorescence-800 intensity for the six experimental groups, normalized by tissue area (tumor and normal brain), is also shown in Fig. 4D-4F, right panel. The T/N ratio of IRDye 800CW-RGD and IRDye 800CW-RAD probes was assessed: 79.7±2.8 and 10.5±1.0 in the U-87 MG model (p<0.01); 31.2±2.8 and 11.6±1.1 in the RCAS-PDGF model (p<0.01); and 16.3±1.3 and 11.6±1.0 in the TS543 model (p<0.01). This further confirms that both IRDye 800CW-RGD and IRDye 800CW-RAD peptides can be delivered to glioblastomas, but only IRDye 800CW-RGD peptide can specifically bind to the integrin receptors.
IRDye 800CW-RGD probe-guided glioblastoma tumor resection
Successful detection of mouse glioblastomas by NIRF imaging using the IRDye 800CW-RGD peptide was shown above both in vivo and ex vivo. Next, we investigated whether this probe could be used to delineate tumor margins and assist in glioblastoma resection. Fig. 5 depicts a representative RCAS-PDGF glioblastoma-bearing brain, 48 hours after intravenous injection of the IRDye 800CW-RGD peptide probe. In the white light channel, it was very difficult to judge the exact location and extent of tumor within the intact brain (Fig. 5A). This is important, as this macroscopic visual determination is the sole means by which non-contrast enhanced intraoperative imaging can be performed. NIRF-800 imaging of the whole brain using a Pearl Impulse imager showed IRDye 800CW-RGD fluorescence in the middle part of the right cerebrum (Fig. 5B). The brain was then sectioned coronally to expose the glioblastoma. The NIRF-800 image showed a high fluorescence signal from the hyperemic tumor area (black arrow heads). A two-step resection was performed to remove the tumor tissue. The resected tissue exhibited very high fluorescence and tumor was confirmed by H&E staining (Fig. 5C). After Resection step 1, significant fluorescence in the resection bed was still observed. A second resection step (Resection 2) was then performed to remove the remaining tissue with strong fluorescence. However, a very small area of residual fluorescence was still observed after Resection 2, suggesting residual tumor tissue. To confirm the presence or absence of tumor in the resection bed after Resection 2, cryosections (10 μm thick) were prepared for ex vivo NIRF imaging by an Odyssey imager and histological examination. The presence of a very small amount of residual tumor tissue in the resection bed after Resection 2 was confirmed, demonstrating that the IRDye 800CW-RGD NIRF imaging can aid in the resection of microscopic tumor foci (Fig. 5D).
Fig. 5
Fig. 5
Fluorescence-guided, step-wise resection of a RCAS-PDGF glioblastoma using IRDye 800CW-RGD peptide NIRF imaging. A representative glioblastoma-bearing brain was collected 48 hours after IRDye 800CW-RGD injection. The tumor (indicated by the black arrow (more ...)
NIRF imaging in the 650-900 nm range has specific advantages: efficient NIRF photon penetration of tissue over several millimeters, minimal intra-tissue photon scattering and low absorption by water and biomolecules, as well as minimal autofluorescence in this spectral range. Different integrin-targeted NIRF optical probes have been developed. The NIR fluorophores include cyanine analogs (such as Cy5 (27), Cy5.5 (15), and Cy7 (28)) and fluorescent nanoparticles (such as semiconductor nanocrystals, i.e. quantum dots) (29). Studies have shown that NIRF dyes conjugated to cyclic RGD peptides were able to visualize subcutaneously inoculated integrin-positive tumors. However, both unconjugated Cy5.5 and Cy7 showed relatively high tumor-nonspecific targeting, which may be due to the high lipophilicity of the cyanine dyes (30). Furthermore, these NIRF probes were unable to visualize U-87 MG orthotopic gliomas noninvasively, although the dissected tissue demonstrated tumor-specific uptake of the NIRF probes. Quantum dots represent an attractive NIRF contrast agent because of their composition-tunable fluorescence emission, good quantum yield, increased brightness, and photostability (31). However, their uptake may be restricted in gliomas (particularly low-grade gliomas) due to their size and the blood-tumor barrier. In addition, the uptake and retention of quantum dots in other organs (e.g., spleen, liver and lung), and the potential toxicity may provide obstacles to further clinical applications (29).
Here, we have evaluated an alternative NIRF dye with excitation and emission characteristics that provide enhanced tissue penetration and thus better sensitivity and ultimately more accurate quantification of integrin expression in vivo. IRDye 800CW (Ex/Em=774/805 nm) is a good candidate because the RGD peptides coupled with IRDye 800CW showed deeper tissue penetration, less scattering, and lower background fluorescence compared to Cy5.5 (Ex/Em=675/694 nm) and Cy7 (Ex/Em=743/767 nm) in a U-87 MG subcutaneous tumor model (30), as well as a M21 melanoma model (31). However, integrin-targeting NIRF probes have not yet been tested in the more clinically relevant spontaneous or induced glioblastoma mouse models. Such models demonstrate a unique intracranial microenvironment and infiltrating character, which cannot be replicated by subcutaneous or intracranial xenografts. In this study, a commercially available IRDye 800CW-conjugated cyclic RGD peptide was investigated in a preclinical mouse model of glioblastoma tumorigenesis, namely, the RCAS-PDGF glioblastoma model, as well as in two human glioblastoma intracranial xenografts models, U-87 MG and TS543.
We first demonstrated specific binding of the IRDye 800CW-RGD peptide to integrin receptors. Conjugation of IRDye 800CW to cRGD peptide did not have significant effect on the optical properties of IRDye 800CW nor on the receptor binding affinity and specificity of the cRGD peptide. Comparing the NIRF images in the 800 nm and 700 nm channels, we observed that the IRDye 800CW-RGD peptide resulted in very low background fluorescence in vivo in the 800 nm channel, whereas significant background and autofluorescence were observed in the 700 nm channel. The NIRF-800 images were not “contaminated” by fluorescence from the 700 nm channel, demonstrating the spectrum-specificity of IRDye 800CW-RGD peptide. This would also allow for fluorescence multiplexing, by combining the IRDye 800CW with other NIR fluorophores with different emission wavelengths. Ex vivo binding assays also showed that the IRDye 800CW-RGD peptide had significantly higher affinity to integrin receptors than the nonspecific peptide (IRDye 800CW-RAD), and ex vivo blocking assays confirmed that the receptor binding of IRDye 800CW-RGD peptide was saturable.
We have also demonstrated the selective retention and long-lasting tumor accumulation of the IRDye 800CW-RGD peptide in the integrin β3-high glioblastomas, both in vivo and ex vivo. Noninvasive dynamic NIRF imaging showed rapid clearance of the IRDye 800CW-RGD peptide in the brains of normal mice and high tumor-targeting of the IRDye 800CW-RGD peptide in the glioblastoma-bearing mice. The maximum tumor-to-normal brain ratio of IRDye 800CW-RGD fluorescence in living animals (T/N= 3.3 in U-87 MG model, T/N=3.0 in RCAS-PDGF model and 1.4 in the TS543 model) was observed at 48 hour post-injection.
NIRF imaging of tumor-bearing brains ex vivo (comparable to a neurosurgical setting) showed high and specific tumor uptake of the IRDye 800CW-RGD peptide in all three glioblastoma models. In the integrin β3-“low” TS543 glioblastomas, tumor-targeting of the IRDye 800CW-RGD peptide was lowest with a T/N ratio of 16. In contrast, the T/N ratios in the other integrin β3-high glioblastomas were considerably higher (T/N=80 in U-87 MG model and T/N=31 in RCAS-PDGF model), reflecting the different expression levels of integrin β3 in these glioblastomas. Overexpression of other integrin family members in the TS543 glioblastomas is also possible, because it has been reported that all five αV integrins, two β1 integrins and αII3 share the ability to recognize ligands containing an RGD tri-peptide active site (32).
The potential for translation to clinical applications exists. For example, intraoperative fluorescence-guided resection emerged as a promising technique to facilitate tumor resection of malignant gliomas in the late 1940s. More recently, 5-ALA/PpIX has been used as a fluorescent probe for visualization of malignant glioma tissues intraoperatively. 5-ALA is a non-fluorescing molecule that is metabolized to fluorescent protoporphyrin IX (PpIX). PpIX accumulates preferentially in glioma cells and emits a red-violet light (635-704 nm) when excited with blue light (400-410 nm). Stummer et al. demonstrated in a Phase III trial that 5-ALA/PpIX fluorescence-assisted neurosurgical resection of gliomas led to a significantly higher frequency of compete resections (MRI contrast-enhancing tumor area) as compared to the conventionally operated white light resection group (65% versus 36%, respectively) (33). However, there remain several inherent limitations in using 5-ALA during surgical resection, including specificity (34, 35) and photobleaching (36).
An advantage of the IRDye 800CW is that both the excitation and emission wavelengths (774 nm and 805 nm) are centered at a wavelength amenable to intraoperative imaging, which allows deeper tissue penetration of both excitation and emission photons. For example, we were able to detect IRDye 800CW-RGD fluorescence from intracranial glioblastomas in intact animals, with photons having to penetrate the skull and skin. We also did not observe problematic photobleaching effects of IRDye 800CW-RGD peptide under ambient room light and NIRF laser excitation.
Another advantage of the IRDye 800CW-RGD peptide, in comparison to fluorescein and 5-ALA, is its tumor-targeting properties (binds specifically to integrin receptors). As the integrin αvβ3 is overexpressed in both malignant and low-grade gliomas (although at lower levels (37)), it should be possible to visualize low-grade gliomas using the IRDye 800CW-RGD probe. The toxicity of IRDye 800CW dye has been tested in a preclinical model with good results (7). IRDye 800CW dye is currently on record with European regulatory authorities and a Drug Master File has been registered with the United States FDA in anticipation of similar clinical trials in the U.S. A cyclic RGD peptide-based drug (cilengitide) for the treatment of glioblastoma is also in Phase III clinical trials (38).
Instrumentation capable of visualizing the IRDye 800CW fluorescence is in existence. The Frangioni group developed the FLARE intraoperative NIRF imaging system, which has been used in clinical trials to map sentinel lymph nodes in breast cancer (39), or image the thoracic duct (40). In addition, the Zeiss Penero (Carl Zeiss) (41) and the Leica FL800 (Leica Microsystems) (42) are adaptable for intraoperative surgical resection of tumor tissue using IRDye 800CW-targeting agents. Thus, the IRDye 800CW-RGD peptide is a potential alternative to 5-ALA for fluorescence-guided glioblastoma resection.
Translational relevance
The integrin receptors are highly expressed on many human solid tumors, including glioblastomas. Treatment of glioblastoma initially involves surgery, and the extent of resection correlates with survival. However, tumor margins are difficult to visualize intraoperatively because of the invasive character of glioblastomas. Here, we show that an IRDye 800CW-conjugated RGD peptide can specifically target the overexpressed integrin receptors in murine RCAS-PDGF-driven/tv-a glioblastomas and two human orthotopic glioblastomas (U-87 MG and TS543), and thereby visualize tumor tissue and tumor margins by near-infrared fluorescent (NIRF) imaging. This has significant implications for optical molecular imaging applications, both in research and potentially clinical settings. We show that IRDye 800CW-RGD NIRF imaging facilitates more sensitive glioblastoma detection and more complete surgical resection. A Drug Master File for the IRDye 800CW dye has been registered with the United States FDA and instrumentation capable of intraoperative visualization of IRDye 800CW fluorescence is in existence.
Supplementary Material
Supplementary Materials and Methods
Acknowledgments
We thank Dr. Daniel Thorek for critical review of the manuscript, Dr. Bisen Ding, Carl Campos, Jim Finney, Theresa Cunningham, Tina Bayer and Olga Strachna for their assistance and Dr. Cameron W. Brennan and Alicia Pedraza Fernandez for deriving TS543 cells. We thank the Small Animal Imaging Core Facility and Molecular Cytology Core Facility of MSKCC for their support.
Grant Support This work was supported by NIH Grants R01-CA102673 (to R.G.B.) and P50-CA086438 (to R.G.B.), the Brain Tumor Center of MSKCC Grants (to R.H. and M.F.K.), and a Radiological Society of North America (RSNA) Scholar Grant, Geoffrey Beene Cancer Research Center Grant, Dana Foundation Brain and Immunoimaging Grant, and Society of MSKCC Grant (to M.F.K.). The Small Animal Imaging Core Facility of MSKCC is supported by NIH Grants R24 CA83084 and P30 CA08748.
Footnotes
The authors declare no conflict of interest.
1. Ye Y, Chen X. Integrin targeting for tumor optical imaging. Theranostics. 2011;1:102–26. [PMC free article] [PubMed]
2. Chen X, Park R, Shahinian AH, Tohme M, Khankaldyyan V, Bozorgzadeh MH, et al. 18F-labeled RGD peptide: initial evaluation for imaging brain tumor angiogenesis. Nucl Med Biol. 2004;31:179–89. [PubMed]
3. Jia B, Liu Z, Shi J, Yu Z, Yang Z, Zhao H, et al. Linker effects on biological properties of 111In-labeled DTPA conjugates of a cyclic RGDfK dimer. Bioconjug Chem. 2008;19:201–10. [PMC free article] [PubMed]
4. Ghoroghchian PP, Therien MJ, Hammer DA. In vivo fluorescence imaging: a personal perspective. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2009;1:156–67. [PMC free article] [PubMed]
5. Keereweer S, Kerrebijn JD, van Driel PB, Xie B, Kaijzel EL, Snoeks TJ, et al. Optical image-guided surgery--where do we stand? Mol Imaging Biol. 2011;13:199–207. [PMC free article] [PubMed]
6. Kitai T, Inomoto T, Miwa M, Shikayama T. Fluorescence navigation with indocyanine green for detecting sentinel lymph nodes in breast cancer. Breast Cancer. 2005;12:211–5. [PubMed]
7. Marshall MV, Draney D, Sevick-Muraca EM, Olive DM. Single-dose intravenous toxicity study of IRDye 800CW in Sprague-Dawley rats. Mol Imaging Biol. 2010;12:583–94. [PMC free article] [PubMed]
8. Adams KE, Ke S, Kwon S, Liang F, Fan Z, Lu Y, et al. Comparison of visible and near-infrared wavelength-excitable fluorescent dyes for molecular imaging of cancer. J Biomed Opt. 2007;12:024017. [PubMed]
9. Houston JP, Ke S, Wang W, Li C, Sevick-Muraca EM. Quality analysis of in vivo near-infrared fluorescence and conventional gamma images acquired using a dual-labeled tumor-targeting probe. J Biomed Opt. 2005;10:054010. [PubMed]
10. Sampath L, Kwon S, Ke S, Wang W, Schiff R, Mawad ME, et al. Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer. J Nucl Med. 2007;48:1501–10. [PubMed]
11. Kovar JL, Volcheck W, Sevick-Muraca E, Simpson MA, Olive DM. Characterization and performance of a near-infrared 2-deoxyglucose optical imaging agent for mouse cancer models. Anal Biochem. 2009;384:254–62. [PMC free article] [PubMed]
12. Tucker GC. Integrins: molecular targets in cancer therapy. Curr Oncol Rep. 2006;8:96–103. [PubMed]
13. Tabatabai G, Weller M, Nabors B, Picard M, Reardon D, Mikkelsen T, et al. Targeting integrins in malignant glioma. Target Oncol. 2010;5:175–81. [PubMed]
14. Shih AH, Dai C, Hu X, Rosenblum MK, Koutcher JA, Holland EC. Dose-dependent effects of platelet-derived growth factor-B on glial tumorigenesis. Cancer Res. 2004;64:4783–9. [PubMed]
15. Chen X, Conti PS, Moats RA. In vivo near-infrared fluorescence imaging of integrin alphavbeta3 in brain tumor xenografts. Cancer Res. 2004;64:8009–14. [PubMed]
16. Vivanco I, Rohle D, Versele M, Iwanami A, Kuga D, Oldrini B, et al. The phosphatase and tensin homolog regulates epidermal growth factor receptor (EGFR) inhibitor response by targeting EGFR for degradation. Proc Natl Acad Sci U S A. 2010;107:6459–64. [PubMed]
17. Koutcher JA, Hu X, Xu S, Gade TP, Leeds N, Zhou XJ, et al. MRI of mouse models for gliomas shows similarities to humans and can be used to identify mice for preclinical trials. Neoplasia. 2002;4:480–5. [PMC free article] [PubMed]
18. Holland EC. Gliomagenesis: genetic alterations and mouse models. Nat Rev Genet. 2001;2:120–9. [PubMed]
19. Dai C, Celestino JC, Okada Y, Louis DN, Fuller GN, Holland EC. PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo. Genes Dev. 2001;15:1913–25. [PubMed]
20. Szerlip NJ, Pedraza A, Chakravarty D, Azim M, McGuire J, Fang Y, et al. Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response. Proc Natl Acad Sci U S A. 2012;109:3041–6. [PubMed]
21. McGillicuddy LT, Fromm JA, Hollstein PE, Kubek S, Beroukhim R, De Raedt T, et al. Proteasomal and genetic inactivation of the NF1 tumor suppressor in gliomagenesis. Cancer Cell. 2009;16:44–54. [PMC free article] [PubMed]
22. Ozawa T, Brennan CW, Wang L, Squatrito M, Sasayama T, Nakada M, et al. PDGFRA gene rearrangements are frequent genetic events in PDGFRA-amplified glioblastomas. Genes Dev. 2010;24:2205–18. [PubMed]
23. Genovese G, Ergun A, Shukla SA, Campos B, Hanna J, Ghosh P, et al. microRNA regulatory network inference identifies miR-34a as a novel regulator of TGF beta signaling in GBM. Cancer Discov. 2012;2:736–49. [PubMed]
24. Silber J, Jacobsen A, Ozawa T, Harinath G, Pedraza A, Sander C, et al. miR-34a repression in proneural malignant gliomas upregulates expression of its target PDGFRA and promotes tumorigenesis. PLoS One. 2012;7:e33844. [PMC free article] [PubMed]
25. Kircher MF, de la Zerda A, Jokerst JV, Zavaleta CL, Kempen PJ, Mittra E, et al. A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle. Nat Med. 2012;18:829–34. [PMC free article] [PubMed]
26. Mulder WJ, Strijkers GJ, Habets JW, Bleeker EJ, van der Schaft DW, Storm G, et al. MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle. FASEB J. 2005;19:2008–10. [PubMed]
27. Jin ZH, Razkin J, Josserand V, Boturyn D, Grichine A, Texier I, et al. In vivo noninvasive optical imaging of receptor-mediated RGD internalization using self-quenched Cy5-labeled RAFT-c(-RGDfK-)(4) Mol Imaging. 2007;6:43–55. [PubMed]
28. Wu Y, Cai W, Chen X. Near-infrared fluorescence imaging of tumor integrin alpha v beta 3 expression with Cy7-labeled RGD multimers. Mol Imaging Biol. 2006;8:226–36. [PMC free article] [PubMed]
29. Cai W, Shin DW, Chen K, Gheysens O, Cao Q, Wang SX, et al. Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects. Nano Lett. 2006;6:669–76. [PubMed]
30. Liu Z, Liu S, Niu G, Wang F, Chen X. Optical imaging of integrin alphavbeta3 expression with near-infrared fluorescent RGD dimer with tetra(ethylene glycol) linkers. Mol Imaging. 2010;9:21–9. [PMC free article] [PubMed]
31. Wang W, Ke S, Wu Q, Charnsangavej C, Gurfinkel M, Gelovani JG, et al. Near-infrared optical imaging of integrin alphavbeta3 in human tumor xenografts. Mol Imaging. 2004;3:343–51. [PubMed]
32. Humphries JD, Byron A, Humphries MJ. Integrin ligands at a glance. J Cell Sci. 2006;119:3901–3. [PMC free article] [PubMed]
33. Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen HJ. Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial. Lancet Oncol. 2006;7:392–401. [PubMed]
34. Ewelt C, Floeth FW, Felsberg J, Steiger HJ, Sabel M, Langen KJ, et al. Finding the anaplastic focus in diffuse gliomas: the value of Gd-DTPA enhanced MRI, FET-PET, and intraoperative, ALA-derived tissue fluorescence. Clin Neurol Neurosurg. 2011;113:541–7. [PubMed]
35. Tonn JC, Stummer W. Fluorescence-guided resection of malignant gliomas using 5-aminolevulinic acid: practical use, risks, and pitfalls. Clin Neurosurg. 2008;55:20–6. [PubMed]
36. Sherman JH, Hoes K, Marcus J, Komotar RJ, Brennan CW, Gutin PH. Neurosurgery for brain tumors: update on recent technical advances. Curr Neurol Neurosci Rep. 2011;11:313–9. [PubMed]
37. Schnell O, Krebs B, Wagner E, Romagna A, Beer AJ, Grau SJ, et al. Expression of integrin alphavbeta3 in gliomas correlates with tumor grade and is not restricted to tumor vasculature. Brain Pathol. 2008;18:378–86. [PMC free article] [PubMed]
38. Schottelius M, Laufer B, Kessler H, Wester HJ. Ligands for mapping alphavbeta3-integrin expression in vivo. Acc Chem Res. 2009;42:969–80. [PubMed]
39. Troyan SL, Kianzad V, Gibbs-Strauss SL, Gioux S, Matsui A, Oketokoun R, et al. The FLARE intraoperative near-infrared fluorescence imaging system: a first-in-human clinical trial in breast cancer sentinel lymph node mapping. Ann Surg Oncol. 2009;16:2943–52. [PMC free article] [PubMed]
40. Ashitate Y, Tanaka E, Stockdale A, Choi HS, Frangioni JV. Near-infrared fluorescence imaging of thoracic duct anatomy and function in open surgery and video-assisted thoracic surgery. J Thorac Cardiovasc Surg. 2011;142:31–8. e1–2. [PMC free article] [PubMed]
41. Kim EH, Cho JM, Chang JH, Kim SH, Lee KS. Application of intraoperative indocyanine green videoangiography to brain tumor surgery. Acta Neurochir (Wien) 2011;153:1487–95. [PubMed]
42. Cordero E, Ensenat J, Macho J, Gonzalez JJ, Sanchez M, Fernandez C, et al. Intraoperative videoangiography using green indocyanine during aneurysm surgery. Neurocirugia (Astur) 2010;21:302–5. [PubMed]