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 αII
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
) 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 αv
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.