Quantitative Molecular Magnetic Resonance Imaging of Tumor Angiogenesis Using cNGR-Labeled Paramagnetic Quantum Dots Marlies Oostendorp, 1,2 Kim Douma, 2,3 Tilman M. Hackeng, 2,4 Anouk Dirksen, 2,4 Mark J. Post, 2,3,5,7 Marc A.M.J. van Zandvoort, 2,3 and Walter H. Backes 1,2,6 1 Department of Radiology, Maastricht University Hospital, 2 Cardiovascular Research Institute Maastricht (CARIM), Departments of 3 Biomedical Engineering, 4 Biochemistry, and 5 Physiology, and 6 Research Institute GROW, Maastricht University, Maastricht, the Netherlands; and 7 Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands Abstract The objective of this study was to develop and apply cyclic Asn-Gly-Arg (cNGR)-labeled paramagnetic quantum dots (cNGR-pQDs) for the noninvasive assessment of tumor angiogenic activity using quantitative in vivo molecular magnetic resonance imaging (MRI). cNGR was previously shown to colocalize with CD13, an aminopeptidase that is highly overexpressed on angiogenic tumor endothelium. Because angiogenesis is important for tumor growth and metastatization, its in vivo detection and quantification may allow objective diagnosis of tumor status and evaluation of treatment response. I.v. injection of cNGR-pQDs in tumor- bearing mice resulted in increased quantitative contrast, comprising increased longitudinal relaxation rate and de- creased proton visibility, in the tumor rim but not in tumor core or muscle tissue. This showed that cNGR-pQDs allow in vivo quantification and accurate localization of angiogenic activity. MRI results were validated using ex vivo two-photon laser scanning microscopy (TPLSM), which showed that cNGR-pQDs were primarily located on the surface of tumor endothelial cells and to a lesser extent in the vessel lumen. In contrast, unlabeled pQDs were not or only sparsely detected with both MRI and TPLSM, supporting a high specificity of cNGR-pQDs for angiogenic tumor vasculature. [Cancer Res 2008;68(18):7676–83] Introduction Angiogenesis, the formation of new capillaries from existing blood vessels, is key to tumor growth and metastatization by providing proliferating tumor cells with oxygen and nutrients (1, 2). Moreover, angiogenic activity is related to tumor malignancy (3, 4). Noninvasive detection of angiogenic activity is therefore highly relevant for adequate tumor diagnosis. Quantification of angio- genesis may furthermore allow objective monitoring of tumor progression, for instance in response to treatment. Currently, molecular imaging techniques are being developed that allow direct visualization and characterization of cellular or molecular activation of angiogenesis-related pathways (5). More specifically, molecular imaging uses contrast agents that home to up-regulated biomolecules (e.g., receptors, enzymes) via interaction with high-affinity ligands coupled to the contrast agent. Ideally, this results in an altered signal intensity at the location of these molecules. Of the different imaging modalities, magnetic reso- nance imaging (MRI) may be the most desirable for molecular imaging due to its excellent spatial resolution and soft tissue contrast. Moreover, molecular MRI potentially allows direct covisualization of tumor angiogenic activity with anatomy. However, the inherently low sensitivity of MRI is a problem due to the typically low abundance of up-regulated biomolecules. This can be overcome by large molecular weight constructs carrying a high payload of gadolinium or iron, and multiple targeting ligands to enhance the relaxivity and targeting efficacy, respectively, of the particle (6). One of the best-defined ligands for molecular imaging of angiogenesis is the cyclic Arg-Gly-Asp (cRGD) peptide, which binds specifically to the a v h 3 -integrin (7, 8). However, for the cyclic Asn-Gly-Arg (cNGR) motif, the tumor-homing capability was shown to be 3-fold higher compared with cRGD (9). The clinical applicability of cNGR as a tumor-homing ligand was previously shown by conjugating cNGR to tumor necrosis factor a (TNFa). Compared with unlabeled TNFa , cNGR-TNFa displayed a signif- icantly increased antitumor activity with similar systemic toxicity (10–12). The vascular address of cNGR is a specific isoform of CD13 (aminopeptidase N), a transmembrane glycoprotein involved in cancer angiogenesis, tumor invasion, and metastasis, which is overexpressed by activated endothelial cells (ECs) of tumor vasculature (9, 13, 14). CD13 is not required for vessel growth during embryonic development and normal adult function, as shown in CD13-null mice (15). In a model of retinal neovascula- rization, these mice had significantly decreased vessel growth, suggesting that CD13 is important in pathologic neovasculariza- tion. In addition, fluorophore-conjugated cNGR allowed detection of the in vivo expression of CD13 in tumors and infarcted myocardium (16, 17). Competition with unconjugated cNGR significantly decreased the fluorescence signal, indicating high specificity of cNGR for CD13 (16, 17). Despite the aforementioned high tumor-homing capability of cNGR, its potency as a targeting ligand for molecular imaging of tumor angiogenesis is currently unknown. Therefore, the objective of this study was to explore cNGR-labeled paramagnetic quantum dots (cNGR-pQDs) for the noninvasive and selective in vivo detection of tumor neovascularization using quantitative molecu- lar MRI. QDs were chosen as contrast agent scaffolds because of their excellent photophysical properties, i.e., broad excitation, small emission spectra, and limited photobleaching (18, 19). Further- more, QDs enabled binding of multiple targeting ligands and gadolinium chelates. The bimodal nature of the particle (i.e., paramagnetic and fluorescent) allowed validation of the results with ex vivo two-photon laser scanning microscopy (TPLSM). With Note: M. Oostendorp and K. Douma contributed equally to this work. Requests for reprints: Walter H. Backes, Maastricht University Hospital, Department of Radiology, P.Debyelaan 25, 6229 HX, Maastricht, the Netherlands. Phone: 0031-43-3876948; Fax: 0031-43-3876909; E-mail: wbac@rdia.azm.nl. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-0689 Cancer Res 2008; 68: (18). September 15, 2008 7676 www.aacrjournals.org Research Article Research. on July 20, 2015. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from