In Vivo Near-Infrared Fluorescence Imaging of Integrin αvβ3 in an Orthotopic Glioblastoma Model
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Expression of cell adhesion molecule integrin αvβ3 is significantly up-regulated during tumor growth, and sprouting of tumor vessels and correlates well with tumor aggressiveness. The purpose of this study was to visualize tumor integrin αvβ3 expression in vivo by using near-infrared fluorescence (NIRF) imaging of Cy5.5-linked cyclic arginine–glycine–aspartic acid (RGD) peptide in an orthotopic brain tumor model.
U87MG glioma cells transfected with the firefly luciferase gene were stereotactically injected into nude mice in the right frontal lobe. Bioluminescence imaging (BLI) using d-luciferin substrate and small animal magnetic resonance imaging (MRI) using gadolinium contrast enhancement were conducted weekly after tumor cell inoculation to monitor intracranial tumor growth. Integrin αvβ3 expression was assessed by using a three-dimensional optical imaging system (IVIS 200) 0–24 hours after administration of 1.5 nmol monomeric Cy5.5-RGD via the tail vein. Animals were injected intravenously with both Texas Red–tomato lectin and Cy5.5-RGD prior to sacrifice to visualize peptide localization to tumor vasculature using histology.
Fluorescence microscopy demonstrated specific Cy5.5-RGD binding to both U87MG tumor vessels and tumor cells with no normal tissue binding. NIRF imaging showed highest tumor uptake and tumor to normal brain tissue ratio two hours postinjection (2.64 ± 0.20). Tumor uptake of Cy5.5-RGD was effectively blocked by using unlabeled c(RGDyK), and injection of Cy5.5 dye alone showed nonspecific binding.
Optical imaging via BLI and NIRF offer a simple, effective, and rapid technique for noninvasive in vivo monitoring and semiquantitative analysis of intracranial tumor growth and integrin αvβ3 expression. This study suggests that NIRF via fluorescently labeled RGD peptides may provide enhanced surveillance of tumor angiogenesis and anti-integrin treatment efficacy in orthotopic brain tumor models.
Key wordsNear-infrared fluorescence (NIRF) Bioluminescence imaging (BLI) Optical imaging Integrin RGD Cy5.5 Orthotopic glioblastoma
We would like to thank Drs. Kai Chen and Weibo Cai for their valuable assistance in the cell preparation and fluorescence microcopy involved in this study. This work was supported, in part, by National Institute of Biomedical Imaging and Bioengineering (NIBIB) Grant R21 EB001785, DOD BCRP IDEA Award W81XWH-04-1-0697, DOD Ovarian Cancer Research Program (OCRP) Award OC050120, DOD Prostate Cancer Research Program (PCRP) New Investigator Award (NIA) DAMD17-03-1-0143, National Cancer Institute (NCI) Small Animal Imaging Resource Program (SAIRP) grant R24 CA93862, NCI R21 CA102123, NCI In Vivo Cellular Molecular Imaging Center (ICMIC) grant P50 CA114747, NCI Centers of Cancer Nanotechnology Excellence (CCNE) U54 Grant 1U54CA119367-01, Stanford University School of Medicine Medical Scholars Award, American Medical Association Foundation Seed Grant, and Chinese American Medical Society Research Grant.
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