Abstract
1172
Purpose: To develop a novel near-infrared fluorescence (NIRF) imaging probe as a visualization tool for image-guided surgical resection of orthotopic glioblastoma.
Methods: We combined RGD (arginine-glycine-aspartic) with anti-cancer peptide, DKL (LKlLKlLlkKLLkLL-NH2; K = Lys, L = Leu, k = D-Lys, l = D-Leu), to generate RKL [COOH-RGDK(DOTA)-(PEG2)-LKlLKkLlkKLLkLL-NH2; K = Lys, L = Leu, k = D-Lys, l = D-Leu], which was labeled by Sulfo-Cy5.5-NHS ester for NIRF imaging. To determine the binding specificity and subcellular localization of Cy5.5-RKL, the probe (50 nM) was incubated with U87MG tumor cells at 37°C for 1 h, followed by laser confocal microscopy. NIRF imaging in nude mice bearing subcutaneous U87MG tumors was performed after intravenous injection of 1.5 nmol Cy5.5-RKL. In the orthotopic tumor model, optical imaging was performed from 1 to 8 h after intravenous injection of 1.5 nmol Cy5.5-RKL.
Results: In this study, Cy5.5-RKL probe was successfully synthesized. Its properties were investigated in vitro and in vivo. The laser confocal microscopy results demonstrated that Cy5.5-RKL bound specifically to U87MG cells. In vivo, Cy5.5-RKL was able to detect U87MG xenografts for at least 8 h p.i.. The maximum tumor to muscle ratios of Cy5.5-RKL was 7.65 ± 0.72. Cy5.5-RKL displayed excellent delineation of the boundaries between orthotopic glioblastomas and normal brain tissue at 8 h p.i.
Conclusions: NIRF imaging using Cy5.5-RKL is promising not only for diagnostic purposes but also for use in image-guided surgery for orthotopic glioblastoma or other superficial tumors. Key Words: Near-infrared fluorescence imaging, glioblastoma, RGD, NGR, anti-cancer peptide Suppl. Fig. 1: Confocal microscopy images of U87MG cells incubated with Cy5.5-RKL (50 nM of Cy5.5 labeled peptides in 500 μL of PBS) (magnification ×60). For the blocking group, the U87MG cells were co-incubated with extra unlabeled peptide (50 μM). The results were scanned under confocal laser-scanning microscopy. Suppl. Fig. 2: In vivo fluorescent imaging of the probes. The mice received 1.5 nmol of Cy5.5-RKL intravenously and subjected to optical imaging at various time points post injection. Suppl. Fig. 3: Time-course fluorescence imaging of the orthotopic glioma mouse model. a: fluorescence images of Cy5.5-RKL in the orthotopic U87MG glioblastoma model from 0.5 to 8h post injection. b: in vivo targeting quantification of Cy5.5-RKL in orthotopic U87MG glioblastoma vs. muscle. c: tumor contrast (tumor-to-muscle) calculated from ROI measurement at different time points after administration of Cy5.5-RKL. Suppl. Fig. 4: Near-infrared fluorescence imaging for the delineation of boundary of glioma. a: NIRF imaging delineates boundary of glioblastoma and normal brain tissue at 8 h after intravenous injection of Cy5.5-RKL. The tumor can be clearly visualized in situ through NIRF imaging. b: H&E staining of the brain containing orthotopic glioblastoma (2×) and the magnification (40×) of the part surrounded by the blue rectangle, which clearly showing the boundaries between tumor tissue and normal brain tissue. c: immunohistochemical analysis of tumor tissue staining by anti-αv antibody (1:100) (40×). d: immunohistochemical analysis of tumor tissue with anti-CD13 antibody (1:100) (40×).