64Cu-cyclam-RAFT-c(-RGDfK-)4 is a novel multimeric positron emission tomography (PET) probe for αVβ3 integrin imaging. Its uptake and αVβ3 expression in tumors showed a linear correlation. Since αVβ3 integrin is strongly expressed on activated endothelial cells during angiogenesis, we aimed to determine whether 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET can be used to image tumor angiogenesis and monitor the antiangiogenic effect of a novel multi-targeted tyrosine kinase inhibitor, TSU-68. Athymic nude mice bearing human hepatocellular carcinoma HuH-7 xenografts, which expressed negligible αVβ3 levels on the tumor cells, received intraperitoneal injections of TSU-68 or the vehicle for 14 days. Antiangiogenic effects were determined at the end of therapy in terms of 64Cu-cyclam-RAFT-c(-RGDfK-)4 uptake evaluated using PET, biodistribution assay, and autoradiography, and they were compared with microvessel density (MVD) determined by CD31 immunostaining. 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET enabled clear tumor visualization by targeting the vasculature, and the biodistribution assay indicated high tumor-to-blood and tumor-to-muscle ratios of 31.6 ± 6.3 and 6.7 ± 1.1, respectively, 3 h after probe injection. TSU-68 significantly slowed tumor growth and reduced MVD; these findings were consistent with a significant reduction in the tumor 64Cu-cyclam-RAFT-c(-RGDfK-)4 uptake. Moreover, a linear correlation was observed between tumor MVD and the corresponding standardized uptake value (SUV) (r = 0.829, P = 0.011 for SUVmean; r = 0.776, P = 0.024 for SUVmax) determined by quantitative PET. Autoradiography and immunostaining showed that the distribution of intratumoral radioactivity and tumor vasculature corresponded. We concluded that 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET can be used for in vivo angiogenesis imaging and monitoring of tumor response to antiangiogenic therapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-012-9281-1) contains supplementary material, which is available to authorized users.

A quartz crystal microbalance technique with dissipation monitoring and a complementary optical microscopy technique were used for monitoring the capture and release of specific cells on a surface displaying a bifunctional molecular device, composed of a molecular scaffold endowed with the cell recognition property of an RGD ligand and a β-CD/Fc redox-switchable system.

(64)Cu-cyclam-RAFT-c(-RGDfK-)4, an αVβ3 integrin-targeting tetrameric cyclic RGD peptide probe, is a potential theranostic compound for positron emission tomography (PET) of tumor angiogenesis and for internal radiotherapy owing to the multiple decay modes of (64)Cu. Since kidneys are dose-limiting organs in internal radiotherapy, we aimed to reduce the renal accumulation of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 by co-injection with Gelofusine (GF), a succinylated gelatin solution, and/or L-lysine (Lys), and to explore, for the first time, the related mechanisms using the noninvasive and quantitative PET imaging technology. Biodistribution assays, dynamic and static PET scans, and metabolism studies with radio-thin-layer chromatography (radio-TLC) were performed in healthy or αVβ3-positive tumor-bearing mice. In the results, co-injection with GF markedly reduced the renal uptake and slightly increased the tumor uptake of (64)Cu-cyclam-RAFT-c(-RGDfK-)4. L-Lysine alone had no effect on the probe biodistribution, but the combined use of Lys and GF tended to enhance the effect of GF. Dynamic PET and metabolite analysis by radio-TLC highly revealed that GF blocks the renal reabsorption of (64)Cu-cyclam-RAFT-c(-RGDfK-)4, but does not interfere with its metabolism and excretion. In conclusion, administration of GF and Lys is a useful strategy for kidney protection in (64)Cu-cyclam-RAFT-c(-RGDfK-)4-based internal radiotherapy.

There is a growing interest in isolating tumor cells from biological samples. Considering that circulating tumor cells can be rare in blood, it appears challenging to capture these cells onto a surface with high selectivity and sensitivity. In the present paper we describe the design of functionalized surfaces aiming to selectively capture tumor cells by using the RGD peptide ligand either with a tetrameric or a monomeric presentation. β-cyclodextrin-coated self-assembled monolayers were used as platforms for the binding of RGD ligands endowed with a redox ferrocene cluster as the dissociation of the inclusion complex on the surface is accounted for the release of the captured cells upon electrochemical oxidation of ferrocene. For this purpose, we determined suitable RGD densities for both monovalent and tetravalent ligand presentations. The present results indicate that the clustered RGD architecture efficiently improves the selective cell capture at a very low RGD-surface density (∼ 10 RGD/μm 2 ) in comparison with the monovalent presentation (∼ 1000 RGD/ μm 2 ). IntroductionMultivalent interactions are ubiquitously observed in biological systems for a wide range of functions including integrinmediated cell adhesion to extracellular matrix (ECM). 1 Among the integrin receptors, the α V β 3 integrin subclass has received special attention as it is involved in tumour progression such as aggressive metastatic cancer.2 Felding-Habermann et al. have shown that this receptor contributes to circulating tumour cell (CTC) arrest. 3 As cells expressing α V β 3 integrin interact with the ECM through the recognition of the ubiquitous triad sequence RGD (Arg-Gly-Asp), a series of peptides containing the RGD sequence were developed for diagnosis and tumour therapy. 4 The cyclopentapeptide derivatives were found to specifically bind the α V β 3 integrin. 5 Additionally, the multimeric presentation of RGD motifs appears to be a prerequisite for efficient integrin targeting. 6 Our group and others have shown that multimeric compounds exhibit attractive biological properties. 7 Recently, we have reported the benefit of tetrameric RGD derivatives for the near-infrared optical guided surgery of solid tumours 8 and for tumour-targeted drug delivery. Although multimeric RGD compounds show better affinity than their corresponding monomers, 7 few reports have tested how important clustered architectures are for cell binding to RGD-functionalized surfaces. Pioneering studies have demonstrated that nanoscale patterning of RGD ligand presentation significantly influences cell adhesion and spreading. 10 Those surfaces were prepared from highmolecular weight RGD-containing polymers or nanoparticles. Journal NameScheme 1 Synthesis of compounds 1 and 3. Reaction conditions: i) TFA/H2O (7:3), rt, 20 min then TFA/H2O (95:5), rt, 120 min, 44%; ii) NaIO4, H2O, rt, 20 min, 46%; iii) TFA/H2O/CH3CN (7:2:1), rt, 20 min, 42%; iv) CuSO4, THPTA, sodium ascorbate, phosphate buffer pH 7.4/DMF (6/4), 45°C, 150 min, 1: 63%, 3 :71 %. SPPS: solid-ph...

(90)Y-RAFT-RGD and (177)Lu-RAFT-RGD are potent agents targeting αvβ3-expressing tumours for internal targeted radiotherapy.

Melanoma is a highly metastatic and deadly form of cancer. Invasive melanoma cells overexpress integrin a v b 3 , which is a well-known target for Arg-Gly-Asp-based (RGD) peptides. We developed a sophisticated method to synthetize milligram amounts of a targeted vector that allows the RGD-mediated targeting, internalization, and release of a mitochondria-disruptive peptide derived from the pro-apoptotic Bax protein. We found that 2.5 mM Bax[109-127] was sufficient to destabilize the mitochondria in ten different tumor cell lines, even in the presence of the anti-apoptotic Bcl2 protein, which is often involved in tumor resistance. This pore-forming peptide displayed antitumor activity when it was covalently linked by a disulfide bridge to the tetrameric RAFT-c[RGD] 4 -platform and after intravenous injection in a human melanoma tumor model established in humanized immuno-competent mice. In addition to its direct toxic effect, treatment with this combination induced the release of the immuno-stimulating factor monocyte chimoattractant protein 1 (MCP1) in the blood and a decrease in the level of the pro-angiogenic factor FGF2. Our novel multifunctional, apoptosis-inducing agent could be further customized and assayed for potential use in tumortargeted therapy.

Integrin a v b 3 expression is upregulated during tumor growth and invasion in newly formed endothelial cells in tumor neovasculature and in some tumor cells. A tetrameric RGD-based peptide, regioselectively addressable functionalized template-(cyclo-[RGDfK])4 (RAFT-RGD), specifically targets integrin a v b 3 in vitro and in vivo. When labeled with indium-111, the RAFT-RGD is partially reabsorbed and trapped in the kidneys, limiting its use for further internal targeted radiotherapy and imaging investigations. We studied the effect of Gelofusine on RAFT-RGD renal retention in tumor-bearing mice. Mice were imaged using single photon emission computed tomography and optical imaging 1 and 24 h following tracer injection. Distribution of RAFT-RGD was further investigated by tissue removal and direct counting of the tracer. Kidney sections were analyzed by confocal microscopy. Gelofusine significantly induced a >50% reduction of the renal reabsorption of 111In-DOTA-RAFT-RGD and A700-RAFT-RGD, without affecting tumor uptake. Injection of Gelofusine significantly reduced the renal retention of labeled RAFT-RGD, while increasing the tumor over healthy tissue ratio. These results will lead to the development of future therapeutic approaches.

The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET) imaging probe, ⁶⁴Cu-labeled cyclam-RAFT-c(-RGDfK-)₄ peptide (⁶⁴Cu-RAFT-RGD), using this model. Varying degrees of αvβ₃ integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP), was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT) comparing ⁶⁴Cu-RAFT-RGD and ¹⁸F-fluoro-2-deoxy-d-glucose (¹⁸F-FDG) accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly αvβ₃ integrin specific. ⁶⁴Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of ⁶⁴Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, ⁶⁴Cu-RAFT-RGD accumulation provided better tumor contrast to background than ¹⁸F-FDG. Our results suggest that ⁶⁴Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of αvβ₃ integrin-expressing pancreatic tumors.