Pet Imaging Of Tumor Angiogenesis

Multivalent biomolecules such as peptides have been explored as a useful stra-tegy to construct molecular imaging probes and drug delivery carriers. It is generally accepted that multivalency has advantages over monovalency for improving binding affinities and even activity. Compared to the monomeric peptides, many multivalent peptides could show improved binding affinities in vitro and tumor targeting and imaging ability in vivo.Macrocyclic chelators are generally used for complexation of metal ions, and they have been widely used in molecular imaging applications when they complex with radioactive, paramagnetic or fluorescent metal ions. Various macrocyclic che-lators such as polyamino polycarboxylate-type macrocycles have been successfully and extensively used as bifunctional chelators (BFCA) for labeling biomolecules.Herein, we present the use of a widely available macrocyclic chelator,1,4,7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), for the assembly of multiple peptides in tumor targeting. Considering that DOTA has four acetic acid side-arms, they could all be easily activated, functionalized and used for preparation of mono, di, tri, tetra-valent biomolecules. For the proof of concept, a small cyclic arginine-glycine-aspartic acid (RGD) peptide [c(RGDyK)] which is a well known ligand for tumor integrin receptors was selected for construction of multivalent molecules. The resulting bioconjugates were radiolabeled with 64Cu and their in vivo performance was then studied using microPET.The multivalent DOTA-peptide bioconjugates demonstrate promising tumor targeting ability. The malignant tumors must establish a neovasculature to grow, to invade, and to metastasize in which inlegrinsαγβ3 play an important role. Dimer-RGD peptoid has been synthesized in this study. It tagged with radionuclides have been demonstrated to be a promising agent for tumor angiogenesis PET imaging and/or peptoid receptor-targeted radionuclide therapy. Herein by using dimer-RGD peptoid, U87MG-bearing mice and microPET imaging technology, we systematically investigated the influence of dimer-RGD peptoid in vivo U87MG tumor targeting profiles.1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the lysine residue of dimer-RGD peptoid for radiolabeling with a PET radioisotope,64Cu. After radiolabeling with 64Cu, the in vivo performances of dimer-RGD peptoid was evaluated in subcutaneous U87MG tumor xenografted mice by micro-PET imaging followed by biodistribution studies.In vivo study demonstrated good performance of dimer-RGD peptoid as an imaging agent due to its high tumor uptake and high kidney accumulation. Dimer RGD effects have complex impact to peptoid's in vivo behaviors. In vivo tumor targeting ability is achieved by dimer-RGD peptoid. Cu-64 labeled dimer-RGD peptoid has been identified as an ideal tumor angiogenesis PET imaging probe. Purpose Noninvasive positron emission tomography (PET) imaging of vascular endothelial growth factor receptor 2 (VEGFR-2) expression could be a valuable tool for evaluation of patients with a variety of malignancies, and particularly for monitoring those undergoing antiangiogenic therapies that block VEGF/VEGFR-2 function. The aim of this study was to develop a VEGFR-2-specific PET tracer.Methods The D63AE64AE67A mutant of VEGF121 (VEGFDEE) was generated by recombinant DNA technology. VEGF121 and VEGFDEE were purified and conjugated with DOTA for 64Cu labeling. The DOTA conjugates were tested in vitro for VEGFR-2 specificity and functional activity. In vivo tumor targeting efficacy and pharmacokinetics of 64Cu-labeled VEGF121 and VEGFDEE were compared using an orthotopic 4T1 murine breast tumor model. Blocking experiments, biodistribution studies, and immunofluorescence staining were carried out to confirm the noninvasive imaging results.Results Cell binding assay demonstrated that VEGFDEE had about 20-fold lower VEGFR-1 binding affinity and only slightly lower VEGFR-2 binding affinity as compared with VEGF121. MicroPET imaging studies revealed that both 64Cu-DOTA-VEGF121 and 64Cu-DOTA-VEGFDEE had rapid and prominent activity accumulation in VEGFR-2-expressing 4T1 tumors. The renal uptake of 64Cu-DOTA-VEGFDEE was significantly lower than that of 64Cu-DOTA-VEGF121 as rodent kidneys expressed high levels of VEGFR-1 based on immunofluorescence staining. Blocking experime-nts and biodistribution studies confirmed the VEGFR specificity of 64Cu-DOTA-VEGFDEE.Conclusion We have developed a VEGFR-2-specific PET tracer,64Cu-DOTA-VEGFDEE-It has comparable tumor targeting efficacy to 64Cu-DOTA-VEGF121 but much reduced renal toxicity. This tracer may be translated into the clinic for imaging tumor angiogenesis and monitoring antiangiogenic treatment efficacy.