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Angiogenesis is a fundamental requirement for tumor growth and therefore it is a primary target for anti-cancer therapy. Molecular imaging of angiogenesis may provide novel opportunities for early diagnostic and for image-guided optimization and management of therapeutic regimens. Here we reviewed the advances in targeted imaging of key biomarkers of tumor angiogenesis, integrins and receptors for vascular endothelial growth factor (VEGF). Tracers for targeted imaging of these biomarkers in different imaging modalities are now reasonably well-developed and PET tracers for integrin imaging are currently in clinical trials. Molecular imaging of longitudinal responses to anti-angiogenic therapy in model tumor systems revealed a complex pattern of changes in targeted tracer accumulation in tumor, which reflects drug-induced tumor regression followed by vascular rebound. Further work will define the competitiveness of targeted imaging of key angiogenesis markers for early diagnostic and image-guided therapy.

Recent evidence supports a role for vascular endothelium growth factor (VEGF) signaling in bladder inflammation. However, it is not clear what bladder cells are targeted by VEGF. Therefore, we determined the nature of cells responding to VEGF in normal and inflamed bladders by tagging such cells in vivo with a targeted fluorescent tracer, scVEGF/Cy, an engineered single-chain VEGF labeled with Cy5.5 dye, which identifies cells with accessible and functionally active VEGF receptors. Inflammation was induced by intravesical instillation of PAR-activating peptides or BCG. In vivo NIRF imaging with intravenously injected scVEGF/Cy revealed accumulation of the tracer in the control mouse bladder and established that inflammation increased the steady-state levels of tracer uptake. Ex vivo colocalization of Cy5.5 dye revealed that in normal and at a higher level in inflamed bladder, accumulation of scVEGF/Cy occurs in both urothelial and ganglial cells, expressing VEGF receptors VEGFR-1 and VEGFR-2, as well as VEGF coreceptots neuropilins (NRP) NRP1 and NRP2. PCR results indicate that the messages for VEGF-Rs and NRPs are present in the bladder mucosa and ChIP/QPCR analysis indicated that inflammation induced up-regulation of genes encoding VEGFRs and NRPs. Our results strongly suggest new and blossoming VEGF-driven processes in bladder urothelial cells and ganglia in the course of inflammation. We expect that molecular imaging of the VEGF pathway in the urinary tract by receptor-mediated cell tagging in vivo will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of bladder-targeting drugs and treatments. bladder inflammation; VEGF coreceptors; interstitial cystitis; VEGF receptor imaging; human urothelium; targeted imaging; scVEGF/Cy fluorescent tracer; Cy5.5

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments., Author(s): Marina V Backer [1]; Zoya Levashova [2]; Vimalkumar Patel [1]; Brian T Jehning [1]; Kevin Claffey [3]; Francis G Blankenberg [2]; Joseph M Backer (corresponding author) [1] Preclinical and [...]

Purpose: Metastatic breast cancer is the second leading cause of cancer-related death in women. The 5-year survival rate for metastatic breast cancer has remained near 26.9 % for over a decade. The recruitment of hematopoietic stem cells with high expression of the vascular endothelial growth factor receptor 1 (VEGFR-1) has been implicated in early stages of metastasis formation. We propose the use of an 18F-labeled single-chain version of VEGF121, re-engineered to be selective for VEGFR-1 (scVR1), as a positron emission tomography (PET) imaging agent to non-invasively image early-stage metastases.Procedures: scVR1 was 18F-labeled via a biorthogonal click reaction between site-specifically trans-cyclooctene functionalized scVR1 and an Al18F labeled tetrazine-NODA (1,4,7-triazacyclononane-1,4-diiacetic acid). The [18F]AlF-NODA-scVR1 was purified using a PD10 column and subsequently analyzed on HPLC to determine radiochemical purity. Animal experiments were performed in 6-8-week-old female BALB/c mice bearing orthotopic primary 4T1 breast tumors or 4T1 metastatic lesions. The [18F]AlF-NODA-scVR1 tracer was administered via tail vein injection; PET imaging and ex vivo analysis was performed 2 h post-injection.Results: The [18F]AlF-NODA-scVR1 was prepared with a 98.2 ± 1.5 % radiochemical purity and an apparent molar activity of 7.5 ± 1.2 GBq/μmol. The specific binding of scVR1 to VEGFR-1 was confirmed via bead-based assay. The ex vivo biodistribution showed tumor uptake of 3.5 ± 0.5 % ID/g and was readily observable in PET images. Metastasis formation was detected with [18F]AlF-NODA-scVR1 tracer showing colocalization with bioluminescent imaging as well as ex vivo autoradiography and immunofluorescent staining of VEGFR-1.Conclusions: The diagnostic capabilities of the [18F]AlF-NODA-scVR1 PET tracer was confirmed in both orthotopic and metastatic murine cancer models. These results support the potential use of [18F]AlF-NODA-scVR1 as a PET tracer that could image metastases, providing clinicians with an additional tool to assess a patient's need for adjuvant therapies. [ABSTRACT FROM AUTHOR]

Purpose: Plaque vulnerability is associated with inflammation and angiogenesis, processes that rely on vascular endothelial growth factor (VEGF) signaling via two receptors, VEGFR-1 and VEGFR-2. We have recently reported that enhanced uptake of scVEGF-PEG-DOTA/Tc-99m (scV/Tc) single photon emission computed tomography (SPECT) tracer that targets both VEGFR-1 and VEGFR-2, identifies accelerated atherosclerosis in diabetic relative to non-diabetic ApoE-/- mice. Since VEGFR-1 and VEGFR-2 may play different roles in atherosclerotic plaques, we reasoned that selective imaging of each receptor can provide more detailed information on plaque biology.Procedures: Recently described VEGFR-1 and VEGFR-2 selective mutants of scVEGF, named scVR1 and scVR2, were site-specifically derivatized with Tc-99m chelator DOTA via 3.4 kDa PEG linker, and their selectivity to the cognate receptors was confirmed in vitro. scVR1 and scVR2 conjugates were radiolabeled with Tc-99m to specific activity of 110 ± 11 MBq/nmol, yielding tracers named scVR1/Tc and scVR2/Tc. 34-40 week old diabetic and age-matched non-diabetic ApoE-/- mice were injected with tracers, 2-3 h later injected with x-ray computed tomography (CT) contrast agent and underwent hybrid SPECT/CT imaging. Tracer uptake, localized to proximal aorta and brachiocephalic vessels, was quantified as %ID from. Tracer uptake was also quantified as %ID/g from gamma counting of harvested plaques. Harvested atherosclerotic arterial tissue was used for immunofluorescent analyses of VEGFR-1 and VEGFR-2 and various lineage-specific markers.Results: Focal, receptor-mediated uptake in proximal aorta and brachiocephalic vessels was detected for both scVR1/Tc and scVR2/Tc tracers. Uptake of scVR1/Tc and scVR2/Tc was efficiently inhibited only by "cold" proteins of the same receptor selectivity. Tracer uptake in this area, expressed as %ID, was higher in diabetic vs. non- diabetic mice for scVR1/Tc (p = 0.01) but not for scVR2/Tc. Immunofluorescent analysis revealed enhanced VEGFR-1 prevalence in and around plaque area in diabetic mice.Conclusions: Selective VEGFR-1 and VEGFR-2 imaging of atherosclerotic lesions may be useful to explore plaque biology and identify vulnerability. [ABSTRACT FROM AUTHOR]