Noninvasive Visualization of the Activated αvβ3 Integrin in Cancer Patients by Positron Emission Tomography and [(18)F]Galacto-RGD

BACKGROUND: The integrin αvβ3 plays an important role in angiogenesis and tumor cell metastasis, and is currently being evaluated as a target for new therapeutic approaches. Several techniques are being studied to enable noninvasive determination of αvβ3 expression. We developed [(18)F]Galacto-RGD, a (18)F-labeled glycosylated αvβ3 antagonist, allowing monitoring of αvβ3 expression with positron emission tomography (PET). METHODS AND FINDINGS: Here we show by quantitative analysis of images resulting from a small-animal PET scanner that uptake of [(18)F]Galacto-RGD in the tumor correlates with αvβ3 expression subsequently determined by Western blot analyses. Moreover, using the A431 human squamous cell carcinoma model we demonstrate that this approach is sensitive enough to visualize αvβ3 expression resulting exclusively from the tumor vasculature. Most important, this study shows, that [(18)F]Galacto-RGD with PET enables noninvasive quantitative assessment of the αvβ3 expression pattern on tumor and endothelial cells in patients with malignant tumors. CONCLUSIONS: Molecular imaging with [(18)F]Galacto-RGD and PET can provide important information for planning and monitoring anti-angiogenic therapies targeting the αvβ3 integrins and can reveal the involvement and role of this integrin in metastatic and angiogenic processes in various diseases

Binding of αvβ3 Integrin-Specific Radiotracers Is Modulated by Both Integrin Expression Level and Activation Status

Open Access via Springer Compact Agreement Acknowledgements We are very grateful to Prof. Calderwood (Yale University, USA) for providing the THD DNA construct, Dr. Massimiliano Baldassarre (University of Aberdeen) for useful discussions on integrin regulation and Charlie Taylor for helping to optimise the THD transfection experiments. We thank the NHS Grampian Endowment Fund for funding this research and CRANES and the Roland Sutton Academic Trust for financial support for AA.Peer reviewedPublisher PD

Positron emission tomography imaging of endometrial cancer using engineered anti-EMP2 antibody fragments.

PurposeAs imaging of the cell surface tetraspan protein epithelial membrane protein-2 (EMP2) expression in malignant tumors may provide important prognostic and predictive diagnostic information, the goal of this study is to determine if antibody fragments to EMP2 may be useful for imaging EMP2 positive tumors.ProceduresThe normal tissue distribution of EMP2 protein expression was evaluated by immunohistochemistry and found to be discretely expressed in both mouse and human tissues. To detect EMP2 in tumors, a recombinant human anti-EMP2 minibody (scFv-hinge-C(H)3 dimer; 80 kDa) was designed to recognize a common epitope in mice and humans and characterized. In human tumor cell lines, the antibody binding induced EMP2 internalization and degradation, prompting the need for a residualizing imaging strategy. Following conjugation to DOTA (1,4,7,10-tetraazacyclododecane-N,N',N',N'″-tetraacetic acid), the minibody was radiolabeled with (64)Cu (t (1/2) = 12.7 h) and evaluated in mice as a positron emission tomography (PET) imaging agent for human EMP2-expressing endometrial tumor xenografts.ResultsThe residualizing agent, (64)Cu-DOTA anti-EMP2 minibody, achieved high uptake in endometrial cancer xenografts overexpressing EMP2 (10.2 ± 2.6, percent injected dose per gram (%ID/g) ± SD) with moderate uptake in wild-type HEC1A tumors (6.0 ± 0.1). In both cases, precise tumor delineation was observed from the PET images. In contrast, low uptake was observed with anti-EMP2 minibodies in EMP2-negative tumors (1.9 ± 0.5).ConclusionsThis new immune-PET agent may be useful for preclinical assessment of anti-EMP2 targeting in vivo. It may also have value for imaging of tumor localization and therapeutic response in patients with EMP2-positive malignancies

A comprehensive evaluation of the activity and selectivity profile of ligands for RGD-binding integrins

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins avß3, avß5, avß6, avß8, a5ß1, aIIbß3, using homogenous ELISA-like solid phase binding assay.Postprint (published version

A Novel Aptamer LL4A Specifically Targets Vemurafenib-Resistant Melanoma through Binding to the CD63 Protein.

Melanoma is a highly aggressive tumor with a poor prognosis, and half of all melanoma patients harbor BRAF mutations. A BRAF inhibitor, vemurafenib (PLX4032), has been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) to treat advanced melanoma patients with BRAFV600E mutation. However, the efficacy of vemurafenib is impeded by adaptive resistance in almost all patients. In this study, using a cell-based SELEX (systematic evolution of ligands by exponential enrichment) strategy, we obtained a DNA aptamer (named LL4) with high affinity and specificity against vemurafenib-resistant melanoma cells. Optimized truncated form (LL4A) specifically binds to vemurafenib-resistant melanoma cells with dissociation constants in the nanomolar range and with excellent stability and low toxicity. Meanwhile, fluorescence imaging confirmed that LL4A significantly accumulated in tumors formed by vemurafenib-resistant melanoma cells, but not in control tumors formed by their corresponding parental cells in vivo. Further, a transmembrane protein CD63 was identified as the binding target of aptamer LL4A using a pull-down assay combined with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. CD63 formed a supramolecular complex with TIMP1 and β1-integrin, activated the nuclear factor кB (NF-кB) and mitogen-activated protein kinase (MAPK) signaling pathways, and contributed to vemurafenib resistance. Potentially, the aptamer LL4A may be used diagnostically and therapeutically in humans to treat targeted vemurafenib-resistant melanoma

Hybrid PET/MRI Nanoparticle Development and Multi-Modal Imaging

The development of hybrid PET/MRI imaging systems needs to be paralleled with the development of a hybrid intrinsic PET/MRI probes. The aim of this work was to develop and validate a novel radio-superparamagnetic nanoparticle (r-SPNP) for hybrid PET/MRI imaging. This was achieved with the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) that intrinsically incorporated 59Fe and manganese iron oxide nanoparticles (MIONs) that intrinsically incorporated 52Mn. Both [59Fe]-SPIONs and [52Mn]-MIONs were produced through thermal decomposition synthesis. The physiochemical characteristics of the r-SPNPs were assessed with TEM, DLS, and zeta-potential measurements, as well as in imaging phantom studies. The [59Fe]-SPIONs were evaluated in vivo with biodistribution and MR imaging studies. The biodistrubution studies of [59Fe]-SPIONs showed uptake in the liver. This corresponded with major MR signal contrast measured in the liver. 52Mn was produced on natural chromium through the 52Cr(p,n)52Mn reaction. The manganese radionuclides were separated from the target material through a liquid-liquid extraction. The αVβ3 integrin binding of [52Mn]-MION-cRGDs was evaluated with αVβ3 integrin solid phase assays, and the expression of αVβ3 integrin in U87MG xenograft tumors was characterized with fluorescence flow cytometry. [52Mn]-MION-cRGDs were used for in vivo PET and MR imaging of U87MG xenograft tumor bearing mice. PET data showed increased [52Mn]-MION-cRGD uptake compared with untargeted [52Mn]-MIONs. ROI analysis of PET and MRI data showed that MR contrasted corresponded with PET signal. Future work will utilize [52Mn]-MION-cRGDs in other tumor models and with hybrid PET/MRI imaging systems