Binding of αvß3 Integrin-Specific Radiotracers Is Modulated by Both Integrin Expression Level and Activation Status.

PURPOSE: Molecular imaging of αvß3 integrin has exhibited real potential to guide the appropriate use of anti-angiogenic therapies. However, an incomplete understanding of the factors that influence binding of αvß3 integrin-specific radiotracers currently limits their use for assessing response to therapy in cancer patients. This study identifies two fundamental factors that modulate uptake of these radiotracers. Procedures Experiments were performed in prostate cancer (PC3) and glioblastoma (U87MG) cells, which differentially express αvß3 integrin. αvß3 integrin-specific radiotracers were used to investigate the effect of manipulating αvß3 integrin expression or activation in cellular binding assays. ß3 integrin and αvß3 integrin expression were measured by western blotting and flow cytometry, respectively. The effect of select pharmacological inhibitors on αvß3 integrin activation and expression was also determined. RESULTS: Radiotracer binding was proportional to αvß3 integrin expression when it was decreased (ß3 knock-down cells) or increased, either using pharmacological inhibitors of cell signalling or by culturing cells for different times. Studies with both small molecule and arginine-glycine-aspartic acid (RGD)-based radiotracers revealed increased radiotracer binding after activation of αvß3 integrin with Mn2+ or talin head domain. Moreover, inhibition of fundamental signalling pathways (mitogen-activated protein kinase kinase (MEK), Src and VEGFR2) decreased radiotracer binding, reflecting reduced αvß3 integrin activity. CONCLUSION: Binding of small molecule ligands and radiolabelled RGD peptides is modulated by expression and activation status of αvß3 integrin. αvß3 integrin-specific radiotracers can provide otherwise inaccessible information of the effect of signalling pathways on αvß3 integrin. This has significant implications for assessing response to anti-angiogenic therapies in clinical studies.

High-Affinity "Click" RGD Peptidomimetics as Radiolabeled Probes for Imaging αv ß3 Integrin.

Nonpeptidic Arg-Gly-Asp (RGD)-mimic ligands were designed and synthesized by click chemistry between an arginine-azide mimic and an aspartic acid-alkyne mimic. Some of these molecules combine excellent in vitro properties (high αv ß3 affinity, selectivity, drug-like logD, high metabolic stability) with a variety of radiolabeling options (e.g., tritium and fluorine-18, plus compatibility with radio-iodination), not requiring the use of chelators or prosthetic groups. The binding mode of the resulting triazole RGD mimics to αv ß3 or αIIb ß3 receptors was investigated by molecular modeling simulations. Lead compound 12 was successfully radiofluorinated and used for in vivo positron emission tomography/computed tomography (PET/CT) studies in U87 tumor models, which showed only modest tumor uptake and retention, owing to rapid excretion. These results demonstrate that the novel click RGD mimics are excellent radiolabeled probes for in vitro and cell-based studies on αv ß3 integrin, whereas further optimization of their pharmacokinetic and dynamic profiles is necessary for successful use in in vivo imaging.

Early changes in [18F]FDG incorporation by breast cancer cells treated with trastuzumab in normoxic conditions: role of the Akt-pathway, glucose transport and HIF-1α.

HER-2 overexpression does not guarantee response to HER2-targeting drugs such as trastuzumab, which is cardiotoxic and expensive, so early detection of response status is crucial. Factors influencing [(18)F]FDG incorporation in the timeframe of cell signalling down-regulation subsequent to trastuzumab treatment are investigated to provide a better understanding of the relationship between growth response and modulation of [(18)F]FDG incorporation. HER-2-overexpressing breast tumour cell lines, MDA-MB-453, SKBr3 and BT474 and MDA-MB-468 (HER2 non-over-expressor) were treated with trastuzumab (4 h) and probed for AKT, pAKT, ERK1/2, pERK1/2 and HIF-1α to determine early signalling pathway inhibitory effects of trastuzumab. Cells incubated with trastuzumab and/or PI3K inhibitor LY294002 and ERK1/2 inhibitor U0126 and glucose transport and [(18)F]FDG incorporation measured. Cell lines expressed AKT, pAKT, ERK1/2 and pERK1/2 but not HIF-1α. Trastuzumab treatment decreased pAkt but not pERK1/2 levels. Trastuzumab did not further inhibit AKT when maximally inhibited with LY294002. Treatment with LY294002 and trastuzumab for 4 h decreased [(18)F]FDG incorporation in BT474 and MDA-MB-453 but not SKBr3 cells. LY294002 inhibited glucose transport by each cell line, but the glucose transport rate was tenfold higher by SKBr3 cells than BT474 and MDA-MB-453 cells. AKT-induced uptake of [(18)F]FDG was found to be HIF-1α independent in breast cancer cell lines. AKT inhibition level and tumour cell glucose transport rate can influence whether or not PI3K inhibitors affect [(18)F]FDG incorporation which may account for the variation in preclinical and clinical findings associated with [(18)F]FDG-PET in response to trastuzumab and other HER-2 targeting drugs.