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.

PurposeMolecular imaging of αvβ3 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 αvβ3 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.ProceduresExperiments were performed in prostate cancer (PC3) and glioblastoma (U87MG) cells, which differentially express αvβ3 integrin. αvβ3 integrin-specific radiotracers were used to investigate the effect of manipulating αvβ3 integrin expression or activation in cellular binding assays. β3 integrin and αvβ3 integrin expression were measured by western blotting and flow cytometry, respectively. The effect of select pharmacological inhibitors on αvβ3 integrin activation and expression was also determined.ResultsRadiotracer binding was proportional to αvβ3 integrin expression when it was decreased (β3 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 αvβ3 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 αvβ3 integrin activity.ConclusionBinding of small molecule ligands and radiolabelled RGD peptides is modulated by expression and activation status of αvβ3 integrin. αvβ3 integrin-specific radiotracers can provide otherwise inaccessible information of the effect of signalling pathways on αvβ3 integrin. This has significant implications for assessing response to anti-angiogenic therapies in clinical studies.Electronic supplementary materialThe online version of this article (doi:10.1007/s11307-017-1100-z) contains supplementary material, which is available to authorized users.

Non-peptidic 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 αvβ3 affinity, selectivity, drug-like logD, high metabolic stability) with a variety of radiolabeling options (e.g. tritium and [ 18 F]fluorine, plus compatibility with radio-iodination), not requiring the use of chelators or prosthetic groups. The binding mode of the resulting triazole RGD-mimics to αvβ3 or αIIbβ3 receptors was investigated by molecular modeling simulations. Compound 12 was successfully radiofluorinated and used for in vivo PET/CT studies in U87-tumour models, which showed only modest tumour 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 αvβ3 integrin, whereas further optimization of their pharmaco-kinetic and dynamic profile would be necessary for a successful use in in vivo imaging.

Abstract. A 1,2,3-triazole-based RGD peptidomimetic having nanomolar affinity for αvβ3 integrin was conjugated to the potent antimitotic paclitaxel via an oxime heterobifunctional linker. The resulting construct maintained nanomolar binding concentration to αvβ3 integrin and showed 11-fold selectivity in terms of cytotoxicity towards highly αvβ3 expressing U87MG cancer cells relative to non αvβ3 expressing MCF7 cells, indicating promising cancer cell targeting capacity. RGD, peptidomimetic, integrin, paclitaxel, click, cytotoxicity, cancer The efficacy of chemotherapeutic agents clinically used for the treatment of malignant tumours is generally limited by their non-specific toxicity against off-target cells, especially those characterised by a high proliferation rate, resulting in a reduced therapeutic index and serious side effects (e.g. alopecia, nausea, vomiting and immunosuppression). Several strategies have been developed over the years in order to overcome these problems and achieve a tumour-targeted delivery of cytotoxic agents. 1 An attractive approach is represented by hybrid compounds, containing a cytotoxic drug bound to a moiety that specifically identifies protein markers overexpressed on cancer cells. Thanks to the development of phage display technologies, several peptide sequences capable of recognising tumour markers have been discovered and extensively investigated as valuable tools for tumour-targeted delivery of chemotherapeutics. 1-2 RGD-containing peptides targeting αvβ3 integrin belong to this class of tumourhoming vectors. The overexpression of αvβ3 on solid tumours, along with its capacity to be internalised via endocytosis after binding of ligands such as RGD peptides, makes it an attractive target for tumour delivery of anticancer agents. 3 A number of cytotoxic drugs have been conjugated to tumour-homing peptides containing the RGD motif and the resulting constructs assessed in animal models. Several studies in this field provided experimental evidence that RGDbased strategies contribute to reduce the toxicity and augment the therapeutic window of existing chemotherapeutics. 1,4,5 Key words

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