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

Purpose Molecular 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. Procedures Experiments 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. Results Radiotracer 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. Conclusion Binding 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 material The online version of this article (doi:10.1007/s11307-017-1100-z) contains supplementary material, which is available to authorized users.

PF573228 and ZM323881 were prepared in DMSO. A range of concentrations of PP2 and ZM323881 was added to the cells and left for 72 h. The cytotoxic effect of UO126 and PF228 was confirmed using established IC50 concentrations. Cell viability was evaluated by incubation in RPMI containing 10 % MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). After 2-4 h incubation medium was aspirated. The formazan product was solubilized in DMSO and detected at A560 using a microplate reader. Inhibitory concentrations 50 (IC50) were calculated using Graph PAD Prism v.5 software.

Cell cytotoxic assays
The efficacy of the various treatments used in this study were initially assessed using cell cytotoxicity assays. The IC50 value for each inhibitor is presented in Table 1. The IC50 values were used to guide mechanistic studies to assess the effect of each agent on its target protein (e.g. Src phosphorylation). and PF228 were taken from published literature [2] and [3].

Inhibitor
Determining total, specific and non-specific binding of αvβ3 integrin radiotracers Radiotracer binding was measured in the presence and absence of 10µM non-radiolabelled cRGDfK peptide (Fig. S1). Binding in the absence of cRGDfK represents total binding. cRDGfK binds to αvβ3 integrin with high affinity, and 10µM was included so that it is present in a large excess compared to the radiotracer under investigation. Radiotracer binding in the presence of cRGDfK therefore 3 represents background (non-specific) binding. Specific binding is the difference between total binding and background binding.

Correlation between αvβ3 integrin radiotracer binding and either αvβ3 integrin expression or activation status
The relationship between αvβ3 integrin radiotracer binding and αvβ3 integrin expression was established using data from Fig 1 and Fig 3. For each datapoint the relative radiotracer binding was plotted against αvβ3 integrin expression (Fig S2). Similarly, the relationship between αvβ3 integrin radiotracer binding and αvβ3 integrin activation status (Fig S2) was investigated by plotting αvβ3 integrin radiotracer binding values from Fig 2 against the established effect of each metal ion on αvβ3 integrin activation, as measured by binding of purified receptor to vitronectin [4]. Correlations were observed in both graphs. The impact of these correlation graphs is, however, restricted by the limited number of independent datapoints in each plot.  (Fig. S3). Based on these values, an optimal transfection time was chosen for each cell line (24 h for PC3 and 48 h for U87MG cells).
Western blot analysis confirmed strong expression of THD-GFP (see Fig. 2b of manuscript) at the predicted molecular weight of the THD-GFP construct. Optimising use of PP2 on Src phosphorylation A range of drug concentrations were tested to determine the maximal effect of drug inhibition of each target of interest (pERK, pFAK, pSrc and pVEGFR2). For example PP2 treatment was used at concentrations ranging from 1 nM to 10 μM (Fig. S4). Cells started to detach from the plates when PP2 concentration higher than 10 µM were employed, and there was also a decrease in total Src expression. Therefore the dose that reduced pTyr 416 Src with minimal effect on total Src expression or cell detachment (i.e.10 μM) was selected for further experiments.  Fig. S5b), αvβ3 integrin expression (Fig. S5c) or radiotracer binding (Fig. S5d) were observed, indicating that FAK inhibition has little effect on αvβ3 integrin activation.