Abstract
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Objectives: We set out to explore [18F]hGTS13 as a novel radiopharmaceutical to measure xC- transporter activity by positron emission tomography (PET) imaging. The xC- transporter is an emerging oncologic target, mediating oxidative stress and providing a growth advantage for a variety of tumors. This study was performed to evaluate the tumor uptake and biodistribution of [18F]hGTS13 compared to [18]FSPG, a previously described xC- transporter substrate.
Methods: Synthesis of [18F]hGTS13 was adopted from US2015/0011773. The cellular uptake of [18F]hGTS13 was first evaluated in H460 and A549 lung carcinoma cell lines. Competition studies were performed using known substrates or inhibitors of the xC- transporter, L-cystine, L-glutamate, (S)-4-carboxyphenyglycine (CPG). To further confirm the specificity of [18F]hGTS13 for the xC- transporter, small inhibitory RNA (siRNA) methodology was used to reduce transporter expression in A549 cells. Human peripheral blood mononuclear cells (PBMCs) were derived from freshly obtained buffy coat fractions and T cells isolated using the Naïve Pan T cell Isolation Kit. T cells were activated with the T cell activation/expansion kit (Miltenyi Biotec) and radiotracer uptake was performed 48 hours after activation. H460 tumor bearing rats were investigated with [18F]FSPG and [18F]hGTS13 PET/CT imaging. Results: Cell assays with [18F]hGTS13 revealed rapid and extensive uptake in both A549 and H460 human lung carcinoma cells. Uptake values at 60 mins were 21.6 ± 3.6 and 32.1 ± 0.8 %uptake/mg protein in A549 and H460 cells respectively. Transient reduction of xC- transporter expression in A549 cells resulted in a significant reduction in [18F]hGTS13 uptake (p<0.0001), indicating specific uptake through the xC- transporter. The specificity of [18F]hGTS13 for this transporter was further confirmed through competition uptake studies. Significant blocking of radiotracer uptake was observed in the presence of excess L-cystine, L-glutamate and CPG but not with L-aspartate, as expected. Uptake of [18F]hGTS13 in activated T cells revealed a 2.8 fold increase relative to resting T cells (p<0.0133), this was in stark contrast to the uptake of [18F]FSPG which revealed a 75 fold increased uptake in activated T cells compared to resting T cells (p<0.0001). Evaluation of [18F]hGTS13 in H460 tumor bearing rats with PET/CT imaging revealed good tumor accumulation at 60 mins post injection, which was significantly higher (p<0.0061) than that of [18F]FSPG (6.5 ± 1.0 vs 3.2 ± 0.4 %ID/g of tissue respectively), albeit a significant increase (p<0.0001) in liver uptake (9.0 ± 0.1 vs 0.6 ± 0.1 %ID/g of tissue respectively). Conclusion: [18F]hGTS13 is a novel radiotracer for measuring xC- transporter activity. It showed high and specific uptake in cell assays with A549 and H460 cells. [18F]hGTS13 showed significantly lower uptake in activated T cells compared to [18F]FSPG. PET/CT imaging of [18F]hGTS13 in H460 tumor bearing rats revealed significantly higher tumor uptake than [18F]FSPG.
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