A novel strategy for imaging multiple sclerosis with positron emission tomography

112 Objectives The cystine/glutamate antiporter (xCT) is modulated by oxidative stress and its expression is reportedly up-regulated in cells from the monocyte-macrophage-microglia lineage in the spinal cord of experimental autoimmune encephalomyelitis (EAE) rodents, as well as in multiple sclerosis (MS) patients. The goal of this study was to evaluate the ability of the novel xCT-targeted positron emission tomography (PET) radiotracer (4S)-4-(3-[18F]fluoropropyl)-L-glutamate (18F-FSPG) to monitor xCT expression in the EAE mouse model of MS at different stages of disease. Specific imaging of this target could improve our understanding of the in vivo role of xCT in MS, and enable monitoring of disease progression and response to novel therapeutics. Methods EAE was actively induced in C57BL/6 mice by subcutaneous injection of myelin oligodentrocyte glycoprotein (MOG) 35-55 peptide fragment in complete Freund’s adjuvant (CFA), followed by intravenous injections of pertussis toxin. Control mice received CFA emulsion (without MOG) and pertussis toxin. Clinical signs of disease were assessed daily according to a standard scoring system. Groups of naive, control, and EAE mice were administered 18F-FSPG (250 μCi each) intravenously. Initial dynamic PET images were acquired over 90 min (n=3-7 per group). Subsequent studies involved 10 min static scans 75 min p.i. (n=6-10 per group). For an anatomic reference, CT images were acquired just before each PET scan. Mice were perfused and sacrificed, and tissue-associated radioactivity was measured with a gamma-counter or tissue lysates were analyzed for xCT expression levels by Western. Results 18F-FSPG-PET/CT allowed clear visual delineation of the spinal cords of EAE mice (see Figure). Time activity curves showed significantly higher 18F-FSPG accumulation in spinal cords of EAE compared to naive mice (p=0.0043), and a trend toward higher uptake in the brain (p=0.0571) at 75 min p.i. Longitudinal PET studies revealed a small but significant difference in 18F-FSPG uptake in EAE versus control mice at the pre-symptomatic stage of disease (1.2±0.1-fold, p=0.0047). This increase in uptake was even more pronounced in symptomatic EAE mice (2.1±0.2-fold, p=0.0002). Uptake was also significantly elevated in specific brain regions, with the most marked fold-changes in the cerebellum of symptomatic EAE mice (1.6±0.2-fold, p=0.005). We confirmed correlation of 18F-FSPG-PET signal with xCT protein expression by Western (R2=0.80 thoracic spinal cord; R2=0.46 lumbar spinal cord; R2=0.67 brain). In vitro studies with primary human immune cells showed markedly elevated 18F-FSPG uptake in activated compared to resting T-cells (88-fold) while uptake in activated monocytes increased only 2.3-fold over a resting control population. Interestingly, the absolute uptake level in activated monocytes was four-fold higher than in T-cells. Conclusions We have demonstrated the ability of 18F-FSPG to reflect xCT expression during the course of disease in a mouse model of MS. Further studies are warranted to characterize if one or multiple immune cell populations are responsible for 18F-FSPG uptake in vivo, and to elucidate the relationship of 18F-FSPG uptake with structural changes typically observed in MS by MRI, oxidative stress levels, and treatment response to antioxidant therapies. Since we have FDA approval to use 18F-FSPG as an Investigational Novel Drug (IND), we furthermore aim to conduct 18F-FSPG-PET imaging in MS versus age-matched healthy control patients in the near-future.