[18F](2S,4R)-4-Fluoroglutamine

Metabolic reprogramming plays an important role in the tumorigenesis of clear cell renal cell carcinoma (ccRCC). Currently, positron emission tomography (PET) reporters are not used clinically to visualize altered glutamine metabolism in ccRCC, which greatly hinders detection, staging, and real-time therapeutic assessment. We sought to determine if (2S,4R)-4-[18F]fluoroglutamine ([18F]FGln) could be used to interrogate altered glutamine metabolism in ccRCC lesions in the lung.

We generated a novel ccRCC lung lesion model using the ccRCC cell line UMRC3 stably transfected with GFP and luciferase constructs. This cell line was used for characterization of [18F]FGln uptake and retention by transport analysis in cell culture and by PET/MRI (magnetic resonance imaging) in animal models. Tumor growth in animal models was monitored using bioluminescence (BLI) and MRI. After necropsy, UMRC3 tumor growth in lung tissue was verified by fluorescence imaging and histology.

In UMRC3 cells, [18F]FGln cell uptake was twofold higher than cell uptake in normal kidney HEK293 cells. Tracer cell uptake was reduced by 60–90% in the presence of excess glutamine in the media and by 20–50% upon treatment with V-9302, an inhibitor of the major glutamine transporter alanine-serine-cysteine transporter 2 (ASCT2). Furthermore, in UMRC3 cells, [18F]FGln cell uptake was reduced by siRNA knockdown of ASCT2 to levels obtained by the addition of excess exogenous glutamine. Conversely, [18F]FGln cellular uptake was increased in the presence of the glutaminase inhibitor CB-839. Using simultaneous PET/MRI for visualization, retention of [18F]FGlnin vivoin ccRCC lung tumors was 1.5-fold greater than normal lung tissue and twofold greater than muscle. In ccRCC lung tumors, [18F]FGln retention did not change significantly upon treatment with CB-839.

We report one of the first direct orthotopic mouse models of ccRCC lung lesions. Using PET/MR imaging, lung tumors were easily discerned from normal tissue. Higher uptake of [18F]FGln was observed in a ccRCC cell line and lung lesions compared to HEK293 cells and normal lung tissue, respectively. [18F]FGln cell uptake was modulated by exogenous glutamine, V-9302, siRNA knockdown of ASCT2, and CB-839. Interestingly, in a pilot therapeutic study with CB-839, we observed no difference in treated tumors relative to untreated controls. This was in contrast with cellular studies, where CB-839 increased glutamine uptake.

The PET radiotracer ¹⁸F-(2S,4R)4-fluoroglutamine (¹⁸F-Gln) reflects glutamine transport and can be used to infer glutamine metabolism. Mouse xenograft studies have demonstrated that ¹⁸F-Gln uptake correlates directly with glutamine pool size and is inversely related to glutamine metabolism through the glutaminase enzyme. To provide a framework for the analysis of ¹⁸F-Gln-PET, we have examined ¹⁸F-Gln uptake kinetics in mouse models of breast cancer at baseline and after inhibition of glutaminase. We describe results of the preclinical analysis and computer simulations with the goal of model validation and performance assessment in anticipation of human breast cancer patient studies. Methods: Triple-negative breast cancer and receptor-positive xenografts were implanted in athymic mice. PET mouse imaging was performed at baseline and after treatment with a glutaminase inhibitor or a vehicle solution for 4 mouse groups. Dynamic PET images were obtained for 1 h beginning at the time of intravenous injection of ¹⁸F-Gln. Kinetic analysis and computer simulations were performed on representative time-activity curves, testing 1- and 2-compartment models to describe kinetics. Results: Dynamic imaging for 1 h captured blood and tumor time-activity curves indicative of largely reversible uptake of ¹⁸F-Gln in tumors. Consistent with this observation, a 2-compartment model indicated a relatively low estimate of the rate constant of tracer trapping, suggesting that the 1-compartment model is preferable. Logan plot graphical analysis demonstrated late linearity, supporting reversible kinetics and modeling with a single compartment. Analysis of the mouse data and simulations suggests that estimates of glutamine pool size, specifically the distribution volume (V_D) for ¹⁸F-Gln, were more reliable using the 1-compartment reversible model than the 2-compartment irreversible model. Tumor-to-blood ratios, a more practical potential proxy of V_D, correlated well with volume of distribution from single-compartment models and Logan analyses. Conclusion: Kinetic analysis of dynamic ¹⁸F-Gln-PET images demonstrated the ability to measure V_D to estimate glutamine pool size, a key indicator of cellular glutamine metabolism, by both a 1-compartment model and Logan analysis. Changes in V_D with glutaminase inhibition support the ability to assess response to glutamine metabolism-targeted therapy. Concordance of kinetic measures with tumor-to-blood ratios provides a clinically feasible approach to human imaging.