[18F]FSPG was shown to provide an indirect measure of the cellular redox state and may be used as an early indicator of therapy response to cancer therapies that cause oxidative stress. A somewhat paradoxical finding was that reduced [18F]FSPG cellular uptake was associated with either lower cellular concentrations of cystine or glutamate, despite opposing the transport of these substances in the Xc antiporter, for which [18F]FSPG is also a substrate. Further studies of the kinetics of [18F]FSPG will help elucidate the factors mediating a decline in [18F]FSPG with oxidative stress.

See related article by McCormick et al, p. 853

Altered tumor cell metabolism is increasingly recognized as a key component of the cancer phenotype (1). Tumors fuel aberrant growth through increased nutrient consumption. Simultaneously, tumors must guard against reactive oxygen species (ROS) and toxic byproducts of rapid growth to maintain redox homeostasis. Certain tumors cells satisfy increased energetic and biosynthetic demands through the catabolism of glutamine and downstream glutamate. Recognition of the importance of glutamine in tumoral metabolism has provided the impetus to develop molecular imaging methods to probe the metabolism of glutamine and glutamate. Analogs of both molecules have been developed, with two fluorinated analogues being the most widely investigated: [18F](2S,4R)4-fluoroglutamine (([18F]F-Gln) (2) and (4S)-4-(3-[18F]fluoropropyl)-l-glutamate ([18F]FSPG)) (3). Glutamine and glutamate have also been recognized as key components of cellular redox homeostasis, prompting the study McCormick and colleagues published in this issue of Cancer Research. These authors hypothesized that [18F]FSPG provides an indirect measure of the cellular redox state and serves as an early indicator of therapy response.

[18F]FSPG has been successfully translated from preclinical studies into early clinical trials. Koglin and colleagues established specific transport of [18F]FSPG via system Xc and demonstrated tracer uptake in tumor, but not in inflammatory lesions (3). [18F]FSPG was studied in humans for the detection of lung, liver, and breast cancers (4, 5). Several lesions were detected with [18F]FSPG PET, but not [18F]FDG PET. IHC staining of Xc transporter in breast and lung cancer correlated with [18F]FSPG uptake, but not with [18F]FDG uptake (4). Together, these studies suggest [18F]FSPG PET gives information distinct from glycolytic rate as provided by [18F]FDG PET. Moreover, the early clinical studies focused on tumor detection with [18F]FSPG, not characterization of the cellular redox state to inform therapy.

McCormick and colleagues investigated the potential utility of [18F]FSPG to measure tumor redox status (6), given the documented importance of the Xc transporter in redox homeostasis (7). The authors used an oxidant, tert-butyl hydroperoxide, and an antioxidant, N-acetylcysteine (NAC), to manipulate the intracellular redox environment. The effect of these perturbations on [18F]FSPG uptake was studied with the hypothesis that [18F]FSPG uptake could measure cellular redox status. The authors found an inverse relationship between [18F]FSPG uptake and cellular ROS levels. A significant decrease in [18F]FSPG uptake was seen in response to oxidative stress and elevated ROS levels caused by THBP, whereas a significant increase in radiotracer uptake was observed with antioxidant NAC treatment. Pretreatment with NAC abrogated the impact of THBP levels on [18F]FSPG uptake, concordant with measurements of total ROS using flow cytometric methods.

The authors then examined the factors mediating the changes in [18F]FSPG with redox status. Upon THBP treatment, the protein level of Xc transporter remained unchanged, suggesting the decrease of [18F]FSPG uptake was not the result of alterations in transporter expression. In addition, THBP did not alter the level of intracellular glutamate, but there was a decrease in intracellular cystine, the oxidized dimeric form of cysteine and rate-limiting component for GSH synthesis. The decreased [18F]FSPG level was thought to be related to increased GSH biosynthesis resulting from oxidative stress, as demonstrated by isotopic tracing studies. Overall, redox-altering treatments resulted in parallel changes in [18F]FSPG and intracellular cystine concentration. This suggested that the decline in [18F]FSPG uptake might be related to a decline in intracellular cystine pool size. In addition, lowering intracellular glutamine concentration by incubating cells in glutamine-free medium reduced the [18F]FSPG uptake. This demonstrated that there was also a close direct relationship between [18F]FSPG uptake and cellular glutamate concentration.

Finally, the authors used an ovarian cancer xenograft model to study the feasibility of using [18F]FSPG as a surrogate marker for redox alterations in response to chemotherapy as an early indicator of therapy response. A decrease in [18F]FSPG levels was observed after just 24 hours of chemotherapeutic treatment with Doxil, correlating with decreased GSH, before observable changes in the tumor size. No change was detected with [18F]FDG PET at the same time point. The authors noted that this finding was in agreement with the known oxidative effect of Doxil, and the decline in [18F]FSPG uptake seen in vivo with Doxil was consistent with the decline in uptake seen with increased oxidative stress in the in vitro cell uptake studies. Taken together, these findings imply that [18F]FSPG imaging might be used as an early marker of treatment effect via the impact of anticancer therapy on tumor cellular redox status.

The intriguing results of the McCormick study suggest that [18F]FSPG is an indicator of cancer cell redox state, with decreased uptake associated with increased oxidative stress and higher levels of ROS. However, some additional studies are needed to better understand the mechanism underlying these findings. Although the uptake of [18F]FSPG clearly involves the Xc system, the levels of uptake in cells at equilibrium and at later times during in vivo imaging would be expected to reflect either intracellular trapping of [18F]FSPG via catabolism, its retention in the cell as a reflection of the glutamate pool size (distribution volume), or some combination of the two. Preclinical studies of [18F]FSPG have not indicated metabolism of the parent compound (4), so catabolic trapping is an unlikely mechanism of [18F]FSPG cellular retention. Therefore, in the absence of binding to intracellular complexes, late [18F]FSPG uptake is likely to be reversible and reflective of its distribution volume in the intracellular pool size of its native biochemical analogue, glutamate. This was, in fact, noted in the McCormick study. In cell uptake studies, removal of glutamine led to decreased [18F]FSPG in parallel with decreased glutamate concentration. This result most likely stems from the role of glutaminolysis in cellular glutamate production, where glutamine is converted to glutamate by the enzyme glutaminase and replenishes the cellular glutamate, which is utilized to produce GSH. However, increased cellular ROS caused by treatment with THBP also resulted in decreased [18F]FSPG uptake that was associated with decreased cellular cystine levels, but not change in glutamate levels. This finding is a bit more perplexing. One might expect that, in the face of elevated ROS level, lower cystine levels would lead to enhanced extracellular–intracellular cystine gradients and resultant efflux of intracellular glutamate due to the antiporter nature of Xc transporter. One would, therefore, also expect, given the similar behavior for both [18F]FSPG and native glutamate, the increase of ROS also leads to both lower [18F]FSPG uptake (observed) and lower cellular glutamate levels (not observed). These somewhat paradoxical findings cannot simply be explained by cellular glutamate levels and the expected impact of cystine and glutamate levels by the Xc system transporter.

The answer to this quandary may lie in the association between glutamine and glutamate. As noted, in the mitochondria, glutamine is converted to glutamate via glutaminase, generating high glutamate levels in cells that catabolize glutamine as part of their metabolic strategy (8). The degree to which the mitochondrial and cytosolic glutamate pools exchange is the subject of ongoing investigation. However, studies suggest this is largely a unidirectional flow of glutamate from the mitochondria to the cytosol (8, 9). As such, the main source of cellular glutamate is likely through mitochondrial metabolism of glutamine, the most abundant amino acid in the blood, and not via uptake of glutamate via the Xc transporter. A reduction in cystine would, therefore, result in a net flow of glutamate from the mitochondria through the cytosol to the extracellular fluid in the presence of decreased cystine level associated with oxidative stress, while maintaining the intracellular glutamate pool. This phenomenon might leave [18F]FSPG, present only in tracer concentrations in the extracellular fluid, “fighting upstream” to gain access to the cell, providing a plausible explanation for a net reduction of [18F]FSPG uptake despite no change of the native cellular glutamate level.

Why then the uptake of [18F]FSPG is reduced when the cell is deprived of glutamine? Lack of glutamine metabolism would lead to a reduction of glutamate production through glutaminase and a resultant decrease in cytosolic glutamate pool size. These cellular effects would reduce the cellular distribution volume for [18F]FSPG and result in decreased tracer uptake, as was observed. Reduced glutamine metabolism likely also impacts the cellular energy balance leading to oxidative stress as reported by Osanai–Sasakawa (10). Using an antibody against alanine–serine–cysteine transporter 2 (ASCT2), a main transporter for glutamine uptake, it was found that this antibody inhibited glutamine uptake, reduced GSH levels, and increased oxidative stress in preclinical models of gastric cancer. It would be of interest to test the effect on [18F]FSPG level by inhibiting ASCT2 as this has not be reported previously.

In summary, the study of McCormick and colleagues suggests the potential utility of [18F]FSPG PET for guiding cancer therapy. High uptake of [18F]FSPG may mark tumors with high GSH levels that might be less susceptible to oxidative stress. Conversely, low uptake on baseline imaging or an early reduction in uptake after treatment might indicate success of treatment with drugs that increase oxidative stress, as shown in the animal model. However, the elucidation of factors mediating [18F]FSPG in relation to the cellular redox state and oxidative stress will need further study to understand what might be expected in translation to in vivo human studies. Early success with [18F]FSPG as a probe for tumor detection, combined with the results of this study linking [18F]FSPG uptake and oxidative stress, suggests additional studies are warranted. Such studies might be carried out in coordination of studies of glutamine uptake kinetics, taking advantage of the existing PET probes. This could represent an important advance in imaging-targeted treatment in the era of precision medicine.

No potential conflicts of interest were disclosed.

This work is supported by Susan G. Komen grant (SAC130060) and NIH grants R01 R01-CA211337 and KL2TR001879.

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