Significance: There is an unmet clinical need for sensitive methods for detecting cell death in vivo, for example, in disease and following tumor treatment. We show here that deuterium magnetic resonance measurements at 7 T of labeled malate production from injected ² H-labeled fumarate provide a sensitive method for detecting tumor cell death in vivo following treatment. Malate production was relatively slow in viable cells but was markedly increased in necrotic tissue.

Deuterium metabolic imaging (DMI) is an emerging clinically-applicable technique for the non-invasive investigation of tissue metabolism. The generally short T₁ values of ²H-labeled metabolites in vivo can compensate for the relatively low sensitivity of detection by allowing rapid signal acquisition in the absence of significant signal saturation. Studies with deuterated substrates, including [6,6'-²H₂]glucose, [²H₃]acetate, [²H₉]choline and [2,3-²H₂]fumarate have demonstrated the considerable potential of DMI for imaging tissue metabolism and cell death in vivo. The technique is evaluated here in comparison with established metabolic imaging techniques, including PET measurements of 2-deoxy-2-[¹⁸F]fluoro-d-glucose (FDG) uptake and ¹³C MR imaging of the metabolism of hyperpolarized ¹³C-labeled substrates.

Metabolic subtypes of glioblastoma (GBM) have different prognoses and responses to treatment. Deuterium metabolic imaging with 2H-labeled substrates is a potential approach to stratify patients into metabolic subtypes for targeted treatment. In this study, we used 2H magnetic resonance spectroscopy and magnetic resonance spectroscopic imaging (MRSI) measurements of [6,6′-2H2]glucose metabolism to identify metabolic subtypes and their responses to chemoradiotherapy in patient-derived GBM xenografts in vivo. The metabolism of patient-derived cells was first characterized in vitro by measuring the oxygen consumption rate, a marker of mitochondrial tricarboxylic acid cycle activity, as well as the extracellular acidification rate and 2H-labeled lactate production from [6,6′-2H2]glucose, which are markers of glycolytic activity. Two cell lines representative of a glycolytic subtype and two representative of a mitochondrial subtype were identified. 2H magnetic resonance spectroscopy and MRSI measurements showed similar concentrations of 2H-labeled glucose from [6,6′-2H2]glucose in all four tumor models when implanted orthotopically in mice. The glycolytic subtypes showed higher concentrations of 2H-labeled lactate than the mitochondrial subtypes and normal-appearing brain tissue, whereas the mitochondrial subtypes showed more glutamate/glutamine labeling, a surrogate for tricarboxylic acid cycle activity, than the glycolytic subtypes and normal-appearing brain tissue. The response of the tumors to chemoradiation could be detected within 24 hours of treatment completion, with the mitochondrial subtypes showing a decrease in both 2H-labeled glutamate/glutamine and lactate concentrations and glycolytic tumors showing a decrease in 2H-labeled lactate concentration. This technique has the potential to be used clinically for treatment selection and early detection of treatment response. Significance: Deuterium magnetic resonance spectroscopic imaging of glucose metabolism has the potential to differentiate between glycolytic and mitochondrial metabolic subtypes in glioblastoma and to evaluate early treatment responses, which could guide patient treatment.

Early detection of tumor cell death in glioblastoma following treatment with chemoradiation has the potential to distinguish between true disease progression and pseudoprogression. Tumor cell death can be detected noninvasively in vivo by imaging the production of [2,3-2H2]malate from [2,3-2H2]fumarate using 2H magnetic resonance (MR) spectroscopic imaging. We show here that 2H MR spectroscopy and spectroscopic imaging measurements of [2,3-2H2]fumarate metabolism can detect tumor cell death in orthotopically implanted glioblastoma models within 48 hours following the completion of chemoradiation. Following the injection of [2,3-2H2]fumarate into tumor-bearing mice, production of [2,3-2H2]malate was measured in a human cell line–derived model and in radiosensitive and radioresistant patient-derived models of glioblastoma that were treated with temozolomide followed by targeted fractionated irradiation. The increase in the [2,3-2H2]malate/[2,3-2H2]fumarate signal ratio posttreatment, which correlated with histologic assessment of cell death, was a more sensitive indicator of treatment response than diffusion-weighted and contrast agent–enhanced 1H MRI measurements, which have been used clinically to detect responses of glioblastoma to chemoradiation. Overall, early detection of glioblastoma cell death using 2H MRI of malate production from fumarate could help improve the clinical evaluation of response to chemoradiation. Significance: 2H magnetic resonance imaging of labeled fumarate metabolism can detect early evidence of tumor cell death following chemoradiation, meeting a clinical need to reliably detect treatment response in glioblastoma.

Purpose: There is an unmet clinical need for direct and sensitive methods to detect cell death in vivo, especially with regard to monitoring tumor treatment response. We have shown previously that tumor cell death can be detected in vivo from ²H MRS and MRSI measurements of increased [2,3‐²H₂]malate production following intravenous injection of [2,3‐²H₂]fumarate. We show here that cell death can be detected with similar sensitivity following oral administration of the ²H‐labeled fumarate.Methods: Mice with subcutaneously implanted EL4 tumors were fasted for 1 h before administration (200 μl) of [2,3‐²H₂]fumarate (2 g/kg bodyweight) via oral gavage without anesthesia. The animals were then anesthetized, and after 30 min, tumor conversion of [2,3‐²H₂]fumarate to [2,3‐²H₂]malate was assessed from a series of 13 ²H spectra acquired over a period of 65 min. The ²H spectra and ²H spectroscopic images were acquired using a surface coil before and at 48 h after treatment with a chemotherapeutic drug (etoposide, 67 mg/kg).Results: The malate/fumarate signal ratio increased from 0.022 ± 0.03 before drug treatment to 0.12 ± 0.04 following treatment (p = 0.023, n = 4). Labeled malate was undetectable in spectroscopic images acquired before treatment and increased in the tumor area following treatment. The increase in the malate/fumarate signal ratio was similar to that observed previously following intravenous administration of labeled fumarate.Conclusion: Orally administered [2,3‐²H₂]fumarate can be used to detect tumor cell death noninvasively following treatment with a sensitivity that is similar to that obtained with intravenous administration.

Purpose: Serine is a major source of one‐carbon units needed for the synthesis of nucleotides and the production of intramitochondrial nicotinamide adenine dinucleotide phosphate (NADPH), and it plays an important role in cancer cell proliferation. The aim of this study was to develop a deuterium (²H) MRS imaging method for imaging tumor serine metabolism.Methods: Sequential (²H) spectra and spectroscopic images were used to monitor the metabolism of [2,3,3‐²H₃]serine in patient‐derived glioblastoma cells in vitro and in tumors obtained by their orthotopic implantation in mouse brain.Results: [14,14‐²H₂] 5,10‐methylene‐tetrahydrofolate, [²H]glycine, [²H]formate, and labeled water were detected in cell suspensions and water labeling in spectroscopic images of tumors. Studies in cells and tumors with variable mitochondrial content and inhibitor studies in cells demonstrated that most of the labeled serine was metabolized in the mitochondria. Water labeling in the cell suspensions was correlated with formate labeling; therefore, water labeling observed in tumors could be used to provide a surrogate measure of flux in the pathway of one‐carbon metabolism in vivo.Conclusion: The method has the potential to be used clinically to select patients for treatment with inhibitors of one‐carbon metabolism and subsequently to detect their early responses to such treatment.