Imaging Glioblastoma Response to Radiotherapy Using 2H Magnetic Resonance Spectroscopy Measurements of Fumarate Metabolism

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.


Introduction
Glioblastoma is the most common of all malignant brain tumors (1,2) and remains the deadliest primary malignant brain tumor, with a median survival of 15 months (3). Standard-of-care treatment comprises debulking surgical resection followed by treatment with temozolomide and fractionated targeted radiation (3,4).
The response of glioblastoma to radiotherapy is typically monitored using contrast agent-enhanced MRI, where response is detected through a decrease in the size of the enhancing lesion using the response assessment in neuro-oncology criteria (5)(6)(7)(8). However, the technique does not measure the activity of the tumor cells but the integrity of the blood-brain barrier (BBB), which when opened leads to increased contrast agent enhancement. Consequently it can be difficult to distinguish disease progression from pseudoprogression, which occurs in approximately 30% of patients and is defined as a new or enlarging area or areas of contrast agent enhancement that occur early after the end of treatment but in the absence of true tumor growth (9). Such increases may result from a transient increase in permeability of the tumor vasculature after irradiation (9), or radio-necrosis, which can also result in a disrupted BBB and oedema and usually occurs 6 to 12 months after radiotherapy (10,11). Thus, changes in BBB permeability can influence the degree and extent of the enhancing area irrespective of the size and activity of the tumor (5,12). Therefore, methods that assess the activity of the tumor more directly are expected to provide a more reliable distinction between true disease progression and pseudoprogression. It is notable that of the advanced imaging techniques that have been developed to distinguish between these two scenarios magnetic resonance spectroscopy (MRS) measurements of tumor cell metabolism have shown the best sensitivity and specificity (13). An alternative to measuring the metabolic activity of the tumor cells posttreatment is to measure tumor cell death directly, which has the potential to indicate longer-term outcomes (14).

Orthotopic tumor implantation
Procedures were performed in compliance with project and personal licenses issued under the United Kingdom Animals Scientific Procedures Act, 1986 and were approved by an Animal Welfare and Ethical Review Body. Twelve-week-old (20 g) female BALB/c nude mice (Charles River Laboratories, RRID:SCR_003792) were anaesthetized by inhalation of 1% to 2% isoflurane in air/O 2 (75%/25%, 2 L/min). Analgesia [0.3 mg/mL buprenorphine hydrochloride and 0.135% w/v chlorocresol diluted 1:10 in 0.9% sodium chloride and 1 mL/kg of subcutaneous Rimadyl LA (Pfizer) containing carpofen (Zoetis; 5 mg/kg diluted in 1:10 in 0.9% sodium chloride] was administered subcutaneously. Animals were placed in a stereotactic surgical frame (Kopf) and the head fixed using ear and bite bars. A 1-mm burr hole was drilled 2-mm anterior and 3-mm lateral to the bregma and a syringe containing 5 mL of 0.5 Â 10 6 cells/mL inserted 3.5 mm intracranially. Upon withdrawal by 0.5 mm, the contents of the syringe were injected into the right frontal lobe at 2 mL/min. The burr hole was filled with bone wax and sutures used to close the wound, which was covered using tissue glue (GLUture). Metacam (10 mg/kg) was provided 48 hours and 72 hours postsurgery as analgesia. Animals were assigned randomly to experimental groups. Three animals developed hydrocephalus $3 weeks after intracranial implantation and were excluded from the study.

Image-guided cranial irradiation
Mice were anaesthetized with isoflurane and placed on an irradiator bed (SARRP, XStrahl Inc.). Treatment planning and radiation delivery were performed using MuriPlan software (XStrahl Inc.). CT images were acquired at 60 kV and 0.8 mA, with a voxel size of 0.275 mm, which delivers a dose of 1.2 cGy (21). Tissue segmentation was adjusted for each animal to define bone, tissue, and air. Field isocenters were applied to the segmented images to target radiotherapy delivery, and a collimator was used to customize the radiotherapy field. The SARRP delivers 225-kVp X-rays at 13-mA current (22), through a 0.15-mm Cu filter, at a dose rate of 3.1 Gy/min, and allows exact anatomic targeting with delivery of beams down to 0.5 mm. Mice received a 90 brain arc field (-45 to 45 ), with a field size of 5Â5 mm 2 . Animals were treated with 5 Gy per day for 4 days. Temozolomide (100 mg/kg) was administered by oral gavage 1 hour prior to irradiation. Treatment planning and delivery simulates the clinical situation (23). Mice were euthanized by cervical dislocation when they developed symptoms. taken at the specified time points, centrifuged at 1,000 g, and the supernatants transferred to 5-mm tubes. A separate flask was used for each time point. Formate-d (5 mmol/L) was added to the samples as a reference and 2 H nuclear magnetic resonance (NMR) spectra were acquired at 310 K using the 2 H coil of a 5-mm 1 H/broadband inverse detection probe in a 14.1 T NMR spectrometer (Bruker Spectrospin Ltd.) with a 90 pulse, a repetition time (TR) of 3 seconds with a 2,000 Hz spectral width into 1,024 data points and were the sum of 1,024 transients. Spectra were phased, baseline corrected, and peak integrals calculated using Topspin (Bruker Spectrospin Ltd., RRID: SCR_014227). The amplitudes of the water, fumarate, and malate resonances were normalized to the formate-d peak, after correction for formate-d resonance saturation. Absolute concentrations were obtained by correcting for the numbers of deuterons per molecule. The rate of deuterated malate production and the rates of water labeling were determined by linear least squares fitting of the concentrations using a MATLAB script (MATLAB, RRID:SCR_001622).

H MR spectroscopy and spectroscopic imaging in vivo
Tumors obtained by orthotopic implantation of A11, S2, and U87 cells were treated when they were >60 mm 3 (at $3.5 weeks for U87, $3.5 months for A11, and $7 months for S2 tumors). Animals were anesthetized by inhalation of 2% isoflurane in air/O 2 (75%/25%, 2 L/min). Breathing rate and body temperature were monitored and body temperature maintained with a stream of warm air. 2 H imaging and spectroscopy were performed at 7 T (Agilent, Palo Alto, CA); 2 H resonance frequency 46.007MHz. Tumors were localized in axial 1 H images acquired using a T 2 -weighted a fast spin echo (FSE) pulse sequence at 7 and 9.4 T (7T: TR, 2 s; TE, 50 ms; field-of-view (FOV), 32Â32 mm 2 , 256Â256 matrix; slice thickness, 1 mm; 10 slices. 9.4 T: TR, 2.2 s; TE, 50 ms; FOV, 35Â35 mm 2 ; 256Â256 matrix; slice thickness, 1 mm; 10 slices). Disodium [2,3-2 H 2 ]fumarate was dissolved in water at a concentration of 312.5 mmol/L and $0.2 mL infused via a tail vein catheter. Infusion started 5 minutes after the start of spectral or image acquisition to give 1 g/kg body weight [2,3-2 H 2 ] disodium fumarate infused over a period of 20 minutes. Serial 2 H spectra were acquired with a 2-ms BIR4 pulse (24), with a nominal flip angle of 67 , and a TR of 140 ms (20). Spectra were zero-and first-order phase corrected, and the peaks fitted with the AMARES toolbox (25,26). Localization of signal to the tumors was achieved by the excitation profile of the surface coil and confirmed by imaging. 3D chemical shift images (CSI) were acquired with a 2-ms BIR4 pulse, with a nominal flip angle of 50 , with phase-encoding gradients encoding a 9Â9Â3 kspace matrix with a FOV of 27Â27Â27 mm 3 . Data were acquired into 256 complex points with a sweep width of 4 kHz and a TR of 140 ms (27).

MR spectroscopy of tissue extracts
A11 and S2 tumor-bearing animals were injected with 1 g/kg [2,3-2 H 2 ]fumarate (n ¼ 6 for each model, 3 treated and 3 untreated) under isoflurane anesthesia and after 20 minutes the animals were killed by cervical dislocation, the brains removed and cut in half transversely. The frontal lobe containing the tumor and the other half of the brain were freeze-clamped in liquid nitrogen-cooled tongs. Frozen samples were homogenized in ice-cold 2-mol/L PCA using a Precellys Cryolys Evolution tissue homogenizer (Bertin Instruments) and neutralized with 2-mol/L KOH. After centrifugation for 15 minutes at 13,000 g, 200 mL of the supernatant was mixed with 300 mL of H 2 O and a formate-d standard added to a final concentration of 4 mmol/L. 2 H NMR spectra were acquired using the same acquisition parameters as used for the media samples. Concentrations were calculated by comparison of the signal intensities with that of the formate-d standard. Following the 2 H NMR measurements, a standard for proton NMR measurements, 3-(trimethylsilyl)-2,2,3,3tetradeuteropropionic acid, was added at a final concentration of 1 mmol/L, together with 50 mL of 2 H 2 O, and 1 H spectra were acquired using a 5-mm 1 H/broadband inverse detection probe in a 14.1 T NMR spectrometer (Bruker Spectrospin Ltd.) with water presaturation and a flip angle of 90 into 16,384 data points, with a spectral width of 7,788 Hz and a repetition time of 8 seconds. Spectra were phased and baseline corrected, and peak integrals calculated using Topspin 4.0.6 (TopSpin, RRID:SCR_014227, Bruker).

Dynamic contrast-enhanced MRI
A separate cohort of A11, S2, and U87 tumor-bearing animals (3 per group) underwent dynamic contrast-enhanced (DCE)-MRI before and 48 hours after the last radio-chemotherapy treatment. Images were acquired at 9.4 T using a Bruker console, an Agilent magnet and a 40-mm diameter 1 H volume coil (Agilent). Tumors were localized in axial (FOV, 35Â35 mm 2 ) and sagittal (FOV, 60Â60 mm 2 ) 1 H images acquired with a FSE pulse sequence (TR, 1,000 ms; TE, 25 ms; 256Â256 matrix; slice thickness, 1 mm; 10 slices). Baseline and dynamic axial T 1 images, acquired following tail vein injection of Dotarem, at 200 mmol/kg (Gadoteric acid, Guerbet), were acquired as described previously (20). Signals from the image series were converted, on a pixel-by-pixel basis, to a contrast-agent concentration by assuming an R 1 relaxivity of the contrast agent of 2.7 s -1 (mmol/L) -1 (29).

Diffusion-weighted magnetic resonance imaging
Diffusion-weighted 1 H images were acquired at 9.4 T using a spin echo pulse sequence with echo planar readout (FOV, 40Â40 mm 2 ; 64Â64 matrix; TR, 2 s; TE, 44.86 ms). Tumors were localized in axial and coronal 1 H images acquired with a FSE pulse sequence (TR, 1,000 ms; TE, 25 ms; FOV, 40Â40 mm 2 ; 256Â256 matrix; slice thickness, 1 mm; 10 slices). Diffusion-sensitizing gradients equivalent to b-values of 16.17, 56.06, 106.06, 206.06, 406.06, and 806.06 s/mm 2 were applied along the slice axis. The apparent diffusion coefficient (ADC) was measured pretreatment and at 2 and 7 days posttreatment in a 1-mm thick slice covering the tumor.

Histology and IHC
Brains were immediately transferred to 10% formalin for 24 hours, then 70% ethanol before embedding in paraffin and sectioning (10-mm thick sections). The tissue was cut in the middle of the frontal lobe transversely and stained with hematoxylin and eosin (ST020 Multistainer-Leica Microsystems, RRID:SCR_008960). TUNEL staining was performed using a DeadEnd Colorimetric System Kit (Promega Benelux BV) and cleaved caspase-3 (CC3) staining using Leica's Polymer Refine Kit (Leica Microsystems, catalog no. DS9800, RRID: AB_2891238) on an automated Bond platform (Leica Biosystems Ltd). The antibody was used at a 1:400 dilution (Cell Signaling Technology). Slides were scanned at 20x magnification with a resolution of 0.5 mm per pixel on an Aperio AT2 (Leica Biosystems, RRID:SCR_021256). Images were analyzed using a CytoNuclear v1.6 algorithm on HALO v3.0.311.293 (RRID:SCR_018350, Indica Labs) to quantify percentage of positive cells.

Statistical analysis
Statistical and graphical analyses were performed using Prism v9 (RRID:SCR_002798, GraphPad). Data are shown as mean AE SD, unless stated otherwise. Analysis of variance was used for multiple comparisons of groups to determine significance. A paired or unpaired Student t test was used for single-parameter comparisons. Power calculations were used to define the number of animals required to detect the effect of interest with a probability of 0.9 assuming a familywise error rate of 5%, using the programme G Ã Power (30). Analysis of recent experiments (31) showed that a group size of 6 to 8 animals would be sufficient to reach this objective, however effect sizes observed here were much larger and we were able to stop at 3 animals per group.

Data availability
The data generated in this study will be made publicly available in a University of Cambridge Data Repository (https://doi.org/10.17863/ CAM.87021).

Deuterated fumarate metabolism detects cell death in glioblastoma cells in vitro
Human glioblastoma (U87) and patient-derived cells (A11, S2) were treated with 50-mmol/L temozolomide and 15 Gy radiation and the concentrations of deuterium labelled water, fumarate and malate were measured following the addition of 5-mmol/L [2,3-2 H 2 ]fumarate using 2 H NMR measurements on samples of culture medium (Fig. 1A and B). Representative spectra from A11 and U87 cells are shown in Supplementary Fig. S2. There was an increase in the rate of labeled malate production following treatment in all three cell lines.
The increase was largest in S2 cells (Fig. 1C-E), consistent with the higher levels of cell death posttreatment, with an increase from 1.84 AE 0.23 fmol/min/cell to 6.76 AE 0.7 fmol/min/cell (P ¼ 0.0028; Fig. 1E). The rates were lower in treated A11 (Fig. 1F-H) and U87 cells (Fig. 1I-K), with malate production rates of 3.01 AE 0.79 and 5.13 AE 1.03 fmol/min/cell, respectively compared with rates in untreated controls of 0.71 AE 0.36 and 1.46 AE 0.74 fmol/min/cell (Fig. 1F and I) The rates of water labelling were not significantly affected by the induction of cell death, as observed previously (20). Cell viability was unchanged in the controls (Fig. 1L) but decreased in the treated group (Fig. 1M).

Deuterated fumarate metabolism detects cell death in glioblastoma tumor models in vivo
Deuterium-labeled fumarate, malate and water concentrations were monitored following intravenous injection of 1 g/kg [2,3-2 H 2 ]-disodium fumarate, using localized 2 H spectroscopy (Fig. 2) and imaging (Fig. 3). The tumor fumarate concentrations were similar in all three tumor models before ( Fig. 2A, B, F, G, K, and L) and at 48 hours after completion of chemoradiotherapy ( Table 1; Fig. 2C, D, H, I, M, and N). However, all showed a significant increase in labeled malate concentration following treatment, which in the A11 and S2 tumors was confirmed by measurements in tumor extracts ( Table 1; Supplementary Fig. S3). In extracts of untreated tumors the concentration of [2,3-2 H 2 ]malate, determined by 2 H NMR, was only 29% (A11) and 30% (S2) of the unlabeled concentration, determined by 1 H NMR, whereas in treated tumors the total malate concentration increased by $3.2 and $4.4x, of which, $36% (A11) and 44% (S2) were deuterium labeled ( Table 1). The malate/fumarate signal ratios measured 48 hours after the last treatment increased significantly in all tumor models, with S2 showing the largest increase (Fig. 2E, J, and O). A fast, dynamic 3D CSI sequence showed no detectable malate signal pretreatment (Fig. 3C, I, and O) whereas increased malate concentrations were observed 48 hours after the last treatment in all tumor models (Fig. 3F, L, and R), with the highest concentration in S2 tumors (Fig. 3L). The tumor malate signal-to-noise ratios posttreatment were 2.05 AE 0.27, 5.64 AE 2.54, and 5.38 AE 2.62 in A11, S2, and U87, respectively and 1.29 AE 0.11, 4.01 AE 2.98, and 3.67 AE 1.12 in the corresponding CSI (data are expressed as mean AE SD, n ¼ 3 for the spectroscopy data and n ¼ 2 for the images). Representative spectra from the 3D CSI dataset are shown in Supplementary Fig. S4. The higher rates of malate production in S2 cells in vitro and S2 tumors in vivo when compared with the other cell lines cannot be explained by higher levels of fumarase activity in these cells, which was similar in all three lines, both in cells in culture and tumors in vivo ( Supplementary Fig. S5). Treatment slowed the growth of all three tumors, which was significant for S2 and U87 tumors (Supplementary Fig. S6; Supplementary Table S1). The greater inhibition of S2 tumor growth was correlated with increased survival ( Supplementary  Fig. S7), although animals with the other tumors also showed evidence of increased survival. Increases in the [2,3-2 H 2 ]malate/ [2,3-2 H 2 ]fumarate signal ratios were correlated with the levels of tumor cell death, as determined from histologic measurements of CC3 and DNA damage (TUNEL) in tumor sections (Fig. 4). Although, there were no decreases in tumor size following treatment there were significant increases in CC3 and TUNEL staining. CC3 staining increased from 2.84 AE 1.29 to 11.66 AE 3.71 in A11 tumors (P ¼ 0.002, n ¼ 4; Fig. 4G), from 9.10 AE 2.60 to 19.67 AE 3.91 in U87 tumors (P ¼ 0.004, n ¼ 4; Fig. 4W) and showed the greatest increase in S2 tumors, from 3.52 AE 2.27 to 22.78 AE 2.78 (P ¼ 0.0008, n ¼ 4; Fig. 4N).        (Fig. 5). However, none of these increases reached statistical significance; with increases of 33.3 AE 1.8% (P ¼ 0.125, n ¼ 4) in A11 (Fig. 5B), 62.9 AE 14.6% in S2 (P ¼ 0.133, n ¼ 4; Fig. 5D) and 51.1 AE 16.2% in U87 (P ¼ 0.08, n ¼ 3) tumors (Fig. 5F) (Fig. 2). The animals used in the 2 H, 13 C, dynamic contrast agent-enhanced and diffusion-weighted imaging experiments are summarized in Supplementary Table S2. 13 C CSI are shown in Supplementary Fig. S8.

Dynamic contrast agent-enhanced MRI measurements
Changes in tumor contrast agent uptake were assessed in separate cohorts of A11 (n ¼ 3), S2 (n ¼ 3), and U87 (n ¼ 3) tumor-bearing mice (Fig. 6). The area under the tumor contrast agent uptake curve increased significantly in S2 tumors at 48 hours posttreatment (Fig. 6D-F) but not in A11 (Fig. 6A-C) or U87 tumors (Fig. 6G-I), although all three tumors showed higher tumor contrast agent concentrations posttreatment with significantly higher Table 1. Deuterium-labelled fumarate, malate, and water concentrations measured in the indicated tumor extracts and in extracts of the corresponding contralateral hemisphere using 2 H NMR.   concentrations in the first 10 minutes post contrast agent injection (Fig. 6B, E, and H).

Diffusion-weighted MRI measurements
The mean ADC of tumor water, measured using diffusion-weighted MRI (DW-MRI; Fig. 7), was unchanged at 48 hours after the last chemoradiotherapy treatment in all tumor models (Fig. 7C, G, and K) and the ADC maps remained largely homogeneous (Fig. 7D, H, and L). At 7 days after treatment, however, the S2 and U87 tumors showed a significant increase in the mean ADC ( Fig. 7G and K).

Discussion
Routine MRI measurements of glioblastoma morphology are unable to distinguish reliably between true disease progression and pseudoprogression, which has prompted a search for MRI methods that are better able to discriminate between the two (32).
Tumor water ADC has been reported to be lower in true disease progression as compared with pseudoprogression, however metaanalyses have shown that while diagnostic performance was superior to that of conventional anatomic imaging it was poorer when compared with MRI measurements of perfusion (13). Dynamic susceptibility contrast (DSC) MRI measurement of relative cerebral blood volume (rCBV) is the most widely used perfusion technique, with numerous studies showing that rCBV is lower in areas of radionecrosis and pseudoprogression when compared with true disease progression. DCE MRI measurements of contrast agent leakage from the tumor vasculature, characterized by a transfer constant (Ktrans), which is a function of vessel permeability, vessel surface area and blood flow, can also distinguish true progression from pseudoprogression, where
Ktrans is lower in regions of radionecrosis and pseudoprogression. However, with no widely established Ktrans thresholds and a dependence of the fitted parameter on the model used to analyze the data this is a less widely used technique than DSC MRI (33). Imaging of proton transfer between protein amide groups and water has shown promise in distinguishing true progression from pseudoprogression, with an almost two-fold higher rate in the case of true progression (34), although currently the technique has not been as widely evaluated as the other MRI methods. 1 H MRS measurements of tumor metabolism can distinguish between true progression and pseudoprogression, with elevated choline concentrations indicating increased tumor cell density and progression and posttreatment necrotic tumors showing elevated lipid and lactate peaks and lower choline concentrations (32,(35)(36)(37)(38). However, while meta-analysis has shown that 1 H MRS is superior to anatomic MRI, DWI-MRI, and MRI-based perfusion measurements in distinguishing true progression from pseudoprogression (13) the low signal-to-noise ratio in spectroscopy means that scan times are long.
The problem of low sensitivity of MRS measurements of tumor metabolism has been addressed using nuclear spin hyperpolarization, which can result in a massive increase in sensitivity (39). We have shown previously that imaging the metabolism of hyperpolarized [1-13 C]pyruvate can detect the response of the A11 PDX used here to chemoradiation by 72 hours after treatment, in an image that took less than 30 seconds to acquire (identified as GB4 in this previous paper; ref . 40). However, the decrease in lactate labeling from the injected pyruvate was relatively small ($29%) and much less than the increase in the [2,3-2 H 2 ]malate/[2,3-2 H 2 ]fumarate signal ratio observed here at 48 hours after treatment despite a similar increase in cell death. The [2,3-2 H 2 ]malate/[2,3-2 H 2 ]fumarate ratio increased in A11 tumors by 340%, in the more radiosensitive S2 tumors by 755%, and in the U87 cell line model by 425%. The increased rate of malate production in S2 tumors cannot be explained by intrinsically higher levels of fumarase activity, which was similar in all three tumor models. The increased rates in A11 and S2 tumors also cannot be explained by increased fumarate delivery because the concentrations of fumarate in the tumors pre-and posttreatment were similar. Moreover, by using the [2,3-2 H 2 ]malate/[2,3-2 H 2 ]fumarate ratio as an indicator of cell death the effects of any possible changes in fumarate delivery are corrected for. We have shown previously, in three drug-treated tumor models, that the accumulation of malate in a tumor posttreatment cannot be explained by metabolism of fumarate elsewhere in the body and wash in of labeled malate (20).
The increases in the hyperpolarized [1,4-13 C 2 , 2,3-2 H 2 ]malate/ [1,4-13 C 2 , 2,3-2 H 2 ]fumarate signal ratio following treatment, following injection of hyperpolarized [1,4-13 C 2 , 2,3-2 H 2 ]fumarate, were much smaller and did not reach statistical significance. This is a reflection of the short half-life of the hyperpolarized 13 C label, which means that labeled malate accumulation can only be measured over a few minutes whereas in the 2 H experiments labeled malate accumulation was  ]fumarate ratio was 0.03 to 0.04 pretreatment and posttreatment ranged from 0.07 to 0.14 at 3 minutes and from 0.3 to 0.5 at 60 minutes. The relative increases in these ratios are similar to those observed in the hyperpolarized 13 C experiments in vivo at 3 minutes and in the 2 H experiments in vivo at 60 minutes. Detection of treatment response using [2,3-2 H 2 ]fumarate was also more sensitive than either of the more advanced imaging techniques used currently to distinguish between true disease progression and pseudoprogression. DW-MRI detected treatment response only in S2 and U87 tumors, which showed higher levels of cell death posttreatment than A11 tumors, and then only at 7 days posttreatment. We have shown previously that both 2 H-(20) and 13 C-labeled (17) fumarate are more sensitive than DW-MRI at detecting low levels of diffuse necrosis. Dynamic contrast agent enhanced images showed an increase in the area under the contrast agent uptake curves in all three tumor models at 48 hours posttreatment but this was only significant in the S2 tumors, which showed the highest levels of cell death posttreatment.
Early detection of glioblastoma cell death following chemoradiation has the potential to distinguish true disease progression from pseudoprogression in the clinic. We have shown here that 2 H MRI of labeled malate production from injected [2,3-2 H 2 ]fumarate can detect cell death in two orthotopically implanted PDX models and a cell line model of glioblastoma within 48 hours of treatment and that it is a more sensitive method of detecting cell death than two MRI methods that have been used in the clinic to distinguish between true progression and pseudoprogression. The clinical feasibility of the technique can be assessed from the measured fumarate and malate concentrations in the tumor, which is more appropriate than comparing SNR because the latter depends not only on magnetic field strength, but also on other factors, such as image resolution and coil performance. De Feyter and colleagues (41) detected 1.5 to 2 mmol/L 2 H-labelled glucose and glutamate/glutamine in the human brain at 4 T. Here we measured deuterated fumarate and malate concentrations of up to 5 mmol/L. Therefore, even with a $40% decrease in SNR at 3T (42), these concentrations coupled with the chemical shift separation of the resonances (20), suggests that this method could be used clinically. Representative diffusion images, in false color, overlaid on the corresponding T 2 -weighted images acquired from A11 (B), S2 (F), and U87 (J) tumors before and at 48 hours and 7 days after the last chemoradiotherapy treatment. The locations of the tumors are outlined by dotted white lines. The underlying T 2 -weighted images acquired at 7 days after treatment are the same as those shown in A, E, and I and the underlying image shown in F at 48 hours posttreatment is the same as that shown in Fig. 3J. The 2 H and diffusion-weighted images were acquired in the same imaging session as the T 2 -weighted images. Mean ADC measured along the slice-selective direction before and at 2 and 7 days after the last chemoradiotherapy treatment in A11 (n ¼ 6; C), S2 (n ¼ 6; G), and U87 (n The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note
Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).