metabolic response of glioma to dichloroacetate measured in vivo

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Metabolic response of glioma todichloroacetate measured in vivo by

hyperpolarized 13C magnetic resonancespectroscopic imaging

Jae Mo Park, Lawrence D. Recht, Sonal Josan, Milton Merchant, Taichang Jang,Yi-Fen Yen, Ralph E. Hurd, Daniel M. Spielman, and Dirk Mayer

Department of Radiology (J.M.P., S.J., D.M.S., D.M.); Department of Electrical Engineering (J.M.P., D.M.S.);

Department of Neurology and Neurological Sciences, Stanford University, Stanford, California (L.D.R., M.M.,

T.J.); SRI International, Neuroscience Program, Menlo Park, California (S.J., D.M.); Applied Science

Laboratory, GE Healthcare, Menlo Park, California (Y.F.Y., R.E.H.)

Background. The metabolic phenotype that derives dis-proportionate energy via glycolysis in solid tumors,includ-ing glioma, leads to elevated lactate labeling in metabolicimaging using hyperpolarized [1-13C]pyruvate. Althoughthe pyruvate dehydrogenase (PDH)mediated flux frompyruvate to acetyl coenzyme A can be indirectly measuredthrough the detection of carbon-13 (13C)-labeled bicar-bonate, it has proven difficult to visualize 13C-bicarbonate

at high enough levels from injected [1-13C]pyruvate forquantitative analysis in brain. The aim of this study is toimprove the detection of13C-labeled metabolites, in par-ticular bicarbonate, in glioma and normal brain in vivoand to measure the metabolic response to dichloroacetate,which upregulates PDH activity.Methods. An optimized protocol for chemical shiftimaging and high concentration of hyperpolarized [1-13C]pyruvate were used to improve measurements of lactateand bicarbonate in C6 glioma-transplanted rat brains.Hyperpolarized [1-13C]pyruvate was injected before and45 min after dichloroacetate infusion. Metabolite ratiosof lactate to bicarbonate were calculated to provide im-

proved metrics for characterizing tumor metabolism.Results. Glioma and normal brain were well differentiatedby lactate-to-bicarbonate ratio (P .002, n 5) as well asbicarbonate (P .0002) and lactate (P .001), and astronger response to dichloroacetate was observed inglioma than in normal brain.

Conclusion. Our results clearly demonstrate for the firsttime the feasibility of quantitatively detecting 13C-bicar-bonate in tumor-bearing rat brain in vivo,permitting themeasurement of dichloroacetate-modulated changes inPDH flux. The simultaneous detection of lactate and bicar-bonate provides a tool for a more comprehensive analysisof glioma metabolism and the assessment of metabolicagents as anti-brain cancer drugs.

Keywords: bicarbonate, dichloroacetate, glioma,hyperpolarized 13C, pyruvate.

Glioblastoma multiforme is one of the most agg-ressive cancers, with less than half of patientssurviving beyond 1.5 years even with optimal

treatment.1 New treatments, such as targeted moleculartherapies and metabolic modulation, with improved ef-fectiveness have been actively developed in the lastdecade, but there is still a need for methods that can beused to gauge the effects of therapy acutely afteradministration.

Unlike normal tissues, which derive the bulk of energyneeds via oxidative phosphorylation (OXPHOS) of mul-tiple energy substrates, solid tumors, including glioma,derive a disproportionate amount of energy via glycolysis(GLY), even when oxygen tension levels are high, alsoknown as the Warburg effect.2,3 Contrary to Warburgsoriginal idea, however, mitochondrial dysfunction is notthe cause of this metabolic change,4,5 raising severalother proposed possibilities, including the capacity toderive energy from glucose more quickly if not more effi-ciently through GLY,6,7 the need to create biomass and tosupport proliferation,4 and the development of a more fa-vorable (for the cancer cell) acidic environment.8 Interest

Corresponding Author:Jae Mo Park, PhD, Stanford University,

Department of Radiology, The Lucas Center for Imaging, 1201 Welch

Road, Stanford, CA, 94305 ([email protected]).

Received March 30, 2012; accepted November 20, 2012.

Neuro-Oncologydoi:10.1093/neuonc/nos319 NEURO-ONCOLOGY

#The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.All rights reserved. For permissions, please e-mail: [email protected].

Neuro-Oncology Advance Access published January 19, 2013


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in the Warburg effect, whatever its underlying function,has been increasing recently due in large part to thehope that a better understanding will lead to novel thera-peutic anticancer strategies.

Until recently, a major difficulty in studying cancermetabolism has been that limited tools exist withwhich to study it repeatedly in the intact organism.Using 2-deoxy-2-(18F)fluoro-D-glucose ([18F]FDG) PET

scanning, the diagnostic effectiveness of which is basedon the Warburg effect (due to increased glucose utiliza-tion by cancer tissue), provides no information as to howglucose is processed within cells because FDG-PETcannot differentiate metabolic products from the inject-ed substrate.9,10 Elevated lactate (Lac) concentration inbrain tumors due to upregulated glycolytic rate can bedetected using proton(1H)andcarbon-13(13C) magneticresonance spectroscopy (MRS).11,12 In 1H-MRS, Lacquantification is hampered by signal overlap from lipidsthat can arise within tumors13 as well as from fatoutside the brain contaminating the spectra due to non-ideal spatial localization. Although spectral editing tech-

niques are available to help differentiate Lac fromlipids,14,15 these methods also reduce the signal-to-noiseratio (SNR),16 can be prone to patient motion duringlong acquisition, and are not routinely used in theclinic. 13C-MRS provides better signal separation due toa wider dispersion of the chemical shift, but because ofits low sensitivity, it requires long acquisition timesthatseverely hinder real-time in vivo investigations.17,18

Hyperpolarized 13C-MRS, using dynamic nuclear po-larization (DNP) in combination with a rapid dissolutionprocess to retain high levels of nuclear spin polarizationin the liquid state, enables the real-time investigation ofin vivo metabolism with more than 10,000-fold signalin-crease over conventional 13C-MRS methods.19,20 Using

[1-13C]pyruvate (Pyr), a substrate occupying a keynodal point in the glucose metabolic pathway, allowsone to quantitatively follow the in vivo conversion ofPyr to Lac, alanine (Ala), or acetyl coenzyme A (CoA).In the conversion to acetyl CoA, the first step toward en-tering the tricarboxylic acid (TCA) cycle, the 13C label isreleased in the form of13CO2, which quickly equilibrateswith 13C-bicarbonate (Bic), thus potentially offering atool to noninvasively track the fate of carbon througheither GLY or entry into the TCA cycle repeatedlywithin the same organism.2123

Hyperpolarized [1-13C]Pyr has shown great potentialfor differentiating cancer from normal tissueand detect-

ing metabolic response to treatment.

2426

Recently,hyperpolarized [1-13C]Pyr has been applied to ratbrain implanted with human gliomas,27 and the earlychanges in tumormetabolism after exposure to temozo-lomide treatment,28 therapy targeted to the phosphatidylinositol 3-kinase (PI3K) pathway,29,30 and radiothera-py31 have been reported. Up to now, however, cancerstudies utilizing 13C-MRS of hyperpolarized Pyr havefocused primarily on Lac production, in large partbecause of the poor SNR of Bic, thus representing aserious hurdle in being able to use this methodology toassess the balance between GLY and OXPHOS.Whereas Bic has been consistently detected in normal

rat brain,32,33 detection in glioma has not been reportedyet. Grant et al.34 recently demonstrated that the reduc-tion of Lac in a transgenic mouse model for prostatecancer after a week of daily administration of dichloroa-cetate (DCA) could be consistently observed, but Bic wasdetected only post-DCA.

In this study we improve detection of Bic in brain oftumor-bearing rats by using single time-point spiral

chemical shifting imaging (CSI)35 to increase SNR andby injecting [1-13C]Pyr with higher concentration.Furthermore, we measure the metabolic response ofglioma and normal-appearing brain tissue to administra-tion of DCA. DCA promotes mitochondrial function byinhibiting pyruvate dehydrogenase kinase (PDK), thusupregulating pyruvate dehydrogenase (PDH),which cat-alyzes the production of acetyl CoA from Pyr.3641 DCAhas beenshown to reduce tumor growth both in vitro andin vivo42,43 and is currently in clinical trials (for details,see clinicaltrials.gov). Due to its small molecular size,DCA can readily transverse the blood brain barrier,and the efficacy of DCA has been demonstrated for pa-

tients with malignant gliomas.

44

Materials and Methods

MR Hardware and Radiofrequency Coils

All experiments were performed on a 3T Signa MRscanner (GE Healthcare) equipped with a high-performance insert gradient coil operating at amaximum amplitude of 500 mT/m with a slew rate of1865 mT/m/ms.45 A custom-built dual-tuned 13C-1Hquadrature volume radiofrequency (RF) coil (50 mm)46 operating at 32.1 MHz and 127.7 MHz,respectively, was used for both RF excitation andsignal reception, respectively.

Animal Preparation and Tumor Model

Implantation of 1 106 C6 glioma cells (derived from anN-methyl-N-nitrosourea induced tumor47 as previouslydescribed48,49) was performed in the right striatum of 5male Wistar rats (body weight 235+10 g) 10.5 daysbefore 13C-MRS imaging. Theestimatedtumor size, mea-sured from hyperintense regions of contrast-enhancedT1-weighted proton images, was 95.3+17.9 mm

3.

Three healthy male Wistar rats (body weight

262+7 g) were also examined, using the same imaging proto-col, to compare the metabolic responses with DCA ofnormal brain of healthy rats and normal-appearingbrain of tumor-bearing rats.

Animals were anesthetized with 1.5% 3.0% isoflur-ane in oxygen (1.5 L/min) followed by tail vein cannu-lation for intravenous administration of hyperpolarized[1-13C]Pyr and DCA. Body temperature was monitoredby a rectal probe and maintained at 378C using awarm water blanket underneath the animal. Vital signsincluding respiration, temperature, and oxygen satura-tion were monitored throughout the MR experiments.

Park et al.: Metabolic response of glioma to dichloroacetate

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All procedures were approved by the local InstitutionalAnimal Care and Use Committee.

Polarization Procedure

A mixture of 14-M [1-13C]labeled pyruvic acid and15 mM trityl radical was polarized using a HyperSense

DNP system (Oxford Instruments Molecular Biotools).The solvent solution consisted of 40 mM tris-hydroxy-methylaminomethane, 125 mM NaOH, 100 mg/L eth-ylene diamine tetraacetic acid, and 50 mM NaCl andwas used to produce a 125-mM solution of [1-13C]Pyr.Using a high concentration exploits the unsaturable com-ponent of Pyr transport into the brain.50 Detailed proce-dures for polarization and dissolution have beendescribed.19,35 Liquid-state polarization at dissolutionwascalculated to be25%based on solid-state polariza-tion and independent calibration experiments. A volumeof 2.6 3.4 mL of the solution with a pH of7.5 was in-jected through the tail vein catheter followed by a flush of0.5 mL of saline with 1% of heparin to clear the catheter

line. Injection rate was maintained at 0.25 mL/s. Theamount of injected solution was adjusted to maintain adose of 1 mmol/kg body weight.

MR Protocol

Single-shot fast spin echo (FSE) images with pulse repeti-tion time (TR)/echo time (TE) of 1492/38.6 ms, slicethickness of 2 mm, and in-plane resolution of 0.47 mmwere acquired for anatomical reference from up to 45slices in axial, coronal, and sagittal planes. A dual-echo T2-weighted FSE sequence (TR/TE1/TE2 5000/11.3/56.7 ms, slice thickness 1 mm, in-plane resolution 0.25 mm, matrix size 256 192,echo train length8) in the axial plane was used to determine the location ofthe tumor. The homogeneity of the B0 field over the regionof the brain that included the tumor was manually opti-mized with a point-resolved spectroscopysequence by min-imizing the line width of the unsuppressed water signalusing the linear shim currents.

Spiral CSI with 4 spatial interleaves (spectralbandwidth 932.8 Hz) was used for metabolic imagingof hyperpolarized [1-13C]Pyr and its metabolic products,[1-13C]Lac, [1-13C]Ala, and 13C-Bic. The 13C transmitRF power was calibratedoverthe targeted slice usinga ref-erence phantom (3.4-M solution of [1-13C]Lac) placed on

top of the animals head.Data were acquired using a var-iable flip angle scheme51 leading up to 90 degrees from a6- to 8-mm slice with a nominal in-plane resolution of2.7 mm 2.7 mm (field of view [FOV] 43.5 mm,matrix size 16 16, TR 125 ms, number ofechoes 96, acquisition time 0.5 s). Furtherdetails ofthe pulse sequence are described by Mayer et al.32 Singletime-point images were acquired 30 s after the start ofthe injection. The time delay was chosen to maximizeBic based on prior dynamic metabolic imaging experi-ments.32 Following the last 13C spiral CSI acquisition,each animal was scanned with T1-weighted protonspin-echo sequence (TE/TR 12/700 ms, 29 slices,

slice thickness 1 mm, number of excitations [NEX] 6, FOV 87 mm, matrix size 256 256, acquisitiontime 9:02 min) both prior to and after injection of0.9 mL of a 1:2 mixture of gadoliniumdiethylene tria-mine pentaacetate (Magnevist, Bayer Schering Pharma)and saline to confirm tumor location and size.

Dichloroacetate Infusion and Histology

13C-MRS of each animal was obtained following a bolusinjection of hyperpolarized [1-13C]Pyr solution beforeand after DCA administration. Beginning 45 min beforethe second scan, 200 mg/kg body weight of sodiumDCA (Sigma-Aldrich) dissolved in saline (30 mg/mL)was injected through the tail vein catheter. About0.5 mL of the DCA solution was injected as a bolus,with the rest infused at a rate of 0.1 mL/3 min.

Data Processing and Analysis

All 13C data sets were first corrected for differencesin po-larization at time of injection estimated from the solid-state polarization level and dissolution-to-injectiondelay using a T1 of 60 s and then processed usingMatLab (Mathworks) as described.35 Briefly, thek-space data were apodized by a 10-Hz Gaussian filterand zero-filled by a factor of 4 in the spectral dimensionand by a factor of 2 in both spatial dimensions. After afast Fourier transform in the time domain, the chemicalshift artifact along the readout direction was removedfollowed by gridding onto a Cartesian grid and a

2-dimensional fast Fourier transform in the 2 spatialfrequency domains. Metabolic maps were calculated byintegrating the corresponding metabolite peaks in ab-sorption mode.

For analysis, the glioma region of interest (ROI) wasselected based on the hyperintense region of thecontrast-enhanced T1-weighted proton images. An ROIof normal-appearing brain was chosen in the contralat-eral hemisphere of the brain. In healthy rats, the ROIswere selected in similar locations to the ROIs of normal-appearing brain in tumor-bearing rats. Metabolite datafor each ROI were evaluated as ratios to total carbon(tC), which was calculated as the sum of the detected

Pyr, Lac, pyruvate hydrate (PyrH), Ala, and Bicsignals. Additionally, the analysis was performed forthe corresponding Lac-to-Bic ratios. For the display ofindividual spectra, a first-order phase correction wasapplied, and the baseline was subtracted by fitting aspline to signal-free regions of each spectrum.

Values are reported as mean+ standard errors.Differences of the Lac/tC, Bic/tC, and Lac/Bic ratiosfrom normal brain and glioma were assessed for statisti-cal significance using a paired Studentst-test unless ex-plicitly stated. Likewise, the metabolic responses ofglioma and normal-appearing brain to DCA were alsoevaluated using a pairedt-test.


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Results

Metabolites in Glioma and Normal-Appearing BrainBefore DCA Infusion

Figure 1A shows the spectra of glioma (red line) andnormal-appearing brain (blue line) from a representativerat (#G1) before DCA infusion. Each spectrum was aver-aged over the voxels within the respective ROIs in theimaged 8-mm axial slice and normalized to the[1-13C]Pyr peak at 173 ppm. [1-13C]Lac and 13C-Bicpeaks, produced from Pyr via Lac dehydrogenase(LDH) and PDH, were detected at 185 and 163 ppm, re-spectively. PyrH at 181 ppm was in dynamic equilibriumwith Pyr and itself not metabolically active. The spectraillustrate the high SNR afforded by hyperpolarization.For example, SNR was 231 for Pyr, 85 for Lac, and 19for Bic in the spectrum from normal-appearing brain.Due to the low Ala aminotransferase concentration inbrain, Ala, which has a chemical shift of 179 ppm, wasundetectable. Increased Lac and decreased Bic of

glioma relative to normal-appearing brain are clearlyevident in the spectra.

Figure 1BD shows the corresponding single time-point metabolic images of Pyr, Lac, and Bic. The posi-tions of the ROIs for glioma and normal-appearingbrain are indicated in Fig.1E. The protruding region atthe skin above the glioma ROI was edema caused bythe tumor cell implantation procedure. Averaged Lacwas 66.1+9.9% higher in the area of tumor, comparedwith the contralateral hemisphere (P .02, n 5). Bycontrast, Bic, mostly from cortex as previously report-ed,32 was 57.3+6.4% lower in glioma than in normal-appearing brain (P .004). Likely due to the increasedperfusion into tumor region, the total carbon signaltrended 16.7+8.7% higher in glioma than in normal-appearing brain (P .06). The hyperintense Pyr signalat the center of the image was from the basilar artery.

Glioma was better differentiated from normal-appearing brain using metabolite ratios. Lac/tC inglioma was 0.44+0.03, whereas it was 0.31+0.02 innormal-appearing brain (P .001). On the otherhand, Bic/tC was 0.018+0.002 in glioma versus0.049+0.004 in normal-appearing brain (P .0002).Lac/Bic was 26.5+3.6 in glioma and 6.4+0.5 innormal-appearing brain (P .002). Lac/tC, Bic/tC,

and Lac/Bic in control rats were comparable to thosevalues in normal-appearing brain in tumor-bearing rats(equal-variance t-test, Ps .5 for Lac/tC, .6 for Pyr/tC, and .2 for Lac/Bic).

Response to DCA

For both pre- and post-DCA infusion, metabolic maps ofLac/tC, Bic/tC, and Lac/Bic from the same animal areshown in Fig. 2. The ratio images were edited toinclude only the brain to provide adequate image con-trast. DCA increased Bic production, measured by Bic/tC, in normal-appearing brain from 0.049+0.004 to

0.1+0.009 (P .002,n 5), whereas Lac/tC was un-changed (0.31+0.02 for pre-DCA, 0.28+0.02 forpost-DCA, P .1). Lac/Bic, which gave the highest con-trast between glioma and normal-appearing brain, de-creased from 6.4+0.5 to 2.9+0.2 (P .0006).Metabolic change in glioma tissue due to the injectedDCA was greater than in normal-appearing tissue.Post-DCA Bic/tC in glioma rose from 0.018+0.002to 0.056+0.005 (P .0008), and post-DCA Lac/tCwas reduced from 0.44+0.03 to 0.39+0.04(P .05). Post-DCA Lac/Bic in glioma recovered to thepre-DCA level of normal brain by decreasing from26.5+3.6 to 7.4+1.1 (P .002). Accordingly, thepost-DCA difference between glioma versus normal-appearing brain became less significant compared withpre-DCA in all the ratio metrics (P .003 for Bic/tC,.008 for Lac/tC, and .005 for Lac/Bic).

Fig. 1. (A) Representative 13C spectra (rat ID: G1) from the ROIs of glioma (red line) and normal-appearing brain (blue line) before DCA

infusion. Each spectrum was normalized by Pyr peak. Metabolic images of (B) Pyr, (C) Lac, and (D) Bic from the rat acquired before

DCA infusion. Higher Lac and lower Bic signals were detected in the glioma region as compared with normal-appearing brain. (E) ROIs

of glioma and normal-appearing brain are shown on the contrast-enhanced T1-weighted proton image.


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The metabolite pattern for the control group wassimilar to the changes in normal-appearing brain in theglioma animals: Bic/tC increased from 0.055+0.002to 0.11+0.003 (P .002), Lac/tC increased from0.27+0.3 to 0.29+0.01 (P .1), and Lac/Bic de-creased from 4.8+0.3 to 2.6+0.08 (P .009).

Detailed results of individual subjects including con-trols are presented in Fig.3A C. The statistical distribu-tions of Lac/tC, Bic/tC, and Lac/Bic are shown inFig. 3DF, with the vertical lines indicating the 95%confidence interval.

The change of total carbon signal due to DCA infu-sion was not significant in either glioma (P .05) ornormal-appearing brain (P .3).

DiscussionLac/Pyr, as measured by hyperpolarized 13C-MRS, hasserved as a common metric to detect GLY-dependenttumor energy metabolism by correlating LDH and Laclabeling.52 However, relative Lac labeling cannot bedirectly used to assess mitochondrial function in tumorcells. On the other hand, Bic production can be usedto monitor PDH activity, which measures Pyr conver-sion to acetyl CoA, so as to potentially serve as a surro-gate marker for cellular mitochondrial metabolism. BothLac and Bic labeling using hyperpolarized [1-13C]Pyr incombination with drug-induced changes in observed

peaks has not been demonstrated previously in any invivo tumor model. However, the primary purpose ofthis work was not to discover a new mechanism bywhich DCA alters tumor metabolism. Rather, the impor-tance of our work is to demonstrate an in vivo imagingtechnique by which changes in energy metabolismfrom GLY toward OXPHOS can be detected and quan-tified. Our results demonstrate for the first time that in

addition to Lac, Bic can be reliably measured both innormal brain and in glioma tissue using an optimizedMR acquisition protocol and high substrate concentra-tion. The effect of DCA on Pyr metabolism was alsoclearly identified by comparing the measured Lac/tCand Bic/tC from normal brain and glioma tissues. Bycontrast, there was no significant change of detectedtotal carbon after DCA infusion in either glioma ornormal brain tissues, suggesting that treatment responseto DCA in balancing metabolic pathways cannot be de-tected by a simple Pyr uptake measurement alone.However, further comparison experiments are necessaryto validate this hypothesis.

Although both Bic/tC and Lac/tC are useful to differ-entiate tumor from normal brain tissue and to assessDCA effects, Lac/Bic was the parameter that was mostsensitive to distinguish glioma from normal brain.More studies of Lac/Bic, or alternatively Bic/Lac, as ametric to evaluate therapeutic responses of othercancers are needed to verify its utility as a biomarkerfor cancer metabolism.

Due to itscapacity to increase PDH flux, DCA has alsoproven efficacious as a Lac-lowering drug in varioustissues,including blood,cerebrospinal fluid, and withina cell.53 Stacpoole et al.41 reported that a single adminis-tration of DCA could decrease Lac by up to 60%, and theeffect was maximized about 10 h after DCA administra-

tion for all tested doses. However, as shown in Fig.3Aand D, Lac labeling did not change significantly inglioma after DCA infusion, which might be due to thehigh dose of infused Pyr that saturated LDH activityeven after PDH activity was elevated or due to the shorttime interval (45 min) between DCA infusion and 13Cimaging. Pyr concentrations at supraphysiologicallevels, typically 2 4 mM at the tissue of interest, are nec-essary in hyperpolarized 13C-labeled Pyr studies toacquire data with adequate SNR, and this representsone of the fundamental limitations of this MRS methodcompared with techniques using tracer doses of injectedsubstrates, such as PET. On the other hand, in combina-

tion with the short time scales associated with the exper-iments, saturating the targeted enzymatic pathways isalso an advantage in that only under such conditionsare the products limited by enzyme activities (theprimary quantities being targeted in these experiments)rather than substrate concentrations. In addition, noadverse hemodynamic event up to 10 mM of plasmaPyr concentration has been reported.54 Using13C-labeled glucose as a substrate instead of Pyr is a po-tential alternative method to observe metabolic reactionto DCA under more physiological conditions.However, the short T1 of

13C-glucose (1 2 s) is highlyproblematic. Recently, metabolic kineticsof hyperpolarized

Fig. 2. Metabolic ratio images of (A) Lac/tC, (B) Bic/tC, and (C)

Lac/Bic from a representative rat, overlaid on contrast-enhanced

T1-weighted 1H-MRI. Overall Bic/tC was increased throughout

the brain, whereas Lac/tC slightly decreased in the tumor-bearing

region after DCA infusion. High contrast between glioma and

normal-appearing brain was visible in the baseline Lac/Bic

images, with this contrast significantly decreasing after DCA

administration.


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[U-2H, U-13C]glucose and products were demonstratedin Escherichia coli,55,56but no invivometabolic productshave been detected thus far in animal models.57

Because of the relatively coarse spatial resolutioncompared with the size of brain tumor, partial volumeaveraging effects were unavoidable in this study, likely

Fig. 3. Summary of metabolic changes in (A) Lac/tC, (B) Bic/tC, and (C) Lac/Bic of all 5 individual animals glioma (solid line) and

normal-appearing brain (dashed line), along with data from the control rats (n 3) shown by the dotted line. Average values of

corresponding parameters from ROIs within the tumor-bearing animals, along with 95% confidence intervals, are presented in (D), (E),

and (F). The differences between normal-appearing brain and tumor for all 3 parameters were significant both before and after

administration of DCA (baseline: P .001, and 0.0002, 0.002; post-DCA: P .008, 0.003, and 0.005 for Lac/tC, Bic/tC, and Lac/Bic,

respectively). With respect to DCA-induced changes, Bic/tC increased in both tumor and normal tissue (P .0008 for glioma, P .002

for normal-appearing brain) as the Lac/Bic ratio (P .002 for glioma, P .0006 for normal-appearing brain) did. In contrast,

DCA-induced changes in Lac/tC ratios were not statistically significant.


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resulting in overestimating Bic and underestimating Lac,especially in rats with small tumors. Therefore, higherSNR would be necessary to reduce the voxel size whilestill being able to reliably detect Bic in tumors. Thiscan be achieved either by using surface coils or improvedpulse sequences or by polarizing at a lower temperatureand/or high magnetic field to increase the polariza-tion.58,59 Alternatively, the higher SNR can be leveraged

to perform dynamic metabolic imaging permitting thecalculation of apparent conversion rate constants thatare less sensitive to experimental parameters such as in-jection time or shape of Pyr bolus than are metaboliteratios.60

Although Bic/tC is a measure of PDH activity, it isnot a direct measure of OXPHOS. Specifically, increasedBic may not necessarily mean increased OXPHOS, asnot all acetyl CoA necessarily enters the TCA cycle.For example, acetyl CoA produced from Pyr might notenter the TCA cycle but instead could be directedtoward other functions, such as fatty acid metabolism.Conversely, low Bic does not necessarily mean low

OXPHOS, as metabolic pathways into the TCA cycleother than oxidative decarboxylation of Pyr via PDHcan contribute relevant molecules.61 One potential strat-egy to assess TCA cycle more directly is to use hyperpo-larized Pyr with the label in the C2 position, since thelabel is then retained in the conversion to acetyl CoA.However, to date, TCA-cycle intermediates have beendetected in the heart when only hyperpolarized[2-13C]Pyr is used in vivo.62 Moreover, the differentialDCA effect between normal-appearing brain andglioma tissues could be affected by the increased perfu-sion of DCA into the tumor because of the disruptionin the blood brain barrier. Nonetheless, MRS measuresof Lac/tC, Bic/tC, and Lac/Bic clearly relate to GLY

and OXPHOS and are likely still useful for measuringin vivo tumor metabolism in both animal modelsandwith increasing availability of polarizers capableof human studiesthe clinic.

We believe the ability to robustly measure such met-abolic shifts in vivo could have major significance in as-sessing the efficacy of multiple antitumor therapies

currently under development that target reversing theWarburg effect as a means of controlling tumorgrowth. For example, the failure of drugs such as DCAin some studies could be due to dosing regimes thatfail to maintain the desired metabolic shift for a suffi-cient duration of time. Observing only Lac, as hasbeen shown in numerous studies of both cancer patientsand animal tumor models, may be inadequate in that

changes in Lac labeling are not necessarily informativewith respect to OXPHOS and the TCA cycle. Futurework will be aimed at assessing the glioma treatmenteffect of DCA in longitudinal studies and estimatingthe appropriate combination of DCA dosage and fre-quency of the treatment. Combining PDH upregulationwith downregulation of LDH activity, which can beachieved by glucose deprivation and ketogenic diets,might also represent an effective treatment for braintumors, which could be monitored using the hyperpolar-ized 13C MRS imaging techniques presented in thisstudy.

Funding

This work was supported by the National Institutes ofHealth (grant nos. P41 EB015891, AA005965, AA018681, AA013521-INIA, EB009070), the Department ofDefense (grant no. PC100427), the Lucas Foundation,and Nadias Gift Foundation.

Acknowledgments

We express our appreciation to GE Healthcare forresearch support. We also thank William Hawkes and

Shara Vinco for assistance with the animal handlingand experiment arrangements.

Conflict of interest statement. Yi-Fen Yen and RalphE. Hurd were employees of GE Healthcare at the timeof this study.

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metabolic response of glioma to dichloroacetate measured in vivo

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