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[Cancer Research 61, 5595-5600, July 15, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

High Glycolytic Activity in Rat Glioma Demonstrated in Vivo by Correlation Peak 1H Magnetic Resonance Imaging1

Anne Ziegler2, Markus von Kienlin3, Michel Décorps and Chantal Rémy

Unité mixte Institut National de la Santé et de la Recherche Médicale/Université Joseph Fourier: Unité 438 "RMN Bioclinique," Laboratoire de Recherche Correspondant du Commissariat à l’Energie Atomique, Centre Hospitalier Universitaire BP 217, 38043 Grenoble, France [A. Z., M. D., C. R.], and Physikalisches Institut V, Universität Würzburg, 97074 Würzburg, Germany [M. v. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High-grade brain tumors are known to have a high rate of glucose (Glc) consumption. Postmortem measurements have suggested that Glc content in experimental brain tumors is relatively low. We used magnetic resonance spectroscopy to investigate this, in vivo, in the brains of seven rats bearing intracerebral C6 gliomas. We combined the high spectral resolution allowed by two-dimensional proton nuclear magnetic resonance with spatial encoding by magnetic field gradient pulses to obtain in vivo maps of Glc, alanine, hypotaurine, aspartate, phosphoethanolamine, Glu/Gln, N-acetylaspartate (NAA), phosphocreatine/creatine (PCr/Cr), choline-containing compounds, and lactate (Lac) (some of which are involved in energy metabolism). Compared with normal brain tissue, the main differences found in the gliomas were that Glc, NAA, PCr/Cr, and aspartate concentrations were much lower, whereas concentrations of alanine, hypotaurine, phosphoethanolamine, and Lac were higher, whatever the extent of necrosis. A striking observation is the similarity of the NAA and Glc images: the concentrations of both metabolites are lower in the tumor than they are in the contralateral brain. If Glc was completely absent from the tumor tissue, and if the residual Glc level was due only to a partial volume effect like that for NAA, a neuronal marker, the ratio [Glc]tumor/[Glc]contralateral tissue, should be similar to that found for NAA. The ratio for Glc was 0.48 ± 0.22 (± SD; n = 6), a ratio similar to that found for PCr/Cr (0.50 ± 0.19) but significantly higher than that obtained for NAA (0.29 ± 0.07). This observation indicates that a measurable Glc concentration is still present in the tumor tissue. Intense glycolysis in tumor cells may explain the increased production of Lac and alanine and decreased amount of Glc. These nuclear magnetic resonance measurements of metabolite concentrations are complementary to positron emission tomography, which measures Glc consumption.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A predominant biochemical characteristic of rapidly growing tumors is their glycolytic activity and high Glc4 consumption rate (1, 2, 3) . Brain Glc consumption can be mapped in vivo in humans using PET after administration of FDG. After transport of FDG from the blood to the brain, FDG is phosphorylated into FDG-6-phosphate by hexokinase. Because no further metabolism of this compound occurs, and because the cells are impermeable to FDG-6-phosphate, the Glc consumption is reflected by the accumulation of FDG-6-phosphate. Di Chiro et al. (4) demonstrated that Glc consumption in brain tumors is correlated with histological grade. Since this initial work, PET has emerged as a reliable method for differentiating benign from malignant primary tumors and for differentiating radiation necrosis from tumor recurrence (5, 6, 7, 8, 9, 10) .

Glucose concentration in tumor tissue is a distinct indicator, which has the potential to improve differential diagnosis, biopsy guidance, or therapy monitoring. Glucose concentration can be determined by enzymatic measurements on postmortem tissue samples dissected under liquid nitrogen (11) , and its distribution in brain slices can be determined by bioluminescence methods (12) . However, both techniques can be applied only to animals or human biopsies and are sensitive to postmortem artifacts. Recent advances in MRS (13) now make possible imaging of the Glc content in vivo in the brain.

In vitro studies (14 , 15) suggest that the high Glc consumption demonstrated by PET techniques in high-grade brain tumors is accompanied by a relatively low Glc content. Our goal was to use MRS imaging of Glc to verify this, in vivo, in the brains of seven rats bearing a C6 intracerebral glioma and to compare Glc concentration in the tumor with that in the contralateral hemisphere. In addition to Glc mapping, the technique used in this work offers the advantage of simultaneous imaging of many other metabolites, such as Lac, alanine, aspartate, HTau, and PE, some of which are involved in energy metabolism.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tumor Model.
Tumors were induced in seven female Sprague Dawley rats by stereotactic injection of 104 C6 glioma cells (16) , 3 mm under the dura, into the striatum (17) . Animal care and all experiments performed on the animals conformed to the guidelines of the French Government (Decree Number 87-848, October 19, 1987, Licenses 006722 and A38071). NMR examinations were performed 24–27 days after cell implantation. Throughout the NMR experiment, the rat was anesthetized with 1.5% isoflurane in oxygen (30 ml/min), and a warming pad was used to avoid hypothermia. After the NMR examinations, the rats were perfused with a 2.5% glutaraldehyde solution in PBS by intracardiac injection under anesthesia. The brain was excised, fixed in a 10% formaldehyde solution in saline, and embedded in paraffin. Three-µm horizontal sections of the brain were stained with H&E-Safran.

NMR Experiments.
Experiments were performed on a Bruker Biospec 7 Tesla/20 cm bore instrument with microimaging accessories. The system included an actively shielded gradient coil assembly. We used a homemade stereotactic probe equipped with a single-turn surface coil (diameter, 17 mm) for transmission and reception. Scout proton images of the rat brain were obtained with a modified FLASH sequence (Ref. 18 ; echo time, 12 ms; one accumulation per encoding step; slice thickness, 2 mm; image matrix, 128 x 128; field of view, 3 x 3 cm). These images were used to localize the tumor and to define the position of the slab in which the subsequent CPI experiment was performed.

The CPI technique and the underlying theory have been described in detail elsewhere (13) . In 2D NMR experiments, a technique that is widely used in chemistry and biochemistry, the molecules are identified by their correlation-peak patterns (i.e., off-diagonal resonances in the 2D frequency map), which are characteristic for scalar coupled spins within the molecule. CPI combines the high spectral resolution allowed by 2D homonuclear correlation spectroscopy with spatial encoding by magnetic field gradient pulses. The acquisition and processing parameters have been described extensively by von Kienlin et al. (13) . Briefly, k-space was covered by 13 x 13 phase encoding steps weighted by a Hanning window. The field of view was 30 x 30 mm, and the slice thickness was 5 mm, resulting in a nominal voxel volume of 75 µl. Both spectral bandwidths were 1562 Hz, sampled by 256 complex data points in the t2 dimension and 64 data points in the t1 dimension. Total acquisition time was 195 min. Data processing was performed on UNIX workstations with home-written software in the IDL programming environment (Interactive Data Language; Research Systems International, Boulder CO). In the t2 dimension, we applied a Gaussian filter, and in the t1 dimension, we applied a squared sinebell filter. After filtering, zero-filling in the spectral dimensions and Four-dimensional-Fourier transformation, a magnitude calculation of the data was performed. Thus, a 2D correlation map was obtained in each of the voxels of the slice. The diagonal peaks and cross-peaks (spots off the diagonal line) were assigned according to Refs. 19 and 20 . In each voxel and for each metabolite, the intensities of the cross-peaks and/or diagonal peaks were measured. Images of metabolite intensities were reconstructed from the cross-peaks for the scalar coupled proton spins or from the diagonal peaks for noncoupled spins. The noise level was measured in a region of the 2D correlation map free of resonance peaks, and a noise image was reconstructed. For all images (metabolites and noise), the spatial matrix was Fourier interpolated to a 256 x 256 matrix.

Intensity measurements were carried out in two ROIs of the metabolic images located in the center of the tumor and in the contralateral hemisphere, respectively (ROI areas = 10 ± 1.3 mm2). Measurements were performed on seven animals, except for measurements of Glc, Ala, HTau, and PE (n = 6): the latter metabolites could not be determined in one of the rats because of a hemorrhage. The mean intensity in each ROI has been normalized to the mean NAA diagonal peak intensity measured in the contralateral ROI. The one-sided nonparametric Wilcoxon test was used to compare the metabolite intensities in the tumor and in the contralateral hemisphere (P = 0.05). To make comparisons between metabolites, the ratio Rx = [x]tumor/[x]contralateral tissue was used.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fig. 1Citation shows typical spectra from voxels located in normal brain tissue, in the center of the tumor, and in a region toward the periphery of the tumor close to normal tissue for a rat brain with a necrotic core tumor. The cross-peaks were assigned to Glc, Lac, Glu/Gln, Ala, Asp, NAA, Tau, HTau, Cho/inositol, and PE. The signal to noise ratio for the Glc cross-peak at (5.3 ppm; 3.5 ppm) was around 16 in the normal brain (maximum cross-peak intensity/noise SD measured before magnitude calculation).



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  		Fig. 1. 2D spectra obtained with CPI on a rat brain bearing a tumor (rat 4) from a voxel localized (a) in the normal brain tissue contralateral to the tumor, (b) in the center of the tumor, and (c) in a region of the tumor close to the normal tissue. Assignments of the cross-peaks are as follows: 1, NAA; 2, Glu/Gln; 3, Glc; 4, Asp; 5, Tau; 6, Cho and inositol; 7, Lac; 8, Ala; 9, HTau; 10, PE. The effective voxel volume for each spectrum is 75 µl.

 
Fig. 2Citation shows the spatial distribution of various metabolites for the same animal as in Fig. 1Citation . They were measured from the different cross-peaks, except for the spatial distributions of NAA, Cho-containing compounds (tCho), and PCr/Cr, which were obtained from the diagonal peaks. The normalized mean signal intensity values and the SDs found in tumor and contralateral ROIs are summarized in Fig. 3Citation .



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  		Fig. 2. Metabolic maps for rat 4. The two ROIs used for intensity measurements are delineated (black circles) on the proton NMR anatomical image (field of view, 30 x 30 mm2; slice thickness, 2 mm). The contours of the brain and the tumor were delineated on the anatomical image and superimposed on the metabolic images. The metabolic maps were reconstructed from the CPI experiment (slice thickness, 5 mm). NAA, PCr/Cr, and tCho were obtained from the diagonal peaks, and NAA, Glc, Lac, Ala, HTau, PE, Asp, and Glu/Gln were obtained from the cross-peaks. The color scale of each map was adjusted to obtain a maximum dynamic range (higher metabolite level is yellow). Hence, peak intensities from different metabolites in this figure cannot be compared directly.

 


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  		Fig. 3. Normalized metabolite intensities in the tumor ROI and in a ROI located in the contralateral brain. The mean intensity in each ROI has been normalized to the mean NAA diagonal peak intensity measured in the contralateral ROI. Data are presented as the mean ± SD of n = 7 rats, with the exception of values for Ala, Glc, PE, and Htau, which are presented as the mean ± SD of n = 6 due to the presence of a hemorrhage in one rat. DP indicates that the measurement was carried out on a diagonal peak instead of a cross-peak (displayed intensities were then divided by 10). Note that the results obtained with the NAA diagonal peak are similar to those obtained with the cross-peak, confirming the robustness of the technique (see also Fig. 2)Citation . For all metabolites except Cho, a significant difference was found between tumor and contralateral brain tissue (*, P < 0.05 as determined by one-sided nonparametric Wilcoxon test).

 
Note that, as expected, the NAA intensity measurements yield similar results for the diagonal peaks and cross-peaks. Lac was detected mainly within the tumor, and NAA and PCr/Cr were detected mainly in normal brain tissue (21, 22, 23, 24, 25, 26) . The Glc signal intensity was significantly lower in the tumor than in the contralateral tissue, as were those for aspartate and Glu/Gln. The Glc ratio (RGlc = 0.48 ± 0.22) was similar to that found for PCr/Cr (RPCr/Cr = 0.50 ± 0.19) but significantly higher than that obtained for the NAA cross-peak (RNAA = 0.29 ± 0.07; Wilcoxon test, P = 0.05). In contrast, higher amounts of PE were found in the tumor than in the contralateral hemisphere (RPE = 1.48 ± 0.65). HTau and alanine were found only in the tumor (in the contralateral ROI, no significant difference was found between HTau or alanine and the noise level). tCho was not significantly different between the tumor ROI and that in the contralateral tissue.

As shown in Fig. 4Citation , the characteristics of the regional distributions of Glc, NAA, and Lac were essentially the same in all of the rats. Some of the tumors were necrotic (animals 2, 4, and 5); others showed only small necrotic foci (animals 1, 3, and 6). A hemorrhage was detected in rat 7 (see the anatomical image and histological slice, Fig. 4Citation ).



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  		Fig. 4. Glucose, NAA and Lac distribution observed in each of the seven rats. Row a, 1H NMR anatomical images. The contour of the brain and that of the tumor were delineated on these images and superimposed on the metabolite maps. The large black spot in the tumor of animal 7 is caused by a hemorrhage. Rows b-d, metabolic maps reconstructed from the CPI experiments for Glc (b), NAA (c), and Lac (d). Row e, H&E-Safran-stained histological sections.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown in this work that in vivo metabolic imaging of Glc and simultaneous imaging of many other metabolites could be carried out using MRS. We used a 2D technique, which offers the advantage of an improved spectral resolution. However, other approaches using one-dimensional rather than 2D MRS can be used. Several attempts have been made to obtain information about brain Glc concentration from the analysis of the 3.44 ppm Glc peak observed by 1H MRS in one-dimensional spectra (27, 28, 29) . However, due to spectral overlap with the myo-inositol and Tau resonances, quantification of this resonance is difficult (30) . Gruetter et al. (31) demonstrated the feasibility of noninvasive quantification of Glc in the human brain using the 5.23 ppm peak, which is well resolved at high magnetic field (4 Tesla). The close proximity of this peak to the water peak (4.8 ppm) may make its observation difficult under routine conditions. Spectral editing techniques have been proposed for Glc detection (32 , 33) and might be used for Glc imaging. In contrast, 2D techniques offer the advantage of simultaneous acquisition of the signals of many metabolites together with Glc, at the expense of an increased experiment time.

The NAA and Glc images were similar in general; the concentrations of both species were lower in the tumor than in the contralateral brain. However, minor differences in the regional distributions were observed; for instance, NAA was detected in the lobe to the lower right of the tumor in Fig. 2Citation , where no Glc was detected. Antibodies to NAA show that it is present only in neurons (34) , and, as expected, only low concentrations have been detected in excised brain tumors and in glioma cells by either chemical analysis (35) or MRS (24 , 36 , 37) . The NAA detected in the tumor is probably due to partial volume effects with healthy tissue. The similarity of the spatial distribution of Glc with that of NAA (Fig. 4)Citation might also be due to partial volume effects and therefore might suggest the absence of Glc in the tumor. However, the reduction of Glc (RGlc = 0.48 ± 0.22) and PCr/Cr (RPCr/Cr = 0.50 ± 0.19) signal in the tumor with respect to the contralateral healthy tissue was less than that of the NAA (RNAA = 0.29 ± 0.07). This result indicates that small but real Glc (and PCr/Cr) concentrations are present in the tumor tissue. Interestingly, the reduction of the Glc concentration was more marked in highly necrotic tumors (RGlc = 0.36 ± 0.15 for rats 2, 4, and 5) than in tumors with small necrotic foci (RGlc = 0.60 ± 0.25 for rats 1, 3, and 6), whereas the NAA ratio was similar in the two groups (RNAA = 0.31 ± 0.07 and 0.28 ± 0.08, respectively). This reinforces the assumption about the presence of Glc in the region of viable tumor cells and highlights the advantage of simultaneous acquisition of images of various metabolites.

The low concentration of Glc in the tumor region demonstrated in vivo in the present study was previously shown in vitro in animal models. Bioluminescence imaging techniques showed that the Glc content of brain slices in rats or cats bearing intracerebral tumors was smaller in the periphery of the tumor than in normal brain, whereas Glc was absent in necrotic regions (14 , 15 , 38) . In F98 gliomas in the rat, the Glc concentration was slightly lower in the solid region of tumors (2.41 ± 0.38 µmol/g wet weight) than in the brain contralateral to the tumor (2.77 ± 0.12 µmol/g in the cortex; Ref. 14 ). A high rate of Glc consumption has been demonstrated in human malignant gliomas by FDG PET studies (5) . The low Glc concentration in the hypoxic and necrotic areas of the tumors might be due to low perfusion and inadequate Glc supply to hypoxic and degenerating cells. The low Glc level in adequately perfused tumor areas might be due to intense glycolysis by proliferating cells.

Pyruvate, the end product of glycolysis, is a major branch point of energy metabolism. In the present work, we detected two of its possible immediate products, Lac and alanine, which may provide additional information about the energy metabolism in tumor. Lac was found in the tumors of all seven animals, despite their different histopathological profiles (Fig. 4)Citation . Lac appeared to be distributed relatively homogeneously over the whole tumor in the presence of large necrotic areas as well as that of small necrotic foci. Thus, in addition to the high glycolytic activity of glioma cells in hypoxic regions and the subsequent Lac accumulation in poorly perfused necrotic areas, Lac also accumulates in regions that are probably adequately perfused (1) . This is in agreement with the NMR data from Terpstra et al. (39) . These authors found that Lac concentration in the C6 glioma model was correlated with neither the extent of necrosis, the extent of inflammatory cell infiltration, nor hypoxia. The high concentration of Lac detected in the present study in adequately perfused tumor might be explained by the high glycolytic activity of the glioma cells (1 , 2) . An alternative cause might be the glial origin of these tumor cells: a high aerobic glycolysis has been found in astrocytes in culture (for review, see Ref. 40 ). The distribution of alanine was very close to that of Lac. This increased alanine concentration in the tumor is in agreement with in vitro results: the alanine concentration has been found to be much higher in brain tumor extracts than in normal human or rat brain extracts (20 , 24 , 25) . The alanine level in normal brain is below the in vivo MRS detection threshold. On the other hand, alanine has been detected in vivo in some meningiomas (41 , 42) . As for Lac, increased glycolytic activity in tumor cells might explain the higher alanine level.

The increased glycolytic flux in tumor cells can be explained by an up-regulation of several glycolytic enzymes (43 , 44) including the fetal isoform of hexokinase (45 , 46) . This hexokinase has a higher affinity for Glc and is bound to mitochondria, which leads to a higher glycolysis rate. It should be noted that although glycolysis is increased in tumors compared to normal tissue, the oxidative metabolism still constitutes the major energy source (47, 48, 49, 50) .

The explanation of the distribution of Glu/Gln is more speculative because these two compounds cannot be resolved by CPI. In normal brain, Glu is the most important compound, whereas in gliomas, the concentration of Gln is higher and that of Glu is lower (24 , 37) . The decrease of Glu in tumors reflects the fact that this neurotransmitter is stored mainly in neuronal synaptic vesicles (50) . Glutamine may serve as an energy fuel through glutaminolyse (51) in tumor cells and is also involved in anabolic pathways. Cultured C6 cells are not able to synthesize Gln due to the lack of glutamine synthetase activity (52) . Bouzier et al. (50) showed that Glu necessary for C6 glioma growth in vivo is taken up from the blood and is used rather for anabolic needs than as an oxidative substrate.

Aspartate was detected only in normal rat brain. A low level of Asp was also described in human brain tumor extracts by Peeling and Sutherland (24) . The decrease of Asp in tumor might be due to the fact that it is a neurotransmitter located mainly in neuronal vesicles.

HTau and PE were detected in the tumor but not in the contralateral brain (Fig. 2)Citation . HTau had already been detected by 1H MRS or biochemical analysis in meningioma and meningeal cells (53) , in glial cells (37 , 54) , in cells of human brain tumor (37) , and in perchloric acid extracts of rat brain tumor and in excised rat brain tumor (20) . However, to our knowledge, HTau was never detected previously in situ and in vivo. Biochemical studies (55) showed that the HTau concentration in normal rat brain is about 0.04 µmol/g wet weight, which is below the detection threshold of MRS. HTau is synthesized only in glial cells (54 , 56) , which may explain its detection in tumor tissue of glial origin. HTau might also be a marker of dedifferentiation because it was found at higher concentrations in 0–2A progenitor cells than in astrocytes and oligodendrocytes (57) . The role of HTau is not clear. It has been suggested that it acts as a free radical scavenger, possibly protecting neuronal membranes from oxidation (56) . Similarly, PE was detected mainly in the tumor. This is in agreement with the data obtained on extracts of water-soluble metabolites from human brain tumors (25) and rat brain gliomas (20) . The PE content of human gliomas has been found to correlate with malignancy (25) . The higher PE level in the tumors might be explained by an activation of the phospholipid metabolism playing a role in the modification of the tumor cell membrane and/or by the breakdown by phospholipase C of phospholipids to diacylglycerol acting as a second messenger system for cellular proliferation (25) . Surprisingly, we did not find any significant difference in Cho-containing compounds between tumor and contralateral brain. It is generally well recognized that the tCho signal reflects cellular proliferation, resulting in a higher Cho-containing compound content in the tumor (58) . However, in necrotic areas, the Cho content can be lower than that in normal brain (25) . The lack of significant tCho difference between tumor and contralateral brain probably results from tissue heterogeneity in the tumor ROI.

With a CPI technique, as with most of the proton MRS approaches, signals are weighted by the transverse relaxation time (T2) and longitudinal relaxation time (T1). Moreover, with CPI, the cross-peaks depend on concentration and on the geometry of the spin system that gives rise to the peaks, making absolute concentration measurements difficult. In this work, the spectroscopic data have been quantified using ratios rather than absolute intensity values. Thus, our results reflect spatial changes in concentrations. Nevertheless, for each metabolite, different T1 or T2 in different tissues could influence the observed spatial changes.

Spatial distributions of several metabolites involved in energy metabolism have been obtained in vivo in rat brain gliomas. The higher level of Lac and alanine in tumor compared to contralateral tissue demonstrated a high glycolytic activity in the tumor. This high glycolytic activity is associated with a relatively low Glc content.


   ACKNOWLEDGMENTS
 
We thank Régine Farion for technical assistance in animal preparation, Christophe Rubin for contributing to the development of the image processing program, Dr. Jonathan Coles for critical reading of the manuscript, and Prof. Axel Haase for providing access to the NMR facilities.


   FOOTNOTES
 
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.

1 Supported by the French-German exchange program PROCOPE, the Ligue Nationale Contre le Cancer, the Association de la Recherche contre le Cancer, the Fondation pour la Recherche Médicale, the Région Rhône-Alpes, the European Union (Biomed Program BMH4-CT 96-0861), and Deutsche Forschungsgemeinschaft Grants Ki 433/2-2 and Ki 433/2-3 (to M. v. K.). Back

2 To whom correspondence should be addressed, at INSERM U438, CHU Pavillon B, BP 217, 38043 Grenoble Cedex 9, France. Back

3 Present address: F. Hoffmann-La Roche AG, PRBT-S Building 68/05, CH-4070 Basel, Switzerland. Back

4 The abbreviations used are: Glc, glucose; PET, positron emission tomography; FDG, 18F-deoxyglucose; CPI, correlation-peak imaging; MRS, magnetic resonance spectroscopy; 2D, two-dimensional; ROI, region of interest; Lac, lactate; NAA, N-acetylaspartate; Tau, taurine; HTau, hypotaurine; Cho, choline; PE, phosphoethanolamine; tCho, total choline; PCr, phosphocreatine; Cr, creatine; Ala, alanine; Asp; aspartate; Glu, glutamate; Gln, glutamine. Back

Received 1/ 5/01. Accepted 5/16/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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