TRANSLATIONAL PHYSIOLOGY
Glycolysis as a metabolic marker in orthotopic breast cancer, monitored by in vivo 13C MRS

Dalia Rivenzon-Segal, Raanan Margalit, and Hadassa Degani

Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Enhanced glycolysis represents a striking feature of cancers and can therefore serve to indicate a malignant transformation. We have developed a noninvasive, quantitative method to characterize tumor glycolysis by monitoring 13C-labeled glucose and lactate with magnetic resonance spectroscopy. This method was applied in MCF7 human breast cancer implanted in the mammary gland of female CD1-NU mice and was further employed to assess tumor response to hormonal manipulation with the antiestrogen tamoxifen. Analysis of the kinetic data based on a unique physiological-metabolic model yielded the rate parameters of glycolysis, glucose perfusion, and lactate clearance in the tumor, as well as glucose pharmacokinetics in the plasma. Treatment with tamoxifen induced a twofold reduction in the rate of glycolysis and of lactate clearance but did not affect the other parameters. This metabolic monitoring can thus serve to evaluate the efficacy of new selective estrogen receptor modulators and may be further extended to improve diagnosis and prognosis of breast cancer.

magnetic resonance spectroscopy; [13C]glucose; [13C]lactate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

GLUCOSE, AN ESSENTIAL COMPOUND for many living organisms, is an important carbon source for energy-producing metabolic processes and for the synthesis of low molecular weight and macromolecular products. For more than 70 years, it has been known that cancer cells utilize and metabolize glucose at high rates, even in the presence of high oxygen concentrations, to form mainly lactate (36). Indeed, lactate was found to be present in tumors at levels much higher than in the corresponding normal tissues (9, 15, 16, 25). Furthermore, quantitative studies of biopsies from human cancers have indicated a positive correlation between tumor lactate concentration and incidence of metastasis (5, 26, 34, 35).

Increased glycolysis is associated with higher glucose uptake and metabolism, and it is now utilized as an indicator of malignancy by applying positron emission tomography (PET) with the glucose analog 2-[18F]fluoro-2-deoxy-D-glucose (FDG) (2, 23, 29, 33). FDG-PET has also been used for prognostic evaluation, for monitoring tumor therapy, and for early detection of recurrent cancer growth (2, 29, 33).

13C magnetic resonance spectroscopy (MRS) with use of enriched [13C]glucose can also serve to monitor in vivo glucose metabolism. Specifically, it monitors glucose uptake and consumption, and it traces other metabolites into which the 13C label is incorporated, including lactate (28). Detailed kinetic MRS studies of 13C-labeled glucose consumption and lactate synthesis were previously performed in cultured breast cancer cells (12, 20, 21, 24). Similar in vivo MRS studies of tumors in animal models focused on evaluating lactate clearance (30) or lactate synthesis and clearance (3, 22). Here, we describe a novel dynamic in vivo 13C MRS study of both glucose metabolism and lactate synthesis in orthotopic MCF7 human breast tumors. The time courses of both [1-13C]glucose and [3-13C]lactate were simultaneously measured and further analyzed by a physiological model, yielding the tumor kinetic parameters of glycolysis, glucose perfusion and lactate clearance. Furthermore, changes in these parameters served to assess the response of the tumors to tamoxifen, the leading antiestrogenic drug in breast cancer management. The results demonstrated the capability of 13C MRS to characterize noninvasively cancer growth and response to treatment.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Animals and tumors. MCF7 human breast cancer cells (~8 × 106 cells in 0.5 ml phosphate-buffered saline solution) were inoculated subcutaneously into the mammary glands of female CD1-NU immunodeficient mice. Before the injection of the cells, a pellet of 17beta -estradiol (0.72 mg/pellet, 60 days release time, Innovative Research of America, Sarasota, FL) was implanted under the skin in the back of the mice. Treatment with tamoxifen was applied by replacing the estrogen pellet with a tamoxifen pellet (5 mg/pellet, 21 days release time, Innovative Research of America). The tumors were investigated 4-6 wk after cell implantation. Before the 13C MRS measurements, the mice were maintained without solid food for 5 h.

In vivo glucose metabolism was measured in 15 tumors implanted in 15 different mice. Percent 13C labeling of glucose and lactate was determined separately in six other tumors after extraction. Percent 13C labeling of glucose and lactate in the plasma was determined separately in plasma samples from a different set of 15 mice. All results are presented as means ± SE. For statistical evaluation, we used a paired two-tailed Student's t-test, unless otherwise stated.

Animal protocols and maintenance were in accord with the guidelines of the Committee on Animals of the Weizmann Institute of Science and were approved by this committee.

Anesthesia. The mice were anesthetized by intraperitoneal injection of 0.06 mg pentobarbital sodium/g body wt. The effect of the anesthetic protocol on blood glucose was evaluated by measuring glucose levels with a blood glucometer (Medisense) in samples of blood (~30 µl) drawn at various times by retroorbital sinus puncture. Blood glucose levels declined very slowly (25% decline at 60 min). Another common anesthetic procedure of a combined intraperitoneal injection of ketamine · HCl (85 µg/g wt, Ketaset, Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (20 µg/g wt, Bayer, Leverkusen, Germany) was found to increase normal glucose levels by two- to threefold and was therefore unsuitable for this metabolic study.

Glucose infusion. After a 5-h fast, mice were anesthetized as described in Anesthesia, and D-[1-13C]glucose (99% enriched, Cambridge Isotope Laboratories, Andover, MA) was infused into the tail vein at a constant rate, 1 ml of 0.3 M solution in 10 min, by use of an infusion pump.

In vivo 1H imaging and 13C spectroscopy. In vivo measurements were performed on a 4.7-Tesla spectrometer (Biospec, Bruker) with a horizontal bore (30-cm) magnet. 1H images were recorded at 200 MHz with a 7.5-cm 1H resonator (Bruker). Initially, T2-weighted axial images of the entire tumor were recorded by use of a multislice spin echo sequence with an echo time (TE) of 68 ms, a repetition time (TR) of 2,400 ms, a slice thickness of 1 mm, a field of view of 5 cm, and a 256 × 256 matrix with four averages. The fraction of necrosis in each tumor was estimated by analyzing the T2-weighted images, as previously described (13).

13C spectra were recorded at 50 MHz with a home-built 13C surface coil (diameter 10 mm) for transmission and reception, placed within the 7.5-cm 1H resonator for decoupling. Each mouse was placed in the magnet with the tumor positioned at the center of the 13C surface coil. 13C spectra were recorded after localized shimming was performed with point-resolved spectroscopy (PRESS) (4); 300 transients were acquired with a 90° adiabatic pulse excitation (sin/cos), a 1-s TR, and a bi-level composite pulse proton irradiation, high only during acquisition, to reduce heating effects. Successive 13C spectra, each for 5 min, were recorded before and during the 13C-enriched glucose infusion period (10 min) and thereafter for 60-80 additional minutes. Conversion of the integrated area of the 13C-1 glucose signals to concentration units was performed using a calibration curve obtained from spectra of increasing concentrations of [1-13C]glucose in a phantom, recorded in the same way as the in vivo spectra. The calculated 13C-enriched glucose concentration served to calibrate lactate concentration by use of the integrated area ratio of lactate to glucose, with a correction factor of 1.5 to account for differences in T1, nuclear Overhauser effect (NOE), and imperfection of the adiabatic pulse.

The in vivo time course data of [1-13C]glucose concentration and [3-13C]lactate concentration in each tumor were fitted using a nonlinear least square algorithm (Levenberg-Marquardt) to model-based equations (see APPENDIX). The quality of the fitting was assessed by calculating the correlation coefficient (R).

Measurement of enriched [13C]glucose and [13C]lactate in plasma. At various times after termination of the infusion, blood samples (one sample of ~1 ml/mouse, no. of mice = 15) were drawn by retroorbital sinus puncture and transferred to ice-cold tubes containing 100 µl of heparin (Elkins-Sinn, Cherry Hill, NJ) at a concentration of 50 U/ml. The blood samples were centrifuged for 10 min at 9,000 g, and the supernatant was separated and diluted with 2H2O to a volume of 500 µl. Methanol (18 µl) was added as a concentration reference.

13C spectra of the plasma samples were recorded using a Bruker DMX-500 spectrometer equipped with a 13C-1H double-tuned probe. The spectra were acquired by 1,800 scans, a 60° pulse, a 2-s TR, and composite pulse proton decoupling (1 W). The fraction of 13C enrichment of glucose in plasma was determined by referencing the integrated area of the enriched 13C-1 glucose to that of 13C-4 glucose at natural abundance (1.108%). The absolute concentration of enriched [1-13C]glucose was determined in reference to the integrated area of a known concentration of methanol at natural abundance (1.108%), with a correction for differences in T1 and NOE. The concentration of 13C-3 lactate and percent labeling were calculated by referencing the integrated area of enriched 13C-3 lactate and 13C-2 lactate signals at natural abundance to the [13C]methanol signal at natural abundance, corrected as for glucose.

The decay of [1-13C]glucose concentration in the plasma was fitted to an equation that describes blood glucose pharmacokinetics by use of a nonlinear least square algorithm (Levenberg-Marquardt).

Tumor extraction. Tumors were resected before and at various times after termination of the [1-13C]glucose infusion. The tumors were immersed instantly in liquid nitrogen and maintained at -80°C. The water-soluble metabolites were extracted using the dual-phase method (32). Before the NMR experiment, the lyophilized extracted samples were dissolved in 500 µl of 2H2O, and 20 µl of methanol were added as a concentration reference. 13C NMR spectra of these extracts were recorded on a Bruker DMX-500 MHz spectrometer with the same probe and parameters as for the plasma samples. Glucose and lactate concentrations and their respective percent labeling were calculated as described above for the plasma samples.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

In vivo studies. Glucose metabolism to lactate was monitored in vivo in 15 MCF7 human breast tumors, each implanted orthotopically in a separate mouse. Initially, the tumors were characterized by high-resolution MR imaging (MRI) by use of a T2-weighted sequence, as demonstrated in Fig. 1. Analysis of the images yielded a measure of the entire volume of each tumor and estimation of the percent necrosis, as previously described (13). The volume of the tumors ranged from 0.35 to 1.5 cm3, with a mean of 0.84 ± 0.1 cm3. Percent necrosis ranged from 1 to 5%, demonstrating the predominance of viable tissue in all of the tumors.


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Fig. 1.   1H magnetic resonance imaging (MRI) of orthotopic MCF7 human breast tumor. a: Image of a central sagittal slice with consecutive axial slices assigned through the entire tumor, which is circled in white. b-g: T2 weighted, spin echo, axial images. Images of every second slice are presented, with the tumor margins circumscribed in white. Bar size, 0.75 cm. Images were recorded as described in MATERIALS AND METHODS.

Sequential 13C spectra of the tumors were recorded before, during, and after termination of [1-13C]glucose infusion. Before the infusion, only acyl-chain residues of lipids, at natural abundance, were detected. However, after the infusion, signals of 13C-1 glucose and of 13C-3 lactate clearly emerged in the spectra (Fig. 2). The time course revealed an initial increase in the alpha  and beta  13C-1 glucose signals during the infusion, followed by a decrease after its termination (Fig. 3). The 13C-3 lactate signal in the tumors reached detectable levels 10-15 min after termination of the infusion and continued to increase for ~50 min, to approach a steady level (Fig. 3).


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Fig. 2.   In vivo 13C spectra of orthotopic MCF7 human breast tumor after infusion of [1-13C]glucose. A and B: spectra were recorded 10 and 70 min, respectively, after termination of the infusion of [1-13C]glucose (1 ml of 0.3 M within 10 min) into the tail vein. Spectra were measured for 5 min each, as described in MATERIALS AND METHODS, and processed using a Gaussian multiplication (exponential factor of -40 and a Gaussian factor of 0.05).



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Fig. 3.   Changes in tumor concentration of [1-13C]glucose and [3-13C]lactate in orthotopic MCF7 human breast cancer during and after infusion of [1-13C]glucose. , Time course of concentration of [1-13C]glucose measured in the tumor with the curve through the data points obtained from the best fit to Eq. A3, yielding kg = 3.3 × 10-2/min, upsilon  = 0.36, a1 = 62 mM, kP = 2.9 × 10-2/min, with R = 0.99. , Time course of concentration of [3-13C]lactate measured in the tumor, with the curve through the data points obtained from the best fit to Eq. A5, yielding Vgly = 48 µM/min, kl = 2.1 × 10-2/min, with R = 0.93.

Quantitative analysis of the time courses of [1-13C]glucose concentration in the tumor, C<UP><SUB>g</SUB><SUP>*</SUP></UP>, and [3-13C]lactate concentration in the tumor, C<UP><SUB>l</SUB><SUP>*</SUP></UP>, was based on the mathematical model described in the APPENDIX. The kinetic data were fitted to Eqs. A3 and A5, respectively (see APPENDIX). This yielded the rate constants of glucose pharmacokinetics in the plasma (a1, kp) and of perfusion in the tumor (kg), and of lactate clearance (kl), as well as the rate of [3-13C]lactate synthesis via glycolysis (Vgly). These parameters converged to similar values, and their means are presented in Table 1.

                              
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Table 1.   Kinetic parameters of glucose perfusion and metabolism derived from time courses of [1-13C]glucose and [3-13C]lactate

Studies of plasma and tumor extracts. Validation of the model and the assumptions was obtained from independent measurements of the blood [1-13C]glucose and [3-13C]lactate concentrations in plasma samples and in tumor extracts. The high-resolution 13C spectra of these samples permitted us to resolve the various 13C-labeled signals, as well as 13C signals at natural abundance. In the 13C MRS spectra of plasma samples, signals of (alpha +beta ) 13C-1 glucose and of 13C-3 lactate, as well as signals due to 13C-2 to 13C-6 of glucose and 13C-2 lactate at natural abundance, could be identified. The plasma concentration of [1-13C]glucose decreased with time as a result of glucose transfer to the tissue. The data were fitted to the plasma glucose decay: (Eq. A1 in APPENDIX), yielding an initial concentration (a1) of 46 mM and an apparent decay rate constant (kp) of 2.3×10-2/min (R = 0.92; Fig. 4). These values were not significantly different from the mean values determined from best-fit in vivo data of the tumors (Table 1). Furthermore, a similar fractional 13C-1 enrichment of glucose was determined in all of the plasma samples, with a mean of 64 ± 3%. The integrated areas of the enriched 13C-3 lactate signal and of the 13C-2 lactate signal at natural abundance in the plasma samples were low. The mean [3-13C]lactate concentration in the plasma was low, 0.18 ± 0.01 mM (Fig. 4), and the enrichment fraction was ~5%.


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Fig. 4.   Changes in the concentration of 13C-labeled glucose and lactate in the plasma of CD1-NU female mice after [1-13C]glucose infusion. , [1-13C]glucose, with the curve obtained by fitting the data to Eq. A1, yielding a1 = 46 mM, kP = 2.3 × 10-2/min, with R = 0.92. , [3-13C]lactate. Nos. of experiments (= no. of mice) per time point were t = 0 min, n = 3; t = 5 min, n = 3; t = 15 min, n = 2; all other 8 time points, n = 1.

The high-resolution 13C spectra of tumor extracts revealed prominent signals of alpha  and beta  13C-1 glucose and 13C-3 lactate (Fig. 5). Other 13C signals of metabolites into which 13C-1 glucose could have been incorporated (13C-3 alanine and 13C-4 glutamate and an unassigned signal at 91 ppm) exhibited much lower signal intensities, as was also demonstrated in the same cells (MCF7) under a culture perfusion condition (12).


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Fig. 5.   13C spectrum of the water-soluble metabolites in MCF7 tumor extract. The extract was obtained from a tumor removed 55 min after end of [1-13C]glucose infusion. Inset: ×24 enlargement of spectrum between 70.3 ppm and 71 ppm, showing the alpha  and beta  13C-4 glucose signals at natural abundance. *Unassigned signal; glu, glutamate. The spectrum was acquired at 125 MHz, as described in MATERIALS AND METHODS. MeOH, methanol added externally, serving as a concentration reference.

The 13C spectra of the tumors also exhibited signals due to 13C-4 of glucose at natural abundance (inset in Fig. 5) and 13C-2 of lactate at natural abundance, which allowed calculation of their corresponding percent enrichment. The 13C enrichment of glucose in the tumors after termination of the infusion remained constant at 73 ± 2% (n = 6), similar to that measured in the plasma of similar mice (64 ± 3%, P = 0.16, unpaired two-tail Student's t-test). The enrichment of lactate after the infusion was lower than that expected from dilution by the breakdown of labeled glucose to labeled and unlabeled lactate (~35%) and ranged from 13 to 19% (mean = 17 ± 1%; n = 6). This lower enrichment could result either from dilution by a preexisting lactate pool at natural abundance or by dilution through fast exchange of the tumor lactate with the plasma pool.

Hormone-induced changes. Evaluation of the ability of this methodology to detect changes in the rate of glycolysis and thereby assess response to therapy was verified by monitoring the effect of tamoxifen after treatment for 10-14 days. Analysis of 1H MRI data showed that tamoxifen induced growth arrest (Fig. 6) and increased percentage of necrosis (from 3.6 ± 0.5 to 13.7 ± 0.5%, P < 0.05). Analysis of the time courses of 13C-enriched glucose and lactate before and after treatment indicated two significant changes: an ~50% reduction in the rate of [3-13C]lactate synthesis via glycolysis (P < 0.008) and an ~50% reduction in the rate constant of lactate clearance (P < 0.06) (Fig. 6). The percent reduction in the rate of glycolysis was higher than that in viable cells (8%, Fig. 6). The parameter associated with glucose perfusion did not change significantly, indicating sufficient transfer of glucose from the blood to the tumor tissue despite the treatment.


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Fig. 6.   Tamoxifen-induced changes in orthotopic MCF7 tumors. A: volume. B: percentage of viable cells. C: rate of glycolysis, Vgly. D: rate of lactate clearance, kl. Open and solid bars, results obtained from tumors growing in the presence of estrogen and tamoxifen, respectively (n = 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

A noninvasive MRS method has been developed and applied to measure the kinetics of glucose perfusion and metabolism to lactate in orthotopic MCF7 human breast cancer. The direct detection of the key metabolites, glucose and lactate, served to determine specific kinetic parameters that are markers of malignancy. To extract these parameters from the time courses, it was necessary to develop a new model that accounted for the multiple physiological and metabolic reactions occurring in vivo. Such a model was thus designed, in line with basic knowledge, introducing assumptions that were validated either by additional measurements or by previous supporting data (see APPENDIX). Although the tumors varied in size, they all appeared to be predominantly viable and nonnecrotic. This was also indicated by the convergence of the kinetic parameters to similar values with a relatively low coefficient of variation (Table 1). The consistency of the model was further reflected by the similarity of the blood glucose pharmacokinetic parameters obtained by fitting the tumor glucose time course and those obtained by direct measurement of the plasma curve.

The time course of [13C]glucose uptake by the tumor was predominantly dependent on the plasma kinetics and perfusion of the tumor and was not sensitive to the rate of glucose metabolism (VGlc) within a range of ±40%. The time course of [13C]lactate was highly sensitive to the rate of [3-13C]lactate synthesis via glycolysis (Vgly) and was therefore determined with a high degree of accuracy. The effective concentration of glucose in the tumor (Fig. 3) was lower than that found in the plasma (Fig. 4), since a large part of free glucose was confined to the extracellular volume fraction of the tumor of ~0.4.

The percentage of 13C-enriched glucose was stable, and therefore the related parameters represented total glucose kinetics. The percentage of 13C-enriched lactate also appeared steady and therefore, despite dilution of 13C-enriched lactate (either by fast exchange with the plasma or by preexisting lactate pools), total rates of lactate synthesis from 13C-enriched glucose could be estimated. From the direct analysis of the data, we calculated the rate of synthesis of 13C-enriched lactate (45 µM/min, Table 1). Thus the rate of total lactate synthesis from 13C-enriched glucose was twice as fast (90 µM/min). The total rate of glycolysis (13C-enriched and nonenriched lactate synthesis) depends also on the percent enrichment. In case of dilution by preexisting pools, this rate remains the same, as the MRS method measures the total amount of 13C-enriched lactate in the detected volume. However, if there is fast exchange of lactate with the plasma, the total rate of lactate synthesis from 13C-enriched glucose should take into account the percentage of labeling (~17%), which is about one-half of that expected without dilution.

Glucose perfusion capacity, determined from the rate constant of glucose transfer from the plasma to the extracellular tumor volume, presumably reflected blood flow, because the glucose membrane permeability of the microcapillaries is not limiting (17). This assumption is further supported by previous investigations of human breast cancer xenografts in nude rats that showed glucose uptake rates to be determined mainly by nutritive blood flow (19). The analysis of the time course of glucose concentration also provided an estimate of the average extracellular volume fraction, ~0.4, in accord with the high viability and low necrosis in these tumors.

Different MRS protocols and modeling approaches of glycolysis were reported in studies of a C6 glioma implanted in rat brain (30) and of a rat radiation-induced fibrosarcoma (RIF-1) tumor (3). In the studies on glioma, glucose was supplied by constant infusion throughout the experiment. Therefore, only changes in lactate were observed, from which the rate constant of lactate clearance was calculated (4.3 × 10-2/min). In the studies on RIF-1 tumors, glucose was administered during a short period but remained persistently high for an extended time. Thus only changes in 13C-3 lactate were analyzed, from which the apparent rate constants of glycolysis (2.2 × 10-2/min) and of lactate clearance (3.4 × 10-2/min) were calculated. Recently, a similar protocol and data analysis were applied to study glycolysis in a murine C3H mammary carcinoma (22). The apparent rate constants of glycolysis and of lactate clearance were 1.6 × 10-2/min and 2.8 × 10-2/min, respectively. In this study, the apparent rate constant of glucose clearance of 8.4 × 10-2/min was determined as well. In our experiments, the rate constant of 13C-enriched lactate clearance (3.2 × 10-2/min) was of the same order of magnitude as rates obtained in the just cited studies. We also estimated for 1 mM 13C-enriched lactate (reached at the steady level) a rate constant of synthesis of ~4.5 × 10-2/min, which is higher than that found in RIF-1 and C3H tumors. This difference could reflect differences in either the nature of the tumor or the model animal, but we cannot exclude variations due to methodological differences.

Changes in the parameters just described can also serve to assess response to treatment and predict its efficacy. We have shown this capability by monitoring the effects of tamoxifen. This selective estrogen receptor modulator (SERM) is used extensively to treat breast cancer patients and has also been assessed as a preventative drug (11). Because other SERMs are currently being developed (10, 18), there is a need for an effective method that can provide an early marker for response. We found that tamoxifen induced a 50% decrease in the rate of glycolysis. This reduction was higher than the percent reduction in viable cells (8%, Fig. 6), indicating a change in the cellular metabolic capacity of the tumor cells. It also preceded a reduction in tumor volume, which is currently the clinical marker of response. Thus reduced glycolysis is an early marker for response to tamoxifen, as was also shown in MCF7 and T47D human breast cancer cells in vitro (12, 21). Because in T47D cells glycolysis was found to be limited by glucose transport (24), it is reasonable to suggest that the tamoxifen inhibition of glycolysis was due to decreased expression and activity of the glucose transporters.

We have also found a decrease in the rate of lactate clearance in tumors treated with tamoxifen. Lactate efflux from the cells to the extracellular environment is predominantly regulated by monocarboxylic acid transporters (MCT1 to MCT5) (14). The clearance rate into the plasma depends on the perfusion of the tumor. In analogy to glucose, it is reasonable to suppose that the reduction in lactate efflux rate is due to downregulation of the MCTs by tamoxifen.

Many previous studies have demonstrated the capacity of tumors to produce lactate from glucose. More recently, significantly higher lactate concentrations were observed in tumors with metastatic spread (5). In addition, studies of dissected breast specimens showed a correlation between lactate concentrations and the histopathological grade of the tumors (7). Comparison of lactate levels in normal and in malignant human mammary epithelial cell extracts showed that lactate levels remained low in normal, immortalized, and oncogene-transformed cells but were significantly higher in the malignant cell lines (1). Furthermore, a negative correlation was found between the state of differentiation of human breast cancer cells and the rate of glycolysis (24). Thus the rate of lactate synthesis measured in vivo by the method described here may also serve as a prognostic parameter.

Recent molecular studies have revealed that there are oncogenes and tumor-suppressor genes that directly affect glucose metabolism and cellular energy (8). For example, the hypoxia-inducible transcription factor HIF-1 increases the level of the glucose transporter GLUT1 (27). Variations in this transporter directly correlate with the rate of glycolysis in breast cancer cells (24). Integration of the kinetics of glycolysis, measured by MRS, with alterations in the expression of the glucose transporters and the glycolytic enzymes may provide novel insights into malignant transformation, as well as development of new diagnostic and therapeutic strategies designed to prevent metabolic adaptation of cancer.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

The Physiological-Metabolic Model

Analysis of the time course of [1-13C]glucose in each tumor was based on a mathematical model that took into account the changes in [1-13C]glucose concentration in the plasma (Cp) after its infusion, by use of the following equation of exponential decay
C<SUB>p</SUB>(<IT>t</IT>)<IT>=a</IT><SUB>1</SUB><IT>×</IT>exp(−<IT>k</IT><SUB>p</SUB><IT>×t</IT>) (A1)
where a1 is [1-13C]glucose concentration immediately after termination of the infusion, and kp is the effective rate constant of the transfer of [1-13C]glucose from the plasma to the tissue, averaged over the entire body.

In the tumor tissue, glucose may be distributed unevenly in the extracellular and intracellular compartments. Thus the detected MRS signal area is related to an average effective concentration in the measured volume. The change with time in this effective [1-13C]glucose concentration in the tumor tissue (C<UP><SUB>g</SUB><SUP>*</SUP></UP>) was influenced by the reversible transfer of [1-13C]glucose from the plasma with an apparent glucose perfusion rate constant kg (min-1), and by the rate of glucose disappearance due to metabolism by the cells VGlc (µM/min).
d<IT>C<SUP>*</SUP></IT><SUB>g</SUB><IT>/</IT>d<IT>t=k</IT><SUB>g</SUB><IT>×</IT>[<IT>C</IT><SUB>p</SUB>(<IT>t</IT>)<IT>−C</IT><SUB>g</SUB>]<IT>−V</IT><SUB>Glc</SUB> (A2)
where Cg is the concentration of [1-13C]glucose in the extracellular tumor volume. A solution to this equation was obtained that took into account the pharmacokinetics of glucose in the plasma (Eq. A1), with the following assumptions. 1) Glucose was rapidly metabolized in the cells; therefore, most of the glucose in the tumor was confined to the extracellular volume. This assumption was based on data indicating negligible intracellular glucose in human breast cancer cells (24). Thus the overall glucose concentration, C<UP><SUB>g</SUB><SUP>*</SUP></UP>, was related to the glucose concentration in the extracellular compartment, according to the equation: C<UP><SUB>g</SUB><SUP>*</SUP></UP>(t) = upsilon  × Cg(t), where upsilon  is the extracellular volume fraction of the tumor accessible to glucose. 2) Glucose transport limited the rate of glucose metabolism. This assumption was based on kinetic studies of human breast cancer cells (24). Because the Km of glucose transport in these cells is ~2.5 mM (6, 24, 37), whereas the available glucose concentration during the measurements was much higher, glucose transport proceeded at the Vmax of transport. Thus
C<SUP>*</SUP><SUB>g</SUB>(<IT>t</IT>)<IT>=b</IT><SUB>1</SUB><IT>×</IT>exp(−<IT>k</IT><SUB>p</SUB><IT>×t</IT>)<IT>−b</IT><SUB>2</SUB><IT>+</IT>(<IT>b</IT><SUB>2</SUB><IT>−b</IT><SUB>1</SUB>)<IT>×</IT>exp(−<IT>m</IT><SUB>1</SUB><IT>×t</IT>)
where
<IT>b</IT><SUB>1</SUB><IT>=k</IT><SUB>g</SUB><IT>×a</IT><SUB>1</SUB><IT>/</IT>(<IT>m</IT><SUB>1</SUB><IT>−k</IT><SUB>p</SUB>); <IT>b</IT><SUB>2</SUB><IT>=V</IT><SUB>Glc</SUB><IT>/m</IT><SUB>1</SUB>;<IT> m</IT><SUB>1</SUB><IT>=k</IT><SUB>g</SUB><IT>/&ugr;</IT> (A3)
The first assumption was also supported by the finding that the overall maximum concentration of [1-13C]glucose in the tumors was about threefold lower than in the plasma (see data in RESULTS). Because glucose in the plasma is rapidly equilibrated in the tissue, this finding suggested localization of the glucose in about one-third of the tumor volume, presumably in the extracellular compartment.

Metabolic conversion of [1-13C]glucose by glycolysis resulted in the production of one molecule of [3-13C]lactate and one molecule of lactate at 13C natural abundance. The change in the concentration of 13C-enriched lactate in the tumor volume (C<UP><SUB><IT>l</IT></SUB><SUP>*</SUP></UP>) was determined by the rate of [3-13C]lactate synthesis via glycolysis, Vgly (one-half the rate of lactate synthesis from 13C-enriched glucose) equal to the apparent change in [3-13C]lactate, V<UP><SUB>gly</SUB><SUP>*</SUP></UP> times the fractional enrichment of [1-13C]glucose, f, and by the rate of [3-13C]lactate transfer to the blood and clearance from the tumor with an apparent clearance constant (kl)
d<IT>C<SUP>*</SUP></IT><SUB>l</SUB><IT>/</IT>d<IT>t=f×V<SUP>*</SUP></IT><SUB>gly</SUB><IT>−k</IT><SUB>l</SUB><IT>×C<SUP>*</SUP></IT><SUB>l</SUB> (A4)
In this equation, lactate was assumed to be localized in the intra- and extracellular tumor compartments. We have neglected any contribution from 13C-labeled lactate in the blood, because its concentration was found to be negligible (see RESULTS). To solve this equation, we applied again the assumption (described above for derivation of Eq. A3) that glucose metabolism to lactate was limited by glucose transport and proceeded at a Vmax of glucose transport yielding the following augmenting curve
C<SUP>*</SUP><SUB>l</SUB>(<IT>t</IT>)<IT>=f×V<SUP>*</SUP></IT><SUB>gly</SUB><IT>×</IT>(1<IT>−</IT>exp(−<IT>k</IT><SUB>l</SUB><IT>×t</IT>))<IT>/k</IT><SUB>l</SUB> (A5)
Usually, 13C-enriched glucose can also be metabolized through the tricarboxylic acid cycle, the pentose phosphate shunt, or to form glycogen. Thus the effective rate of [3-13C]lactate synthesis, Vgly is less than VGlc. On the basis of MRS studies of the same cells, we have used the relation VGlc = Vgly/0.7 (31). In the final simultaneous fitting of the data to Eqs. A3 and A5, we had therefore as free parameters: the plasma pharmacokinetic parameters (a1, kp), the rates of glucose transfer and lactate clearance (kg, kl), the extracellular volume fraction of the tumor accessible to glucose (upsilon ), and the rate of 13C-enriched lactate synthesis (Vgly).


    ACKNOWLEDGEMENTS

The help of Dr. Peter Bendel and the editing by Myra Kaye are gratefully acknowledged.


    FOOTNOTES

This work was supported by National Cancer Institute Grant RO1 CA-42238 and by a joint grant of the Pasteur-Weizmann-Negri Institute.

H. Degani is the incumbent of the Fred and Andrea Fallek Professorial Chair in Breast Cancer Research and Head of the Willner Center for Vascular Biology.

Address for reprint requests and other correspondence: H. Degani, Dept. of Biological Regulation, Weizmann Institute of Science, Rehovot, 76100 Israel (E-mail: hadassa.degani{at}weizmann.ac.il).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

May 21, 2002;10.1152/ajpendo.00050.2002

Received 5 February 2002; accepted in final form 13 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
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