Metabolism of [U-13 C]glucose in human brain tumors in vivo

Abstract

Glioblastomas and brain metastases demonstrate avid uptake of 2-[(18) F]fluoro-2-deoxyglucose by positron emission tomography and display perturbations of intracellular metabolite pools by (1) H MRS. These observations suggest that metabolic reprogramming contributes to brain tumor growth in vivo. The Warburg effect, excess metabolism of glucose to lactate in the presence of oxygen, is a hallmark of cancer cells in culture. 2-[(18) F]Fluoro-2-deoxyglucose-positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation relative to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain cancers to oxidize glucose in the tricarboxylic acid cycle is unknown. Here, we studied the metabolism of human brain tumors in situ. [U-(13) C]Glucose (uniformly labeled glucose, i.e. d-glucose labeled with (13) C in all six carbons) was infused during surgical resection, and tumor samples were subsequently subjected to (13) C NMR spectroscopy. The analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the tricarboxylic acid cycle, anaplerosis and de novo glutamine and glycine synthesis contributed significantly to the ultimate disposition of glucose carbon. Surprisingly, less than 50% of the acetyl-coenzyme A pool was derived from blood-borne glucose, suggesting that additional substrates contribute to tumor bioenergetics. This study illustrates a convenient approach that capitalizes on the high information content of (13) C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse cancers growing in their native microenvironment.

Copyright © 2012 John Wiley & Sons, Ltd.

Figures

Fig. 1. Tumor glucose metabolism in a GBM patient infused with [U-13C]glucose at 4 grams/hour

(a) Pre-operative T1-weighted post-gadolinium coronal image demonstrating a large right temporoparietal ring-enhancing mass lesion with a central cystic component (X) and surrounding edema. (b)18FDG-PET scan demonstrating uptake of 18FDG along the tumor margin and inferior to the cyst (arrow). The expected high rate of cortical 18FDG signal with reduced uptake in white matter is apparent in the left hemisphere. The blunted 18FDG signal in the right hemisphere superior to the tumor is likely a consequence of tumor-associated edema. Histological analysis of the tumor sample revealed nearly 100% malignant cells. (c) Percent 13C enrichment in plasma glucose (Glc) over the 200-minute infusion of [U-13C]glucose at 4g/hr. Enrichment is the fraction of plasma glucose that was uniformly labeled with 13C. Tumor samples were collected during the final 35 minutes (blue bar). Proton-decoupled 13C NMR spectra of (d) a tumor sample corresponding to the arrow in (b); and (e) cyst fluid. The presence of multiplets at many of the highlighted resonances is the result of 13C–13C coupling. Assignments for all spectra throughout the paper: (1) Lactate C2; (2) Glutamate C2; (3) Glutamine C2; (4) Aspartate C2; (5) Alanine C2; (6) Taurine C1; (7) Glycine C2; (8) N-Acetylaspartate C3; (9) GABA C4; (10) Creatine C2; (11) Aspartate C3; (12) Taurine C2; (13) GABA C2; (14) Glutamate C4; (15) Glutamine C4; (16) Glutamate C3; (17) Glutamine C3; (18) GABA C3; (19) N-Acetylaspartate C6; (20), Lactate C3; (21) Alanine C3. The insets, from left to right, are Lactate C2; Glycine C2; Glutamate C4; and Glutamate and Glutamine C3. Abbreviations: S, singlet; D, doublet (e.g. D12, doublet arising from 13C in carbons 1 and 2); Q, quartet (doublet of doublets).

Fig. 2. Summary of glucose metabolism in human brain tumors in vivo

Filled symbols are 13C, open symbols are 12C. Numerals represent carbon positions within metabolites analyzed directly or indirectly by 13C NMR in this study, and are included to aid in the interpretation of the spectra. Labeling beyond the first turn of the TCA cycle can be inferred from the 13C distribution shown for citrate/isocitrate; additional detail is available [47]. Every pathway indicated in the diagram was observed in multiple tumors. The overall metabolic network involved metabolism of glucose to lactate through glycolysis and through more complex metabolic pathways involving part of the TCA cycle and/or the pentose phosphate pathway (data not shown). Pyruvate dehydrogenase (PDH), citrate synthase (CS), complete turnover of the TCA cycle, anaplerosis, and synthesis of glutamate and glutamine from glucose were evident in all eleven tumors. De novo glycine synthesis was apparent in the astrocytoma, the metastatic breast tumor, and in 7 of 8 glioblastomas. GS, glutamine synthetase.

Fig. 3. Tumor glucose metabolism in a patient with WHO Grade III astrocytoma infused with [U-13C]glucose at 8 grams/hour

(a) Pre-operative T1-weighted post-gadolinium axial image demonstrating nodular enhancement in the right temporal lobe. (b) Hematoxylin and eosin staining of a histological section prepared from the resected specimen. Cellularity: ~80% neoplastic nuclei and ~20% nuclei from nonmalignant cells (neurons, astrocytes, oligodendrocytes, and endothelial cells). (c) Time course of enrichment of plasma glucose (Glc). This patient received an 8g bolus of [U-13C]glucose followed by an infusion of 8g/hr. The samples of tumor tissue were removed between 150 and 200 minutes (blue bar) and the infusion was discontinued after the last tumor sample was collected, 30 minutes prior to the final plasma sample. (d) Proton-decoupled 13C NMR spectrum of tumor tissue. Abbreviation: T, triplet due to enrichment in carbons 2, 3 and 4 of glutamate or glutamine. Assignments and other abbreviations are the same as in Fig. 1.

Fig. 4. Tumor glucose metabolism in a GBM patient infused with [U-13C]glucose at 8 grams/hour

a, Pre-operative T1-weighted post-gadolinium sagittal image demonstrating an enhancing lesion in the left temporal lobe. b,18FDG-PET scan demonstrating uptake of 18FDG in the region corresponding to T1 enhancement. c, Representative hematoxylin and eosin-stained section (200× magnification) of the tumor sample revealing features of GBM. Virtually 100% of the live cells in the sample were neoplastic. d, Percent 13C enrichment in plasma glucose (Glc). This patient received an 8g bolus followed by an 8g/hr infusion of [U-13C]-glucose. Note that this patient had steroid-induced hyperglycemia, and this resulted in a relatively low final enrichment in plasma glucose. The blue bar corresponds to removal of the tumor samples. e, Proton-decoupled 13C NMR spectrum of metabolites extracted from the tumor. Assignments and abbreviations are the same as in Figure 1.

Fig. 5. Correlation between plasma glucose enrichment and content of [1,2-13C]acetyl-CoA in tumor TCA cycle

13C enrichment in plasma glucose near the time of tumor sampling was determined by mass spectrometry for each patient. This value was plotted against the value of Fc3 (i.e. the fraction of acetyl-CoA entering the TCA cycle that was 13C-labeled on both carbons, [1,2-13C]acetyl-CoA) determined from each tumor spectrum using a non-steady state analysis. Note that in every patient, 13C enrichment in plasma glucose far exceeded Fc3. The blue symbols are from two of the glioblastoma patients analyzed using the 4 gram per hour infusion with [U-13C]-glucose (Fc3 could not be calculated from the spectrum of the third patient infused on this protocol). The black symbols are from the five glioblastoma patients analyzed using the 8 gram bolus of [U-13C]-glucose followed by the 8 gram per hour infusion. The red symbol is the patient with WHO Grade III astrocytoma and the green symbols are from the two patients with brain metastases.

Fig. 6. Tumor glucose metabolism in a patient with metastatic breast cancer

Pre-operative imaging included (a) T1-weighted post-gadolinium coronal image demonstrating a large, solitary right cerebellar mass and (b)18FDG-PET scan demonstrating uptake of 18FDG within the mass. (c) Hematoxylin and eosin staining of a histological section prepared from the resected specimen. Immunoperoxidase stains demonstrated expression of mammaglobin and BRST-2 (not shown) in neoplastic cells consistent with origin in the breast. This tumor was positive for the HER-2 oncogene, and was negative for expression of the estrogen and progesterone receptors. Tumor cellularity was >99%. (d) Proton-decoupled 13C NMR spectrum of the tumor. Assignments and abbreviations are the same as in Fig. 1.

Fig. 7. Tumor glucose metabolism in a patient with metastatic non-small cell lung cancer

(a) Preoperative imaging included T1-weighted post-gadolinium axial image demonstrating a large right occipital mass. (b) Hematoxylin and eosin staining of a section prepared from the resected specimen. Immunoperoxidase stains demonstrated expression of thyroid transcription factor-1 (TTF-1) in neoplastic cells (not shown), consistent with origin in the lung. Tumor cellularity was ~95%. (c) Proton-decoupled 13C NMR spectrum of the tumor. Assignments and abbreviations are the same as in Fig. 1.