Glucose metabolism via the pentose phosphate pathway, glycolysis and Krebs cycle in an orthotopic mouse model of human brain tumors

Abstract

It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using orthotopic mouse models of human glioblastoma (GBM) and renal cell carcinoma metastatic to brain, we estimated the activity of the PPP relative to glycolysis by infusing [1,2-(13) C(2) ]glucose. The [3-(13) C]lactate/[2,3-(13) C(2) ]lactate ratio was similar for both the GBM and brain metastasis and their respective surrounding brains (GBM, 0.197 ± 0.011 and 0.195 ± 0.033, respectively (p = 1); metastasis: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than the PPP flux in these tumors, and that the PPP flux into the lactate pool is similar in both tumors. Remarkably, (13) C-(13) C coupling was observed in molecules derived from Krebs cycle intermediates in both tumor types, denoting glucose oxidation. In the renal cell carcinoma, in contrast with GBM, (13) C multiplets of γ-aminobutyric acid (GABA) differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. In addition, the orthotopic renal tumor, the patient's primary renal mass and brain metastasis were all strongly immunopositive for the 67-kDa isoform of glutamate decarboxylase, as were 84% of tumors on a renal cell carcinoma tissue microarray of the same histology, suggesting that GABA synthesis is cell autonomous in at least a subset of renal cell carcinomas. Taken together, these data demonstrate that (13) C-labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy.

FIGURE 1

Diagram depicting [1,2-¹³C₂]-glucose metabolism through glycolysis and the pentose phosphate pathway (PPP) with the resulted labeling pattern of lactate C3. As a consequence of the higher activity of glycolysis relative to PPP in normal cells, the contribution of the doublet (D23) to the total NMR signal of lactate C3 is significantly more prominent than the singlet (S3). HK: hexokinase, G-6PDH: glucose 6-phosphate dehydrogenase, 6PGDH: 6-phosphogluconate dehydrogenase, PGI: phosphoglucose isomerase, LDH: lactate dehydrogenase, Glc: glucose, Glc-6-P: glucose 6-phosphate, F6P: fructose 6-phosphate, G3P: glyceraldehyde 3-phosphate, 6PG: 6-phosphogluconate, R5P: ribose 5-phosphate, PYR: pyruvate, LAC: lactate. Open circles denote carbon 12, filled circles denote carbon 13.

FIGURE 2

Orthotopic primary GBM mouse tumors share MR and PET imaging features seen in original patient. In panel A, both human and orthotopic mouse GBM tumors appear as a large hyperintense T2 mass, with significant mass effect (mouse MRI). Clinical and orthotopic tumor FDG-PET images are consistent with enhanced glucose uptake relative to the surrounding brain tissue. Panel B shows hematoxylin-eosin (H&E) and Ki67 staining from the orthotopic GBM and orthotopic CCRCC brain metastasis. GBM tumors were characterized by a high mitotic index, pleomorphic nuclei, tortuous microvasculature and diffuse single cell infiltration. In contrast, the CCRCC showed brisk proliferation, monomorphic nuclei and scant cytoplasm. Magnification of histological images is 20×.

FIGURE 3

Section of the forebrain NMR spectrum from a non-tumor bearing NOD/SCID mouse infused with [1,2-¹³C₂]-glucose. Spin-coupled ¹³C multiplets in metabolites exchanging with Krebs cycle intermediates (i.e., glutamate, glutamine, GABA and aspartate) is observed in the ¹³C spectrum as a consequence of glucose oxidation in the brain. Of note, the labeling pattern of glutamate C4 (GLU4) and GABA C2 (GABA2) were comparable as GABA derives primarily from glutamate in normal brain. Insets display labeling patterns of lactate C3 and from aspartate C3 to glutamine C4. GLU: glutamate, GLN: glutamine, ASP: aspartate, LAC: lactate, TAU: taurine. 1: N-acetylaspartate C6, 2: GABA C3, 3: unassigned, 4: glutamine C3, 5: glutamate C3, 6: unassigned, 7: unassigned, 8: GABA C4, 9: N-acetylaspartate C3, 10: taurine C1, 11: aspartate C2, 12: N-acetylaspartate C2, 13: unassigned, 14: glutamine C2, 15: glutamate C2. S: singlet, Dxx: doublet, Q: quartet.

FIGURE 4

Section of the ¹³C-NMR spectrum illustrating the labeling pattern of glutamate C4 (GLU4), GABA C2 (GABA2), and Lactate C3 (LAC33) in normal forebrain (A), surrounding brain of GBM (B), surrounding brain of CCRCC metastatic to the brain (C). The labeling pattern between both isotopomers is notably consistent throughout all brain tissues. S: singlet, Dxx: doublet, Q: quartet.

FIGURE 5

Section of the ¹³C-NMR spectrum showing the labeling pattern of glutamate C4 (GLU4) and GABA C2 (GABA2) in GBM (A) and CCRCC orthotopic tumors (B) and their respective immunohistochemical staining for GAD 67 (20×). In contrast to GBM, in the CCRCC orthotopic tumor GABA2 showed a lower singlet-to-doublet ratio relative to its precursor GLU4, consistent with compartmentalization of GABA synthesis in the tumor mass. In addition, CCRCC was homogeneously immunopositive for GAD67 throughout the tumor whereas GBM cells were immunonegative, suggesting that CCRCC cells are able to produce GABA, and that the GABA2 observed in the GBM ¹³C spectrum is derived from GABAergic interneurons (blue arrows) and GABAergic projections (green arrowheads) interspersed within the tumor tissue.

FIGURE 6

Primary CCRCC in the kidney and the CCRCC brain metastasis show strong GAD67 immunoreactivity The histopathological features of the CCRCC were well preserved in the metastatic brain lesion, including GAD67 immunoreactivity. This observation suggests that GABA synthesis was likely present in the primary renal mass and is not an idiosyncratic reaction to the brain microenvironment neither in the original patient or when transplanted to the mouse brain. H&E, hematoxylin and eosin.