13C-labeling in lactate during infusion of [1,2-13C2]glucose was used to assess the relative activity of the PPP compared to glycolysis in orthotopic mouse models of GBM and CCRCC that had metastasized to the brain. There was no significant difference in the singlet/doublet ratio in carbon 3 of lactate between either the GBM or CCRCC and surrounding non-tumor bearing brain tissue. Interestingly, all tumors showed excess uptake of 18FDG on PET scan. Taken together and assuming that increased 18FDG signal indicates increased glucose metabolism, these results indicate that the relative flux through the PPP compared to glycolysis is not altered in these tumors but the combined rate of glucose metabolism is increased. This estimation of the PPP relative to glycolysis was based on the singlet-to-doublet ratio of lactate C3 derived from [1,2-13C2]glucose. This approach assumes that the singlet arising from lactate C3 is the product of the oxidative branch of the PPP plus the background natural abundance signal (1.1% of total carbon). In both the GBM and CCRCC brain metastasis, the ratio of singlet to doublet in lactate C3 was similar between the tumor and the surrounding brain. Although the current data could be interpreted as excess flux through the PPP in both tumor and surrounding brain relative to normal brain, it is not possible to draw that conclusion with confidence because of possible compartmentation of lactate. Specifically, lactate in the tumor mass is present in at least three potential compartments: in blood, in a slowly-exchanging pool in poorly perfused or metabolically inactive tumor cells, and in a rapidly-exchanging pool that reflects metabolism of exogenous glucose. We have shown that the majority of lactate in blood is not enriched, thus metabolism of lactate from blood does not contribute to the PPP. Alternatively, PPP flux can be underestimated using this approach, as ribose 5-phosphate generated through the oxidative branch of PPP may not reenter glycolysis. Instead, it can be used for nucleotide synthesis, reducing the abundance of lactate C3 singlet.
Previous studies had suggested that while the bioenergetic demands of malignant cell growth are met by enhanced glycolysis[1
], the PPP is preferentially activated to support macromolecule biosynthesis that is necessary for cellular growth[23
]. It is of interest to note, however, that estimates of maximum flux through PPP relative to glycolysis reported here for GBM and CCRCC tumors (19% for and 12%, respectively) are comparable to those reported in traumatic brain injury in a rodent model (9-12%) and humans (19.6%)[12
]. These estimates are significantly higher than the approximate 5% estimates of PPP activity in brain under unstressed conditions [25
]. One possible explanation for why PPP activity may be increased by TBI or by the presence of a large intracranial mass and TBI tissue is that both circumstances are associated with vasogenic/cytotoxic edema. This could produce a compressive force as the brain expands inside a rigid cranium compromising perfusion and limiting oxygen diffusion as result of interstitial edema. Although speculative, it is possible that the resulting oxidative stress (generation of ROS) may explain why both intracranial tumor tissue and non-tumor bearing regions of the brain show increased PPP activity.
C NMR analysis of both GBM and CCRCC brain metastasis, revealed 13
C coupling in glutamate and GABA, suggesting that glucose was metabolized to acetyl-CoA and further oxidized in the Krebs cycle. Although [1,2-13
] labeled glucose is a suboptimal tracer for assessing Krebs cycle intermediates since only 50% of acetyl-CoA becomes 13
C labeled, nevertheless the presence of multiplets in glutamate and GABA denotes presence of Krebs cycle activity. This observation suggests that malignant tumors growing in the microenvironment of the brain rely on oxidative metabolism in addition to glycolysis and increased production of lactate, typical of the Warburg effect[2
An unexpected observation in the CCRCC brain metastasis was that 13
C multiplets in GABA C2 displayed a lower singlet-to-doublet ratio (0.172) relative to its precursor glutamate C4 (0.346), suggesting that GABA derived from a subset of the total glutamate pool. This was not observed in spectra from normal brain or GBM. Conversion of glutamate to GABA requires GAD67 (or GAD65) which, in the brain, is exclusively expressed by a critically important subset of GABAergic inhibitory interneurons [22
]. Although the orthotopic metabolic data presented here was derived from a single CCRCC brain metastatic tumor, further support for this novel finding comes from the demonstration that the patient’s brain metastasis, from which the orthotopic tumor line was derived, as well as the patient’s primary renal tumor mass, all showed strong GAD67 immunoreactivity. Moreover, the importance of this discovery and its potential clinical relevance is demonstrated by the finding that 86% of an independently generated, clinically annotated, tissue microarray of 96 individual renal tumors with CCRCC histopathology also showed GAD67 immunoreactivity. Since the expression level of GAD67 did not appear to correlate with tumor grade or prognosis, it raises the possibility that upregulation of GAD67 expression is an early event in the genesis of CCRCC tumors. The fact that elevated GAD67 expression persists as CCRCCs accrue mutations and evolve into higher grade local and metastatic disease, including brain, suggests that the ability to synthesize GABA may play an important role in the pathophysiology of this disease. In support of the speculation that GABA signaling may promote tumor growth, there is growing evidence that a wide variety of non-CNS tumors express GAD67, synthesize GABA and express GABA-specific receptors (GABA-A and GABA-B) [27
]. What specific role GABA plays in supporting renal tumor growth, particularly brain metastasis remains to be elucidated.