Introduction: Meningiomas are the most frequently reported central nervous system (CNS) tumors, accounting for 37% of all CNS tumors [1]. They arise from meningeal membranes surrounding the brain and spinal cord. About 80% of meningiomas are slow growing and are benign (WHO grade-I). On the other hand, atypical meningiomas (WHO grade-II), accounting for ~18.0%, show increased high cellular proliferation and recurrence. Anaplastic meningiomas (WHO grade-III) are very rare contributing only up to 2.0% to the total meningiomas [2,3]. High recurrence is common in grade-II meningiomas and it is associated with lower survival rates. Currently, no chemotherapy is part of the standard of care for meningiomas, as a result, surgery followed by radiotherapy is the mainstay of treatment [1-3]. Metabolism is the key to many biological processes including tumorigenesis [4-7]. Both benign and atypical meningiomas are known to have altered metabolic profiles [8]. Quantification of metabolic fluxes of essential nutrients provide information to derive valuable biological insights [4]. Unravelling metabolic phenotypes of various histological subtypes of meningiomas will identify new targets for the therapeutic intervention of these tumors. Here, we are reporting the in vivo metabolism of [U-¹³C]glucose and [1,2-¹³C]acetate in meningioma patients investigated by ¹³C NMR based isotopomer analysis.
Methods: Meningioma patients were enrolled at the Department of Neurosurgery, Houston Methodist Hospital, following an IRB protocol approved by Houston Methodist Hospital Institutional Review Board. 20% stock solutions of [U-¹³C]glucose and [1,2-¹³C]acetate were prepared by a research pharmacist at the investigational drug services (IDS) of Houston Methodist Hospital. On the day of the surgery, intravenous (i.v.) bag containing [U-¹³C]glucose or [1,2-¹³C]acetate was delivered to the OR and the patients were infused with either [U-¹³C]glucose or [1,2-¹³C]acetate, 2-3 hours prior to the surgical resection of the tumor [9,10]. Blood samples were collected at different time intervals (t = 0, 15, 45, 90, 120 min.) during the infusion and used to calculate plasma enrichment of [U-¹³C]glucose or [1,2-¹³C]acetate. Tumor tissues were collected after 120 min of infusion. Both blood and tumor samples were snap-frozen on dry-ice and transported to the laboratory. Blood and tumor tissue specimens were extracted in 5% perchloric acid, supernatants were lyophilized and reconstituted in 180 µL D₂O containing 1.0 mM DSS-d₆ (pH = 7.4). The reconstituted sample-solutions were transferred to a 3-mm NMR tubes, and 1D ¹H and ¹H-decoupled ¹³C NMR data were collected on a Bruker 800 MHz spectrometer equipped with a cryo-probe.
Results: Pathways involved in the metabolism of [U-¹³C]glucose and [1,2-¹³C]acetate are schematically shown in Figure 1. Figure 2 shows the ¹³C NMR spectral comparison of tumor tissue extracts of grade-I (A) and grade-II (B) meningioma patients who were infused with [U-¹³C]glucose during the surgical resection of tumors. Both grades of tumors showed ¹³C-¹³C spin-coupled multiplets in various carbons of glycolytic intermediates lactate (Lac) and alanine (Ala) (i.e. Lac C3 and Ala C3) and TCA cycle metabolites such as glutamate (Glu), glutamine (Gln) and aspartate (Asp) (for example, Glu C3, Glu C4, Gln C4, Asp C2, Asp C3). Levels of Lac C3 were similar in both grades of tumors (Figure 2) whereas Glu C4 which is the readout of the levels of acetyl-CoA are higher in grade-II compared to the grade-I tumors (mean values: 10.10% vs. 4.20 ± 1.02%, inset in upper plot). This suggests that grade-II tumors utilize relatively more glucose than grade-I. Since the amount of acetyl-CoA generated from glucose in meningiomas is only about 10%, these tumors could be utilizing nutrients other than glucose. Acetate is another bioenergetic substrate utilized by tumors [10,11], and we wanted to investigate if meningiomas utilize acetate to fill the bioenergetic gap. We infused meningioma patients with [1,2-¹³C]acetate and determined the levels of [1,2-¹³C]acetyl-CoA generated from acetate. Figure 3A shows the comparison of 13C NMR spectral regions of Glu C4 of meningioma tumor extracts from patients infused with [U-13C]glucose (from grade-I and II) and [1,2-13C]acetate (from grade-I), and the chart in Figure 3B shows the levels of [1,2-¹³C]acetyl-CoA generated from [U-¹³C]glucose and [1,2-¹³C]acetate. We can clearly see that meningioma tumors (grade-I) generated 43.83 ± 2.17% of acetyl-CoA from acetate which is ~10 more than that generated from [U-¹³C]glucose.
Discussion: Although glucose is an abundant nutrient in human body, tumor utilize glucose to a smaller extent and depend on other bioenergetic substrates such as acetate. Most of our patients were from grade-I, we are enrolling more patients and want to include more patients from grade-II to have a better comparison of glucose and acetate metabolism in both groups.
Conclusion: The current study suggest that human meningiomas preferentially utilize acetate. Acetyl-CoA synthetase 2 (ACSS2) is the enzyme responsible for the metabolism of acetate, targeting this enzyme may have therapeutic potential in the treatment of meningiomas.

Acknowledgements

We thank all the patients who participated in this study. This study was supported by the Donna and Kenneth R. Peak Foundation, The Kenneth R. Peak Brain and Pituitary Tumor Treatment Center at Houston Methodist Hospital, The Houston Methodist Foundation, The Taub Foundation, The Pauline Sterne Wolff Foundation, The Veralan Foundation, The Marilee A. and Gary M. Schwarz Foundation, The John S. Dunn Foundation, Contributions in honor of Will McKone. We also thank NMR and Drug Metabolism Core (Baylor College of Medicine, Houston, TX) for providing access to 800 MHz NMR spectrometer.