1. Volume 159, Issue 7, Pages 1603-1614 (December 2014)
Acetate Is a Bioenergetic Substrate for Human Glioblastoma and Brain Metastases 
Tomoyuki Mashimo, Kumar Pichumani, Vamsidhara Vemireddy, Kimmo J. Hatanpaa, Dinesh Kumar Singh, Shyam Sirasanagandla, Suraj Nannepaga, Sara G. Piccirillo, Zoltan Kovacs, Chan Foong, Zhiguang Huang, Samuel Barnett, Bruce E. Mickey, Ralph J. DeBerardinis, Benjamin P. Tu, Elizabeth A. Maher, Robert M. Bachoo 
Cell 
Volume 159, Issue 7, Pages (December 2014)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Metabolism of Coinfused [1,6-13C]Glucose and [1,2-13C]Acetate
(A) Low-power hematoxylin & eosin (H&E) images of a GBM HOT mouse brain at the time of coinfusion. (a) A large tumor (T) mass is seen in the right hemisphere. The left hemisphere is designated nontumor (NT) brain. Scale bar, 3 mm. High-power images of (b) tumor (T), (c) NT brain (scale bars, 10 μm), and (d) tumor that infiltrates brain at the edge of the mass (scale bar, 20 μm).
(B) Schema showing the fate of individual carbons from infused [1,6-13C]glucose (blue-filled circles, 13C) and [1,2-13C]acetate (red filled circles, 13C) through the first turn of the CAC and labeling in α-KG, GLU, and GLN after multiple turns. Open circles, 12C. Numbers refer to carbon positions. Abbreviations: LAC, lactate; Ac-CoA, acetyl CoA; CIT, citrate; α-KG, α-ketoglutarate; GLU, glutamate; GLN, glutamine; OAA, oxaloacetate; PDH, pyruvate dehydrogenase; LDH, lactate dehydrogenase; PYR, pyruvate.
(C) 13C-NMR spectrum from NT brain after coinfusion. Insets are GLU4 and GLN4. Singlet (S) and doublet 3,4 (D34) in blue are generated from 13C-glucose metabolism, and doublets 4,5 (D45) in red are generated from 13C-acetate metabolism. The color scheme is the same in all figures. Chemical shift assignments are the same in Figures 2 and 3: 1, alanine C3; 2, lactate C3; 3, N-acetylaspartate (NAA) C6; 4, gamma-aminobutyric acid (GABA) C3; 5, glutamine C3; 6, glutamate C3; 7, glutamine C4; 8, glutamate C4; 9, GABA C2; 10, aspartate C3; 11, GABA C4; 12, taurine (?); 13, aspartate C2; 14, glutamine C2; 15, glutamate C2.
See also Table S1.
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4. Figure 2
Metabolism of Coinfused [1,6-13C]Glucose and [1,2-13C]Acetate in GBM versus NT Brain
(A) 13C-NMR GBM tumor spectrum after coinfusion. Inserts are GLU4 and GLN4. Chemical shift assignments are the same as in Figure 1.
(B) Relative percent labeling of GLU4 by acetate and glucose in five replicates of NT brain and T from UT-GBM1. Peak areas in GLU4 were measured from the 13C-NMR spectra. The contribution of 13C-glucose (S+D34) (blue bars) and 13C-acetate (D45+Q) (red bars) is expressed as a percent of the total peak area in GLU4.
See also Figure S1 and Table S1.
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5. Figure 3
Oxidation of 13C-Acetate, but Not 13C-Glutamine, in Brain Metastases
(A and B) Coinfusion of [1,6-13C]glucose and [1,2-13C]acetate in HOT models from breast cancer brain metastasis (ER−, PR−, HER2+) (A) and melanoma (BRAFV600E mutant) (B). Scale bars, 10 μm. Patient tumor (left) is compared with the HOT mouse tumor generated from the same patient tumor (right) for HER2 in breast (A) and melanin in melanoma (B). The GLU4 and GLN4 13C-NMR profiles from the HOT tumors show similar labeling patterns with prominent D45 generated from 13C-acetate oxidation. The presence of S and D34 indicates that 13C-glucose was oxidized simultaneously. Note the similar pattern in GLN4 (prominent D45) in both tumor spectra.
(C) Infusion of [U-13C]glutamine in the melanoma HOT model. Insets are GLU4 and GLN4. Note the different labeling pattern in GLU4 and GLN4. Prominent labeling was not detected in aspartate (C3 in position 10 and C2 in position 13 in the full spectrum) or malate (MAL) (not labeled).
See also Figure S2 and Table S1.
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6. Figure 4
Immunoreactivity to ACSS2 Is Correlated with Glioma Grade and Survival of Grade II and II Gliomas
(A) Representative sections from glioma TMA showing the range of ACSS2 staining. Low, fewer than 50% of tumor cells are positive, and intensity of staining is one (scale 0–3); moderate, 75% positive, intensity 1–3; high, 100% positive and intensity 2–3.
(B) Box and whisker plot of ACSS2 histoscore for WHO grades II, III, and IV gliomas. ∗∗grade II versus grade IV (p < 0.001), and ∗grade III versus grade IV (p < 0.01).
(C) Kaplan-Meier curve of grade II and III astrocytomas and oligoastrocytomas. High versus low ACSS2 staining (n = 25 each group) based on the median histoscore. ∗∗p <
See also Figure S3A.
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7. Figure 5
Expression of ACSS2 Is Linked to GBM Growth and Malignant Potential
(A) Comparison of primary GBM cultures infected with retroviruses expressing ACSS2 shRNAi (KD) or scrambled shRNAi (SCR). Two independent cultures are shown. Neurospheres are visible in the SCR, cultures but not in KD cultures. Scale bars, 250 μm.
(B) Fold change (qRT-PCR) of ACSS1 and ACSS2 mRNA in primary conditional (floxed) astrocyte cultures after infection with adenocre to produce (a) p53−/−, (b) p53−/−, PTEN−/−, (c) p53+/−, PTEN−/−, BRAFV600E, and (d) p53−/−, PTEN−/−, BRAFV600E. ∗p < 0.01 versus b or c; ∗∗p < versus a.
(C) Glutamate C4 multiplets from 13C-NMR spectrum of an intracranial tumor arising from p53−/−, PTEN−/−, and BRAFV600E astrocytes after coinfusion of 13C-acetate and 13C-glucose.
(D) Fold change (qRT-PCR) of ACSS1 and ACSS2 mRNA in primary astrocytes in culture at passages 2 (P2), 5 (P5), and 6 (P6). ∗p < 0.01; ∗∗p < versus P2.
See also Figure S3B.
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8. Figure 6 Oxidation of [1,213C]-Acetate in a Patient with GBM
(A) Preoperative sagittal image from gadolinium-enhanced MRI shows a large enhancing tumor in the left frontotemporal region (arrow).
(B) Strong ACSS2 immunoreactivity in the tumor. Scale bar, 10 μm.
(C) 13C-NMR spectrum with GLU4 and GLN4 insets. Note the prominent D45 and Qs reflecting robust 13C-acetate oxidation. Abbreviations are the same as in Figure 2. Chemical shift assignments are the same in Figure 7: 1, alanine C3; 2, lactate C3; 3, NAA C6; 4, acetate C2; 5, unassigned; 6, glutamine C3; 7, glutamate C3; 8, unassigned; 9, glutamine C4; 10, glutamate C4; 12, NAA C3; 16, glutamine C2; 17, glutamate C2.
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9. Figure 7
Infusion of [1,213C]Acetate in a Patient with a Non-Small Cell Lung Cancer Brain Metastasis
(A) Preoperative MRI sagittal image shows a gadolinium-enhancing tumor in the left cerebellum (yellow arrow) with a cystic component (orange arrow).
(B) Moderate ACSS2 immunoreactivity in the tumor (T) with a lack of staining in the surrounding stroma (S). Scale bar, 10 μm.
(C) 13C-NMR spectrum with GLU4, GLN4, and ASP3 insets. Abbreviations are the same as in Figure 2. Chemical shift assignments are the same as in Figure 6, with the following additions: 11, aspartate C3; 13, glycine C2; 14, alanine C2; 15, aspartate C2.
See also Figure S4 for 13C-spectra from two additional patient tumors.
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10. Figure S1
Oxidation of Acetate in Paired Initial and Recurrent GBM, Related to Figure 2
(A) Coronal image from brain MRI with gadolinium shows a heterogeneously enhancing mass. UT-GBM6 HOT line was derived from this tumor at the time of surgical resection.
(B) Coronal images from MRI with gadolinium at the time of completion of treatment (upper image – post-treatment) and at the time of surgery for recurrent tumor (lower image). Note that the recurrent tumor was in the same location and had the same radiographic features of the initial tumor. UT-GBM7 HOT line was derived from the recurrent tumor at the time of repeat resection. Mice from both lines were coinfused with [1,6-13C]glucose and [1,2-13C]acetate and the 13C-NMR profiles are shown. The insets, GLU4 and GLN4, correspond to peaks 7 and 8 in each full spectrum. The pattern of labeling was strikingly similar in both tumors with a higher D45 peak than D34 peak and similar pattern in GLU4 and GLN4. Abbreviations and chemical shift assignments are the same as in Figure 1.
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11. Figure S2
Glutamine Is Not Directly Oxidized in the CAC, Related to Figure 3
(A and B) [U-13C]glutamine infusion in HOT mice from 2 brain metastasis tumor lines: non-small cell lung cancer (A) and endometrial adenocarcinoma (B). The labeling patterns in GLU4 and GLN4 are different reflecting glutamine uptake into the tumor producing large Qs in GLN4, smaller Qs in GLU4 from exchange with 13C-glutamine. However, the D45 in GLU4 is more prominent than in GLN4. These patterns are the same as in the spectrum from the melanoma HOT (Figure 3C). Abbreviations same as Figure 2.
(C) 13C-NMR spectral region showing the labeling pattern in lactate (LAC3) and alanine (ALA3) in the same mouse from the lung cancer brain metastasis HOT line infused with [U-13C]glutamine. The labeling pattern in LAC3 and ALA3 are similar in all 3 tissue compartments (liver, NT brain, and tumor). (D) Full 13C-NMR spectrum from the liver of the mouse in (C), showing prominent labeling in all carbons of glucose.
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12. Figure S3
ACSS2 Is Upregulated in Brain Tumors and May Be a Critical Enzyme for Enabling Acetate Oxidation in These Tumors, Related to Figures 4 and 5
(A) ACSS2 IHC in the 5 HOT tumors used for the coinfusion studies. Shown here are representative sections (scale bar 10 um) from melanoma, clear cell renal cell carcinoma, endometrial carcinoma, breast cancer and non-small cell lung cancer. In each case, classic histopathological architecture is seen in the tumors and there is moderate to strong ACSS2 staining.
(B) Uptake of 13C-acetate into glutamate in ACSS2 null MEFs in the presence of vector alone (VA) or re-introduction of the WT ACSS2 gene into the MEFs.
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13. Figure S4
13C-NMR from Resected Patient Tumors after Infusion of [1,213C]Acetate, Related to Figure 7
(A and B) Two patients underwent infusion of [1,213C]acetate during surgery to resect (A) a right parietal GBM and (B) left cerebellar brain metastasis from breast cancer (ER and PR positive, HER2 negative). The 13C-NMR spectra from the tumors are remarkably similar to each other and to the other 2 patient tumors shown in Figures 6 (GBM) and 7 (lung cancer brain metastasis). Insets GLU4 and GLN5 (numbers 10, 9, respectively, in the full spectra) show prominent D45 labeling and the presence of Qs consistent with robust acetate oxidation. The labeling and chemical shift assignments are the same as in Figure 7.
Cell  , DOI: ( /j.cell )
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