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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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j, Quantification of TME-deconvolved populations (x axis, population; y axis, normalized weight per spot for each phenotype).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Box plot median and range as in h. k, Spatial maps showing that areas enriched for tumour cells display all three metabolic states.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Highlighted areas show that these metabolic phenotypes arise from cells displaying strong tumour signals rather than from cells of the TME.Source data a, Heatmap showing the average intensities of the seven metabolites used for segmentation of the three metabolic regions.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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b, Representative example of three tumour sections segmented into three clusters.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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The region containing state 3 cells (blue) showed high [U-C]lactate and [U-C]pyruvate labelling and was considered to have a glycolytic phenotype.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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The region containing state 2 cells (yellow) showed high labelling of [C2]glutamate and was considered to have an oxidative phenotype.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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The region containing state 1 cells (red) showed low labelling of glycolytic and TCA cycle metabolites.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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c, Metabolic segmentation based on spatial RNA sequencing of sections contiguous with those shown in b. Blue spots correspond to a glycolytic phenotype, and yellow spots correspond to an oxidative phenotype based on Hallmark gene sets.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Red spots correspond to cells with low glycolytic and oxidative gene signatures.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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d, ATP/ADP and PCr/ATP signal intensity ratios (mean; error bars, s.d.)
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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in normal-appearing brain (n = 4) and in the three metabolically defined regions (region 1, n = 22; region 2, n = 14; region 3, n = 17); every dot is a unique region.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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No statistical significance was identified using one-way ANOVA with Tukey’s multiple comparisons test.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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e, Redox status (mean; error bars, s.d.)
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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in normal-appearing brain tissue and GB regions quantified using AsA:DHA, GSH:GSSG and [U-C]lactate/pyruvate ratios (normal brain, n = 4; region 1, n = 22; region 2, n = 14; region 3, n = 17).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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No statistical significance was identified using one-way ANOVA with Tukey’s multiple comparisons test.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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f, Quantification of Ki67 cells, immune cells and blood vessels (mean; error bars, s.d.)
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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in the three metabolically defined regions based on immunohistochemical analysis (region 1, n = 17; region 2, n = 4; region 3, n = 8).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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No statistical significance was identified using one-way ANOVA with Tukey’s multiple comparisons test.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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g, Spatial co-assignment of deconvolved tumour/TME signals (first row) and MSI labels (second row).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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h, Violin plots showing tumour signal distribution by metabolic phenotype.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Box plots display the median (50th percentile) as the central line, with boxes spanning the 25th and 75th percentiles.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Whiskers extend to the minimum and maximum values within 1.5× the interquartile range; Glyco, n = 5,440; Low, n = 8,038; Oxphos, n = 4,508.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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i, Spatial uniform manifold approximation and projection (UMAP) plot of tumour-enriched spots from all metabolic phenotypes.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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j, Quantification of TME-deconvolved populations (x axis, population; y axis, normalized weight per spot for each phenotype).
|
PMC12116388
|
Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
|
Box plot median and range as in h. k, Spatial maps showing that areas enriched for tumour cells display all three metabolic states.
|
PMC12116388
|
Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
|
Highlighted areas show that these metabolic phenotypes arise from cells displaying strong tumour signals rather than from cells of the TME.
|
PMC12116388
|
Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Source data To exclude perfusion and hypoxia as explanations for the observed metabolic heterogeneity, we assessed cellular energy status from measurements of the ATP, ADP and PCr concentrations.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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All tumour regions had ATP/ADP and PCr/ATP ratios comparable to those in normal brain (Fig. 2d).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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ADP was more abundant in the more oxidative tumour regions (Extended Data Fig. 4c), consistent with a high intramitochondrial ADP concentration.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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The three metabolic states and normal brain showed similar redox status, as assessed from measurements of the ascorbic acid to dehydroascorbic acid (AsA:DHA) and reduced to oxidized glutathione (GSH:GSSG) ratios, reflecting the NADPH/NADP ratio, and the [U-C]lactate/[U-C]pyruvate ratios, reflecting the NADH/NAD ratio (Fig. 2e).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Next, we analysed contiguous sections by imaging mass cytometry (IMC) for the presence of immune cells, blood vessels and proliferating cells.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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We defined five immune phenotypes: CD3CD45CD4 (helper T cells); CD3CD45CD8 (cytotoxic T cells); CD3CD45CD8GZMB (activated T cells); CD45GZMB (natural killer or neutrophils); and CD68 (macrophages or microglia).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Vascular phenotypes included large vessels (ASMACollagenIpanCKCD31) and small vessels or capillaries (CD31CollagenI).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Regions occupied by the different metabolic states showed similar numbers of Ki67 cells, immune cell phenotypes and blood vessels (Fig. 2f and Extended Data Fig. 5a,b), with the only significant difference being slightly fewer CD68 cells in the oxidative regions.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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However, the proportion of immune cells was small, representing less than 10% of the total cell population.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Cell density could also not account for the observed metabolic heterogeneity (Extended Data Fig. 5c).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Regions of necrosis, pseudopalisading necrosis and microvascular proliferation showed a lack of correlation with metabolically distinct areas (Extended Data Fig. 5d).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Tumour areas identified by a strong malignant signal in the spatial transcriptomics data showed the three metabolic states in spatially coherent areas on co-registered MS images (Fig. 2g–i), whereas regions identified as containing immune cells were predominantly glycolytic and those containing neurons were predominantly oxidative (Fig. 2j).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Regions that showed a strong malignant signal and that were largely devoid of TME signals displayed all three metabolic states in the MS images (Fig. 2k).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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There were no differences in carbonic anhydrase IX (CAIX) transcript levels between the three metabolic states (Extended Data Fig. 6a), suggesting similar levels of hypoxia, or in immune, vascular, neuron, astrocyte and oligodendrocyte cell populations (Extended Data Fig. 6b).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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The bioenergetic status and microenvironment of the three metabolic states indicate that differences in metabolic activity are unlikely to have been influenced by differences in tissue perfusion, the presence of necrosis, differences in cell proliferation or the presence of immune cell infiltrates, but rather represent tumour-cell-intrinsic metabolic phenotypes.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Despite there being no significant correlation between metabolic phenotype and blood vessel density, we nevertheless investigated a possible relationship between metabolic phenotype and proximity to the vasculature.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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We selected large vessels (Collagen I and αSMA) (Extended Data Fig. 7a), co-registered these with contiguous MSI sections and measured C-labelled glycolytic and TCA cycle metabolites at 65 μm intervals from the blood vessel lumen.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Regardless of distance, there were no differences in lactate and glutamate labelling (Extended Data Fig. 7b), the number of proliferating cells, as indicated by Ki67 staining (Extended Data Fig. 7c), the NADPH/NADP ratio (AsA:DHA ratio) or the NADH/NAD ratio ([U-C]lactate/[U-C]pyruvate ratio) (Extended Data Fig. 7d,e).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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However, there was a decrease in the number of immune cells with increasing distance from the vessel wall (Extended Data Fig. 7f,g).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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To confirm that the metabolic phenotypes are tumour-cell-intrinsic and not a consequence of differences in the TME, we derived 30 primary cell lines from 26 patients with GB (two patients had two cell lines derived from multi-regional tumour sampling).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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These were grown as neurospheres with [U-C]glucose before snap-freezing and sectioning for MSI analysis (Fig. 3a–c).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Three-dimensional culture has been shown to more closely approximate the behaviour of the primary tumour.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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The MS images were segmented using the same k-means clustering as for the human data (Fig. 3d).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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The neurospheres derived from each cell line showed distinct metabolic states (Fig. 3a).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Neurosphere diameter was similar for all three metabolic phenotypes, which showed similar AMP, ADP, ATP and PCr signal intensities and ATP/ADP ratios (Fig. 3e).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Although the segmentation allowed us to group the spheres and the cells from which they were derived into distinct metabolic phenotypes, these nevertheless represent a continuum of glycolytic and TCA cycle activities (Extended Data Fig. 8a).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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RNA sequencing showed that spheres with a glycolytic phenotype had an upregulation of glycolytic genes and those regulated by hypoxia (Extended Data Fig. 8b).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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However, there was no difference in the oxidative or TCA cycle gene expression profiles.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Fig. 3Primary neurospheres recapitulate metabolic phenotypes in patients with GB.a, k-means clustering map of 30 cell lines grown as neurospheres in Matrigel domes (outlined in grey) using the seven C-labelled metabolites from glycolysis and the TCA cycle.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Red corresponds to metabolic state 1 (low glycolytic, low TCA cycle activity), yellow corresponds to metabolic state 2 (high TCA cycle activity) and blue represents metabolic state 3 (high glycolytic activity).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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b, Representative H&E-stained sections of spheres displaying one of the three metabolic states.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Red, yellow and blue outlines correspond to the sphere domes in a. c, Signal intensity maps for [U-C]lactate, [C2]glutamate and the ATP/ADP ratio in spheres shown in b. d, Heatmap showing the average signal intensities of the seven metabolites used for segmentation of the three metabolic states.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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e, Top: sphere diameter for each metabolic phenotype.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Each point represents a single sphere.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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State 3 (glycolytic phenotype) had a higher average sphere diameter than state 1 (low glycolytic, low TCA cycle activity) (P = 0.027).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Bottom: ATP, ADP, AMP and PCr signal intensities in neurospheres with different metabolic phenotypes.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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f, k-means segmentation of MS images of spheres derived from multi-regional tumour sampling (GTP2 and AT21).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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These results are part of the k-means analysis performed on all 30 neurosphere lines.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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g, Signal intensities of [U-C]lactate and [C2]glutamate in the indicated neurospheres (****P < 0.0001).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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The colour of the bar corresponds to the metabolic phenotype.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Data are means; error bars, s.d.;
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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each dot represents a single neurosphere.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Asterisks refer to P values obtained from one-way ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test (*P < 0.05, ***P < 0.0005, ****P < 0.00005).Source data a, k-means clustering map of 30 cell lines grown as neurospheres in Matrigel domes (outlined in grey) using the seven C-labelled metabolites from glycolysis and the TCA cycle.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Red corresponds to metabolic state 1 (low glycolytic, low TCA cycle activity), yellow corresponds to metabolic state 2 (high TCA cycle activity) and blue represents metabolic state 3 (high glycolytic activity).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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b, Representative H&E-stained sections of spheres displaying one of the three metabolic states.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Red, yellow and blue outlines correspond to the sphere domes in a. c, Signal intensity maps for [U-C]lactate, [C2]glutamate and the ATP/ADP ratio in spheres shown in b. d, Heatmap showing the average signal intensities of the seven metabolites used for segmentation of the three metabolic states.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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e, Top: sphere diameter for each metabolic phenotype.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Each point represents a single sphere.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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State 3 (glycolytic phenotype) had a higher average sphere diameter than state 1 (low glycolytic, low TCA cycle activity) (P = 0.027).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Bottom: ATP, ADP, AMP and PCr signal intensities in neurospheres with different metabolic phenotypes.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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f, k-means segmentation of MS images of spheres derived from multi-regional tumour sampling (GTP2 and AT21).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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These results are part of the k-means analysis performed on all 30 neurosphere lines.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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g, Signal intensities of [U-C]lactate and [C2]glutamate in the indicated neurospheres (****P < 0.0001).
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PMC12116388
|
Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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The colour of the bar corresponds to the metabolic phenotype.
|
PMC12116388
|
Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
|
Data are means; error bars, s.d.;
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PMC12116388
|
Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
|
each dot represents a single neurosphere.
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PMC12116388
|
Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Asterisks refer to P values obtained from one-way ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test (*P < 0.05, ***P < 0.0005, ****P < 0.00005).
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PMC12116388
|
Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Source data Next, we looked at neurospheres derived from multi-regional sampling of the same tumour to determine whether the neurospheres captured the regional metabolic heterogeneity observed in the tumour samples.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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GTP2 Med (medial tumour) and GTP2 Lat (lateral tumour) formed similar-sized neurospheres but showed different metabolic phenotypes, with GTP2 Med being more glycolytic and clustering into state 3 and GTP2 Lat clustering into state 1.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Similarly, AT21 Ant (anterior) and AT21 Post (posterior), from another patient, displayed an oxidative and mixed metabolic phenotype, respectively (Fig. 3f,g).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Therefore, metabolic heterogeneity observed in vivo was retained following tissue dissociation and growth outside of the native TME.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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To test the robustness of the tumour-cell-intrinsic metabolic phenotype, we grew a subset of neurospheres, representative of the highly glycolytic to the more oxidative phenotypes, under normoxic and hypoxic conditions (0.5% O2) and compared their transcriptomes.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Despite prolonged exposure to hypoxia (160 h), there was minimal change in the transcriptomes (Extended Data Fig. 8c).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Gene set enrichment analysis of the PC1 loadings showed E2F and MYC targets and mTORC1 pathway genes in the top ten under both normoxic and hypoxic conditions, with hypoxia and glycolysis genes under normoxic conditions and oxidative phosphorylation, and epithelial mesenchymal transition genes under hypoxic conditions (Supplementary Table 4).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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To further test the cell-intrinsic nature of the metabolic phenotypes, we implanted A11, AT8, S2 and AT5 into the brains of athymic rats and found that the cells retained the metabolic phenotypes that were observed when they were grown as neurospheres.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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AT8 and S2 formed more glycolytic xenografts, as demonstrated by higher [U-C]lactate labelling, whereas AT5 xenografts were more oxidative with higher abundance of [C2]glutamate (Fig. 4a,b).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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There were no differences in cell proliferation (Ki67 staining), cell death (CC3 staining) or vascularization (CD3 staining) between these three models, which paralleled the finding in the human data that proliferation rate and vascularization were not responsible for the differences in the observed metabolic phenotypes (Extended Data Fig. 8d,e).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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We have shown previously that S2 cells are more sensitive than A11 cells to irradiation (referred to as GB1 and GB4, respectively, in the earlier publication) and that S2 xenografts are more sensitive than A11 xenografts to treatment with temozolomide plus irradiation.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Although when evaluated alongside all the other neurospheres, A11 and S2 appeared similarly glycolytic (Extended Data Fig. 8a), S2 xenografts showed greater TCA cycle activity than A11 xenografts (Fig. 4c,d).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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We have shown previously that A11 cells show higher glycolytic activity and S2 cells higher oxygen consumption and lower glycolytic activity, and demonstrated this in the corresponding xenografts using deuterium magnetic resonance spectroscopy and spectroscopic imaging measurements of deuterium-labelled glucose metabolism in vivo.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Treatment of cells with AZD2014 (mTOR1 and mTOR2 inhibitor), imatinib (PDGFR; tyrosine kinase inhibitor) and gefitinib (EGFR; tyrosine kinase inhibitor) showed that cell viability was correlated with metabolic phenotype, where oxidative cells were more drug resistant (Extended Data Fig. 8f–h).Fig.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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4Neurospheres retain their metabolic signatures as orthotopically implanted xenografts, and these signatures correlate with drug response.a, Representative MSI sections from rat brains implanted with AT8, S2 and AT5 (n = 3 independent tumours per model).
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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Relative signal intensities for [U-C]lactate (top) and [C2]glutamate (bottom); H&E-staining of the corresponding sections.
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PMC12116388
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Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.
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b, The relative signal intensities of [U-C]lactate (****P < 0.0001) and [C2]glutamate (AT8 vs AT5, P = 0.0012; S2 vs AT5, P = 0.0406; AT8 vs AT5, P = 0.0012) in neurospheres (AT8, n = 4; S2, n = 5; AT5, n = 4) and the respective xenografts (AT8, n = 3; S2, n = 3; AT5, n = 3) expressed as mean values; error bars, s.d. (
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