adp was more abundant in the more oxidative tumour regions ( extended data fig. 4c ), consistent with a high intramitochondrial adp concentration. 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 oxi diz ed 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 ). next, we analysed contiguous sections by imaging mass cytometry ( imc ) for the presence of immune cells, blood vessels and proliferating cells. we defined five immune phenotypes: cd3 cd45 cd4 ( helper t cells ); cd3 cd45 cd8 ( cytotoxic t cells ); cd3 cd45 cd8 gzmb ( activated t cells ); cd45 gzmb ( natural killer or neutrophils ); and cd68 ( macrophages or microglia ). vascular phenotypes included large vessels ( asma collageni panck cd31 ) and small vessels or capillaries ( cd31 collageni ). 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. however, the proportion of immune cells was small, representing less than 10 % of the total cell population. cell density could also not account for the observed metabolic heterogeneity ( extended data fig. 5c ). regions of necrosis, pseudopalisading necrosis and microvascular proliferation showed a lack of correlation with metabolically distinc t areas ( extended data fig. 5d ). 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 glyco lytic and those containing neurons were predominantly oxidative ( fig. 2j ). 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 ). 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 ). 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. 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. we selected large vessels ( collagen i and αsma ) ( extended data fig. 7a ), co - registered these with contiguous msi sections and measured c - labelled glyco lytic and tca cycle metabolites at 65 μm intervals from the blood vessel lumen. 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 ). however, there was a decrease in the number of immune cells with increasing distance from the vessel wall ( extended data fig. 7f, g ). 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 ). these were grown as neurospheres with [ u - c ] glucose before snap - freezing and sectioning for msi analysis ( fig. 3a – c ). three - dimensional culture has been shown to more closely approximate the behaviour of the primary tumour. the ms images were segmented using the same k - means clustering as for the human data ( fig. 3d ). the neurospheres derived from each cell line showed distinc t metabolic states ( fig. 3a ). 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 ). although the segmentation allowed us to group the spheres and the cells from which they were derived into distinc t metabolic phenotypes, these nevertheless represent a continuum of glyco lytic and tca cycle activities ( extended data fig. 8a ). rna sequencing showed that spheres with a glyco lytic phenotype had an upregulation of glyco lytic genes and those regulated by hypoxia ( extended data fig. 8b ). however, there was no difference in the oxidative or tca cycle gene expression profiles. 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. gtp2 med ( medial tumour ) and gtp2 lat ( lateral tumour ) formed similar - sized neurospheres but showed different metabolic phenotypes, with gtp2 med being more glyco lytic and clustering into state 3 and gtp2 lat clustering into state 1. similarly, at21 ant ( anterior ) and at21 post ( posterior ), from another patient, displayed an oxidative and mixed metabolic phenotype, respectively ( fig. 3f, g ). therefore, metabolic heterogeneity observed in vivo was retained following tissue dissociation and growth outside of the native tme. to test the robustness of the tumour - cell - intrinsic metabolic phenotype, we grew a subset of neurospheres, representative of the highly glyco lytic to the more oxidative phenotypes, under normoxic and hypoxic conditions ( 0. 5 % o 2 ) and compared their transcriptomes. despite prolonged exposure to hypoxia ( 160 h ), there was minimal change in the transcriptomes ( extended data fig. 8c ). 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 glyco lysis genes under normoxic conditions and oxidative phosphorylation, and epithelial mesenchymal transition genes under hypoxic conditions ( supplementary table 4 ). 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. at8 and s2 formed more glyco lytic xenografts, as demonstrated by higher [ u - c ] lactate labelling, whereas at5 xenografts were more oxidative with higher abundance of [ c 2 ] glutamate ( fig. 4a, b ). 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 ). 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. although when evaluated alongside all the other neurospheres, a11 and s2 appeared similarly glyco lytic ( extended data fig. 8a ), s2 xenografts showed greater tca cycle activity than a11 xenografts ( fig. 4c, d ).