cell - intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of c - labelled glucose metabolism source paper: pmc12116388 transcriptomic studies have attempted to classify glioblastoma ( gb ) into subtypes that predict survival and have different therapeutic vulnerabilities. here we identified three metabolic subtypes: glyco lytic, oxidative and a mix of glyco lytic and oxidative, using mass spectrometry imaging of rapidly excised tumour sections from two patients with gb who were infused with [ u - c ] glucose and from spatial transcriptomic analysis of contiguous sections. the phenotypes are not correlated with microenvironmental features, including proliferation rate, immune cell infiltration and vascularization, are retained when patient - derived cells are grown in vitro or as orthotopically implanted xenografts and are robust to changes in oxygen concentration, demonstrating their cell - intrinsic nature. the spatial extent of the regions occupied by cells displaying these distinc t metabolic phenotypes is large enough to be detected using clinically applicable metabolic imaging techniques. a limitation of the study is that it is based on only two patient tumours, albeit on multiple sections, and therefore represents a proof - of - concept study. gb is the most common primary adult brain cancer. transcriptomic analyses have attempted to classify gb into subtypes that could predict treatment response, and a recent study that used a pathway - based classification defined metabolism - associated subtypes with distinc t therapeutic vulnerabilities. these included a mitochondrial subtype, which is associated with a more favourable clinical outcome and is sensitive to inhibitors of oxidative phosphorylation, and a glyco lytic, plurimetabolic subtype that is resistant to multiple treatment types. an important question is the extent to which the metabolism displayed by tumour cells in vivo is cell - intrinsic and how much is defined by the tumour microenvironment ( tme ). we addressed this question by using mass spectrometry imaging ( msi ) of rapidly excised tumour sections from patients with gb who were infused with [ u - c ] glucose immediately before surgery to image tumour cell metabolic activity in vivo and from a spatial transcriptomic analysis of adjacent sections. we infused three patients, two with gb and a third with an adenocarcinoma metastasis, with [ u - c ] glucose and performed msi on rapidly excised tumour tissue that was dissected during tumour debulking surgery ( fig. 1a ). we sampled 16 regions from two patients with gb and seven regions from the patient with a metastasis, the latter containing samples from normal - appearing cortex ( fig. 1b ). there were no significant differences in lactate and glutamate c labelling between the tumour mass and tumour margin ( extended data fig. 1a ). tissue sampling began 90 min after the start of infusion, during which time the plasma glucose fractional enrichment reached a steady state ( fig. 1c ). fractional labelling of lactate, an end product of the glyco lytic pathway, and of [ c 2 ] glutamate, which is labelled via α - ketoglutarate in the tricarboxylic acid ( tca ) cycle ( extended data fig. 1b ) in gb1 and gb2, were less than that of tumour glucose ( fig. 1d ), suggesting that neither had reached isotopic steady state and were therefore a measure of glyco lytic and tca cycle activity, respectively. there was a direct correlation between [ c 2 ] glutamate signal intensities and the intensities of the tca cycle intermediates [ c 2 ] malate, [ c 2 ] fumarate and [ c 2 ] succinate ( extended data fig. 1c ). the signal from ceramide - 1-phosphate ( 752. 596 m / z ) confirmed minimal variation in instrument performance during acquisition and uniformity of tissue preparation ( extended data fig. 1d ). mass errors on the metabolite ions and the mass isotopologue distributions of all detectable labelled metabolites are given in supplementary tables 1 and 2, respectively. the sampling technique rapidly arrested tissue metabolism, as indicated by the atp / adp and phosphocreatine pcr / atp ratios ( fig. 1e, f ), which decline rapidly in hypoxic or ischaemic brain tissue. preservation of the atp, adp and pcr concentrations was also demonstrated using p nuclear magnetic resonance ( nmr ) measurements ( extended data fig. 2a, b ). the metabolite c labelling observed was assumed, therefore, to be similar to that present in vivo. the atp and pcr signal intensities were similar in the two gb tumours but significantly higher than those in the metastasis ( extended data fig. 2c ). the concentrations of plasma amino acids and lactate did not change significantly during the infusion protocol. the plasma concentrations of c - labelled lactate and glutamine, although less than 10 % of the unlabelled concentrations, could have contributed to some of the labelled lactate and, via glutamine, the labelled glutamate observed in the tumour sections ( extended data fig. 2d and supplementary table 3 ). the metastatic adenocarcinoma occupied small, discrete areas surrounded by gliotic and normal - appearing brain parenchyma ( fig. 1g ) and therefore provided apparently normal brain tissue for comparative analysis. we segmented the mass spectrometry ( ms ) images using seven c - labelled metabolites from glyco lysis ( [ u - c ] pyruvate, [ u - c ] lactate ) and the tca cycle ( [ c 2 ] fumarate, [ c 2 ] succinate, [ c 2 ] malate, [ c 2 ] glutamate and [ c 2 ] glutamine ). this separated the tumour, gliotic and normal - appearing brain parenchyma and agreed with tissue classification performed by a histopathologist ( extended data fig. 2e ). next, we assessed glyco lytic activity and tca cycle activity in the tumours and in normal - appearing brain. [ u - c ] lactate signals were significantly lower in gb1 than in gb2 and the metastasis, whereas the [ c 2 ] glutamate signals in gb1 were significantly higher than in the metastasis ( fig. 1h ), reflecting higher glyco lytic and lower tca cycle activities in the metastasis. glutamate labelling in the gb tumours was comparable to that in normal brain. we segmented the gb ms images using the same seven c - labelled metabolites from glyco lysis and the tca cycle. we reasoned that the activity of these two pathways could result in four cellular states; however, the msi spectra did not contain a high glyco lytic, high tca cycle phenotype, and images were better segmented assuming only three metabolic states ( fig. 2a and extended data fig. 3a ): state 3 ( high glyco lysis, low tca cycle ), state 2 ( low glyco lysis, high tca cycle ) and state 1 ( low activity in both pathways ). segmentation assuming three or four metabolic states showed that these states occupied distinc t and spatially extensive regions ( fig. 2b and extended data fig. 3b – e ). as observed previously from transcriptomic data, each tumour had a predominant metabolic phenotype, with gb1 containing more of state 2 and gb2 containing more of state 3 ( extended data fig. 3f ). nevertheless, regions occupied by one of these three metabolic states co - existed in both tumours, sometimes within a single tumour section. spatial rna sequencing of adjacent tumour sections and segmentation using hallmark oxidative and glyco lytic gene sets also found three metabolic states: glyco lytic, oxidative and mixed ( extended data fig. 4a ), which showed a strong correlation with the msi data ( fig. 2c ) and an overall concordance of 65 % ( extended data fig. 4b ). 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. all tumour regions had atp / adp and pcr / atp ratios comparable to those in normal brain ( fig. 2d ).