Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	[C2]glutamate AT8 vs AT5, P = 0.0015; S2 vs AT5, P = 0.0049). *
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	P < 0.05; **P < 0.005.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	c, Relative abundance (mean values; error bars, s.d.)
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	of labelled malate and fumarate in sections of A11 and S2 xenografts.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	d, Top: labelled malate and fumarate signal intensities in MS images of the brains of rats implanted with A11 (n = 12) and S2 (n = 12) xenografts.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Bottom: H&E-staining of the corresponding sections.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Asterisks refer to P values obtained from one-way ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test (****P < 0.0001).Source data a, Representative MSI sections from rat brains implanted with AT8, S2 and AT5 (n = 3 independent tumours per model).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Relative signal intensities for [U-C]lactate (top) and [C2]glutamate (bottom); H&E-staining of the corresponding sections.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	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. (
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	[C2]glutamate AT8 vs AT5, P = 0.0015; S2 vs AT5, P = 0.0049). *
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	P < 0.05; **P < 0.005.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	c, Relative abundance (mean values; error bars, s.d.)
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	of labelled malate and fumarate in sections of A11 and S2 xenografts.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	d, Top: labelled malate and fumarate signal intensities in MS images of the brains of rats implanted with A11 (n = 12) and S2 (n = 12) xenografts.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Bottom: H&E-staining of the corresponding sections.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Asterisks refer to P values obtained from one-way ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test (****P < 0.0001).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Source data The concentrations of unlabelled serine, threonine, glutamine and glutamate were significantly higher in oxidative regions in the patient tumour sections (ANOVA, Tukey’s P < 0.05), and in the neurospheres, the concentrations of unlabelled leucine/isoleucine, glutamine, glutamate, histidine and phenylalanine were significantly higher (ANOVA, Tukey’s P < 0.005) (Extended Data Fig. 9).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The oxidative phenotype in GB has been associated with increased fatty acid oxidation, and we observed that the concentrations of unlabelled fatty acids were higher in the more glycolytic regions in both the human and neurosphere data, as were intermediates in the pentose phosphate pathway.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The extent to which tumour metabolism is driven by cell-intrinsic mechanisms or microenvironmental pressures is an open question in tumour biology.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	To address this question, we measured the metabolic activity of GB within its native microenvironment using MS imaging of isotope labelling in rapidly quenched tissue from patients with GB.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	A similar approach has been used to map heterogeneity in fatty acid synthesis in gliomas implanted orthotopically in mice, to map metabolic activities in kidney and brain in mice and to image glycolytic activity in a lung metastasis model.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Using a targeted approach, we identified distinct glycolytic and oxidative metabolic phenotypes.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Although recent reports have identified metabolic phenotypes from a transcriptomic analysis, we describe here the classification of metabolic phenotypes based on measurements of metabolic activity in patient tumours in vivo at relatively high spatial resolution (65 μm).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The metabolic phenotypes occupied distinct territories that did not show significant differences in the cellular composition of their microenvironments, including immune cell infiltration, proliferative index and vascularization.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The presence of these metabolic phenotypes in distinct territories and their cellular composition was confirmed by spatial transcriptomics.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Importantly, we have shown that these tumour-cell-intrinsic metabolic phenotypes can be independent 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.	Previous studies have also shown that tumour subtype-specific protein and gene expression profiles can be independent of the tumour niche.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	We observed no change in lactate and glutamate labelling with distance from the blood vessels.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	This appears to be inconsistent with a study in an orthotopically implanted glioma cell line model (U87MG) in immunocompromised mice that showed high mitochondrial activity in cells adjacent to vessels and increased expression of hypoxia-related genes at increasing distance from the vessels.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	However, there were no significant increases in lactate concentration or differences in α-ketoglutarate concentrations in the cells with distance from the vessels in this study, although these concentrations may have been affected by the flow cytometric method used to sort the cells.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	An MSI study on an orthotopically implanted syngeneic murine model of isocitrate dehydrogenase 1 mutant GB in animals fed for 48 h with [U-C]glucose showed a remarkable degree of metabolic homogeneity in the tumours, in contrast to what was observed here in tumours from patients with GB, emphasising the significance of the observed metabolic phenotypes.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The cell-intrinsic nature of the metabolic phenotypes was confirmed using neurospheres grown in vitro, which reproduced the metabolic phenotypes observed in the patients and were preserved following their orthotopic implantation in rats.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Exposing the neurospheres to chronic hypoxia did not lead to significant changes in the neurosphere transcriptomes, demonstrating the robustness of these phenotypes and further underlining their independence from the TME.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	This is in contrast to a similar study in patients with lung cancer who were infused with [U-C]glucose, which concluded that given the observed differences in glucose oxidation in the TCA cycle in well-perfused versus poorly perfused regions, tumour perfusion in this case overrides tumour-cell-intrinsic metabolic phenotypes.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	A recent study in mouse models of leukaemia, pancreatic, lung and colon cancer showed that these tumours suppress TCA cycle activity relative to normal tissue.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	By contrast, the glutamate labelling observed here in the GB tumours was similar to that in normal-appearing brain.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Similar observations of substantial glucose oxidation in the TCA cycle have been made previously in human lung tumours and in non-imaging studies of patients with GB who were infused with [U-C]glucose.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Metabolic phenotypes with distinct therapeutic vulnerabilities have been identified in several cancers, including GB, in which GB cells with an oxidative phenotype were shown to be more sensitive to inhibitors of mitochondrial complex I and to radiation treatment.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	We have shown previously that S2 cells are more sensitive to irradiation in vitro and in vivo and have shown here, and previously, that they have a more oxidative phenotype with higher TCA cycle activity than A11 cells, which show a more glycolytic phenotype.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Previous RNA sequencing studies have shown that A11 has a mesenchymal phenotype, whereas S2 cells have a neural progenitor cell-like phenotype, consistent with a previously identified association between the mesenchymal and glycolytic phenotypes.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Cells displaying these distinct metabolic phenotypes occupy territories that are sufficiently large to be imaged clinically using techniques such as hyperpolarized C magnetic resonance imaging (MRI) and deuterium metabolic imaging.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	These metabolic phenotypes show a correlation with treatment responsiveness, suggesting that a personalized therapy approach may be possible in which metabolic imaging and phenotyping could be used to guide subsequent treatment selection.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	We have demonstrated here high-resolution imaging of isotope labelling of cellular metabolites in a human tumour in vivo.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	In conjunction with studies on patient-derived neurospheres and orthotopically implanted xenografts, we have demonstrated the presence of different metabolic phenotypes within GB that are tumour-cell-intrinsic and largely independent 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.	Three male patients from Addenbrooke’s Hospital, Cambridge, were infused with [U-C]glucose.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The selection criteria included first clinical presentation, MRI consistent with GB and no significant co-morbidities.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Following induction of anaesthesia, a pyrogen-free 5% solution of [U-C]glucose in sterile saline (Merck) was administered as a bolus of 8 g over 10 min followed by 8 g h continuous infusion, as described previously for patients with GB and several other tumour types.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Arterial blood was collected by a peripheral arterial line before bolus administration and then every 15 min following the start of infusion and at 15 min after the end of the infusion.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Tumour sampling was guided by intra-operative Stealth navigation and assessment of 5-ALA fluorescence.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Tumours were sampled between 90 and 150 min.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	A pituitary ronguer was used to transfer tumour samples directly into liquid nitrogen.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The freezing time was <5 s between tissue devascularization and immersion in liquid nitrogen.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The study was approved by the Central Cambridge Research Ethics Committee and was compliant with the Health Research Authority.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The study adhered to the principles of the Declaration of Helsinki and the Guidelines for Good Clinical Practice.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Participants did not receive financial compensation and gave informed consent.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Frozen tumour samples were embedded in a hydroxypropyl methylcellulose/polyvinylpyrrolidone hydrogel, and 10 µm-thick cryo-sections were obtained.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Sections were thaw-mounted onto Superfrost microscope slides for desorption electrospray ionization (DESI) and IMC experiments (Thermo Scientific), while sections prepared for MALDI-MSI were thaw-mounted onto conductive ITO-coated slides (Bruker Daltonik).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The sections were dried immediately and sealed in vacuum pouches for storage at −80 °C.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Human tumour samples were treated with UV-C light before MSI analysis to minimize aerosolization of potential pathogens.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Decontamination was performed in a sensor-controlled UV chamber (Opsytec Dr. Gröbel) at 250 mJ cm.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	DESI-MSI analysis was performed on a Q-Exactive mass spectrometer (Thermo Scientific) equipped with an automated 2D-DESI ion source (Prosolia).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The spectrometer was used with a home-made Swagelok DESI sprayer and a mixture of 95% methanol, 5% water delivered at a flow rate of 1.5 µl min and nebulized with nitrogen at a backpressure of 6 bar.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Human tumour samples were analysed in the mass range from 70 to 280 m/z with a mass resolution of 140,000 at 200 m/z.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The injection time was set at 500 ms, and data were acquired with a pixel size of 65 µm.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Following analysis, sections were stained with H&E and co-registered with the DESI-MS images.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The resulting .raw files were converted into .mzML files using ProteoWizard msConvert (v.3.0.4043) with the built-in peak picking algorithm and subsequently compiled to an .imzML file (imzML converter, v.1.3).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	All subsequent data processing was performed in SCiLS Lab (v.2022b, Bruker Daltonik).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	MALDI-MSI analysis was performed using a RapifleX Tissuetyper instrument (Bruker Daltonik) operated in negative ion detection mode.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	9-Aminoacridine, prepared in an 80:20 methanol-to-water ratio, was used as a matrix and spray-deposited using an automated spray system (M3-Sprayer, HTX technologies).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Mass spectra were acquired from 180 to 1,000 m/z with a pixel size of 40 µm.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	A total of 350 laser shots were summed up per pixel.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	For all experiments, the laser was operated with a repetition rate of 10 kHz.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Raw data were directly uploaded and processed with SCiLS Lab (v.2022b) software.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	DESI and MALDI data and images were normalized to the total ion current to compensate for signal variation during the course of the experiments, and acquisition parameters and data processing were identical for human tissue, neurospheres embedded in Matrigel and xenograft sections.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Snap-frozen tumour samples were homogenized with 5 µl mg of 2 M perchloric acid.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The extract was centrifuged at 13,000g for 15 min, and the pH of the supernatant was adjusted to 7.0 using 2 M KOH.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Extracts were lyophilized and dissolved in 550 µl deuterium oxide containing methylenediphosphonic acid at 100 nmol g tissue, which was added as a chemical shift and intensity standard.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Proton-decoupled P NMR spectra were acquired with more than 8,000 repetitions into 32,768 data points with an 11 µs 90° pulse, a repetition time of 7.2 s and a spectral width of 57 ppm (14,006 Hz).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Arterial blood collected during intra-operative infusion was centrifuged at 2,000g and 4 °C for 20 min to collect the plasma, which was snap-frozen and stored at −80 °C.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Samples were thawed on wet ice and aliquots diluted 50-fold with cold methanol:acetonitrile:water (50:30:20) in chilled tubes, vortexed for 10 min and centrifuged at 21,100g for 10 min at 4 °C, and then the supernatants stored at −80 °C.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	On the day of analysis, supernatants were centrifuged and aliquots transferred to a 96-well plate for analysis by hydrophilic interaction liquid chromatography (HILIC) high-resolution mass spectrometry (HRMS).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The HILIC–HRMS system consisted of a Shimadzu Nexera X2 UHPLC and Sciex 6600 Triple TOF mass spectrometer, using a SeQuant ZIC-pHILIC 5 µm 150 × 2.1 mm column (with a ZIC-pHILIC 20 × 2.1 mm guard column) at 45 °C.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The liquid chromatography gradient started at 80% acetonitrile and 20% 20 mM ammonium carbonate (pH 9.4), changing to 20% acetonitrile over 17 min at 200 µl min, with a further 11.5 min of column re-equilibration.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Each sample was injected twice for analysis in both positive and negative electrospray ionization with a full-scan m/z range of 75–1,000.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Data were acquired using Sciex Analyst TF and processed using Sciex MultiQuant software.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Extracted ion chromatograms were generated from the theoretical m/z ± 20 ppm.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Peak integration was reviewed manually, and the peak area of each metabolite and isotope was exported.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Primary human cell lines were derived at Addenbrooke’s Hospital, Cambridge, UK, as described previously.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Tissue collection was approved by a Regional Ethics Committee (REC18/EE/0283) and was compliant with the UK Human Tissue Act 2004.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Resected tissue samples were washed with Hanks’ balanced salt solution and minced using sterile razor blades, followed by digestion with Accutase (Sigma-Aldrich) for 60 min at 37 °C.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Single cells were isolated by filtration through a 40 µM filter (Falcon).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Cells were centrifuged at 350g, 21 °C, and the pellet was incubated with 2–3 ml of Red Blood Cell Lysis Buffer (Sigma-Aldrich) for 5 min at room temperature (21 °C).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Following centrifugation, cells were seeded at a density of 15,000 cells per cm in serum-free Neurobasal A medium (Gibco) supplemented with B27, N2, EGF and FGF growth factors (Sigma-Aldrich).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Cells were allowed to form aggregates, and the medium was changed 3 days post derivation.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	For neurosphere formation, cells were seeded in Ultra Low Attachment 96-well plates (Corning) at a density of 10,000 cells per well.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Sphere diameter was monitored using an IncuCyte microscope (Sartorius).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Spheres were embedded in 150 μl Matrigel (Corning) domes in 24-well plates (Corning).
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The domes were covered with 2 ml of fresh Neurobasal medium and grown for 24 h. The domes were then washed three times with PBS before 2 ml of fresh glucose-free Neurobasal medium supplemented with 25 mM [U-C]glucose was added.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	Following incubation for 24 h, the medium was removed and the domes were washed three times with ice-cold PBS and then snap-frozen in liquid nitrogen.
PMC12116388	Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of <sup>13</sup>C-labelled glucose metabolism.	The domes were sectioned to a thickness of 10 µm and analysed using DESI-MSI and MALDI-MSI, as described above.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

[C2]glutamate AT8 vs AT5, P = 0.0015; S2 vs AT5, P = 0.0049). *

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

P < 0.05; **P < 0.005.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

c, Relative abundance (mean values; error bars, s.d.)

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

of labelled malate and fumarate in sections of A11 and S2 xenografts.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

d, Top: labelled malate and fumarate signal intensities in MS images of the brains of rats implanted with A11 (n = 12) and S2 (n = 12) xenografts.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Bottom: H&E-staining of the corresponding sections.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Asterisks refer to P values obtained from one-way ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test (****P < 0.0001).Source data a, Representative MSI sections from rat brains implanted with AT8, S2 and AT5 (n = 3 independent tumours per model).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Relative signal intensities for [U-C]lactate (top) and [C2]glutamate (bottom); H&E-staining of the corresponding sections.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

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. (

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

[C2]glutamate AT8 vs AT5, P = 0.0015; S2 vs AT5, P = 0.0049). *

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

P < 0.05; **P < 0.005.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

c, Relative abundance (mean values; error bars, s.d.)

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

of labelled malate and fumarate in sections of A11 and S2 xenografts.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

d, Top: labelled malate and fumarate signal intensities in MS images of the brains of rats implanted with A11 (n = 12) and S2 (n = 12) xenografts.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Bottom: H&E-staining of the corresponding sections.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Asterisks refer to P values obtained from one-way ANOVA followed by Tukey’s multiple comparisons test or unpaired t-test (****P < 0.0001).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Source data The concentrations of unlabelled serine, threonine, glutamine and glutamate were significantly higher in oxidative regions in the patient tumour sections (ANOVA, Tukey’s P < 0.05), and in the neurospheres, the concentrations of unlabelled leucine/isoleucine, glutamine, glutamate, histidine and phenylalanine were significantly higher (ANOVA, Tukey’s P < 0.005) (Extended Data Fig. 9).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The oxidative phenotype in GB has been associated with increased fatty acid oxidation, and we observed that the concentrations of unlabelled fatty acids were higher in the more glycolytic regions in both the human and neurosphere data, as were intermediates in the pentose phosphate pathway.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The extent to which tumour metabolism is driven by cell-intrinsic mechanisms or microenvironmental pressures is an open question in tumour biology.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

To address this question, we measured the metabolic activity of GB within its native microenvironment using MS imaging of isotope labelling in rapidly quenched tissue from patients with GB.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

A similar approach has been used to map heterogeneity in fatty acid synthesis in gliomas implanted orthotopically in mice, to map metabolic activities in kidney and brain in mice and to image glycolytic activity in a lung metastasis model.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Using a targeted approach, we identified distinct glycolytic and oxidative metabolic phenotypes.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Although recent reports have identified metabolic phenotypes from a transcriptomic analysis, we describe here the classification of metabolic phenotypes based on measurements of metabolic activity in patient tumours in vivo at relatively high spatial resolution (65 μm).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The metabolic phenotypes occupied distinct territories that did not show significant differences in the cellular composition of their microenvironments, including immune cell infiltration, proliferative index and vascularization.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The presence of these metabolic phenotypes in distinct territories and their cellular composition was confirmed by spatial transcriptomics.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Importantly, we have shown that these tumour-cell-intrinsic metabolic phenotypes can be independent of the TME.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Previous studies have also shown that tumour subtype-specific protein and gene expression profiles can be independent of the tumour niche.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

We observed no change in lactate and glutamate labelling with distance from the blood vessels.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

This appears to be inconsistent with a study in an orthotopically implanted glioma cell line model (U87MG) in immunocompromised mice that showed high mitochondrial activity in cells adjacent to vessels and increased expression of hypoxia-related genes at increasing distance from the vessels.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

However, there were no significant increases in lactate concentration or differences in α-ketoglutarate concentrations in the cells with distance from the vessels in this study, although these concentrations may have been affected by the flow cytometric method used to sort the cells.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

An MSI study on an orthotopically implanted syngeneic murine model of isocitrate dehydrogenase 1 mutant GB in animals fed for 48 h with [U-C]glucose showed a remarkable degree of metabolic homogeneity in the tumours, in contrast to what was observed here in tumours from patients with GB, emphasising the significance of the observed metabolic phenotypes.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The cell-intrinsic nature of the metabolic phenotypes was confirmed using neurospheres grown in vitro, which reproduced the metabolic phenotypes observed in the patients and were preserved following their orthotopic implantation in rats.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Exposing the neurospheres to chronic hypoxia did not lead to significant changes in the neurosphere transcriptomes, demonstrating the robustness of these phenotypes and further underlining their independence from the TME.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

This is in contrast to a similar study in patients with lung cancer who were infused with [U-C]glucose, which concluded that given the observed differences in glucose oxidation in the TCA cycle in well-perfused versus poorly perfused regions, tumour perfusion in this case overrides tumour-cell-intrinsic metabolic phenotypes.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

A recent study in mouse models of leukaemia, pancreatic, lung and colon cancer showed that these tumours suppress TCA cycle activity relative to normal tissue.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

By contrast, the glutamate labelling observed here in the GB tumours was similar to that in normal-appearing brain.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Similar observations of substantial glucose oxidation in the TCA cycle have been made previously in human lung tumours and in non-imaging studies of patients with GB who were infused with [U-C]glucose.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Metabolic phenotypes with distinct therapeutic vulnerabilities have been identified in several cancers, including GB, in which GB cells with an oxidative phenotype were shown to be more sensitive to inhibitors of mitochondrial complex I and to radiation treatment.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

We have shown previously that S2 cells are more sensitive to irradiation in vitro and in vivo and have shown here, and previously, that they have a more oxidative phenotype with higher TCA cycle activity than A11 cells, which show a more glycolytic phenotype.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Previous RNA sequencing studies have shown that A11 has a mesenchymal phenotype, whereas S2 cells have a neural progenitor cell-like phenotype, consistent with a previously identified association between the mesenchymal and glycolytic phenotypes.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Cells displaying these distinct metabolic phenotypes occupy territories that are sufficiently large to be imaged clinically using techniques such as hyperpolarized C magnetic resonance imaging (MRI) and deuterium metabolic imaging.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

These metabolic phenotypes show a correlation with treatment responsiveness, suggesting that a personalized therapy approach may be possible in which metabolic imaging and phenotyping could be used to guide subsequent treatment selection.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

We have demonstrated here high-resolution imaging of isotope labelling of cellular metabolites in a human tumour in vivo.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

In conjunction with studies on patient-derived neurospheres and orthotopically implanted xenografts, we have demonstrated the presence of different metabolic phenotypes within GB that are tumour-cell-intrinsic and largely independent of the TME.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Three male patients from Addenbrooke’s Hospital, Cambridge, were infused with [U-C]glucose.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The selection criteria included first clinical presentation, MRI consistent with GB and no significant co-morbidities.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Following induction of anaesthesia, a pyrogen-free 5% solution of [U-C]glucose in sterile saline (Merck) was administered as a bolus of 8 g over 10 min followed by 8 g h continuous infusion, as described previously for patients with GB and several other tumour types.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Arterial blood was collected by a peripheral arterial line before bolus administration and then every 15 min following the start of infusion and at 15 min after the end of the infusion.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Tumour sampling was guided by intra-operative Stealth navigation and assessment of 5-ALA fluorescence.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Tumours were sampled between 90 and 150 min.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

A pituitary ronguer was used to transfer tumour samples directly into liquid nitrogen.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The freezing time was <5 s between tissue devascularization and immersion in liquid nitrogen.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The study was approved by the Central Cambridge Research Ethics Committee and was compliant with the Health Research Authority.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The study adhered to the principles of the Declaration of Helsinki and the Guidelines for Good Clinical Practice.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Participants did not receive financial compensation and gave informed consent.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Frozen tumour samples were embedded in a hydroxypropyl methylcellulose/polyvinylpyrrolidone hydrogel, and 10 µm-thick cryo-sections were obtained.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Sections were thaw-mounted onto Superfrost microscope slides for desorption electrospray ionization (DESI) and IMC experiments (Thermo Scientific), while sections prepared for MALDI-MSI were thaw-mounted onto conductive ITO-coated slides (Bruker Daltonik).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The sections were dried immediately and sealed in vacuum pouches for storage at −80 °C.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Human tumour samples were treated with UV-C light before MSI analysis to minimize aerosolization of potential pathogens.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Decontamination was performed in a sensor-controlled UV chamber (Opsytec Dr. Gröbel) at 250 mJ cm.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

DESI-MSI analysis was performed on a Q-Exactive mass spectrometer (Thermo Scientific) equipped with an automated 2D-DESI ion source (Prosolia).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The spectrometer was used with a home-made Swagelok DESI sprayer and a mixture of 95% methanol, 5% water delivered at a flow rate of 1.5 µl min and nebulized with nitrogen at a backpressure of 6 bar.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Human tumour samples were analysed in the mass range from 70 to 280 m/z with a mass resolution of 140,000 at 200 m/z.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The injection time was set at 500 ms, and data were acquired with a pixel size of 65 µm.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Following analysis, sections were stained with H&E and co-registered with the DESI-MS images.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The resulting .raw files were converted into .mzML files using ProteoWizard msConvert (v.3.0.4043) with the built-in peak picking algorithm and subsequently compiled to an .imzML file (imzML converter, v.1.3).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

All subsequent data processing was performed in SCiLS Lab (v.2022b, Bruker Daltonik).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

MALDI-MSI analysis was performed using a RapifleX Tissuetyper instrument (Bruker Daltonik) operated in negative ion detection mode.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

9-Aminoacridine, prepared in an 80:20 methanol-to-water ratio, was used as a matrix and spray-deposited using an automated spray system (M3-Sprayer, HTX technologies).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Mass spectra were acquired from 180 to 1,000 m/z with a pixel size of 40 µm.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

A total of 350 laser shots were summed up per pixel.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

For all experiments, the laser was operated with a repetition rate of 10 kHz.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Raw data were directly uploaded and processed with SCiLS Lab (v.2022b) software.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

DESI and MALDI data and images were normalized to the total ion current to compensate for signal variation during the course of the experiments, and acquisition parameters and data processing were identical for human tissue, neurospheres embedded in Matrigel and xenograft sections.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Snap-frozen tumour samples were homogenized with 5 µl mg of 2 M perchloric acid.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The extract was centrifuged at 13,000g for 15 min, and the pH of the supernatant was adjusted to 7.0 using 2 M KOH.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Extracts were lyophilized and dissolved in 550 µl deuterium oxide containing methylenediphosphonic acid at 100 nmol g tissue, which was added as a chemical shift and intensity standard.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Proton-decoupled P NMR spectra were acquired with more than 8,000 repetitions into 32,768 data points with an 11 µs 90° pulse, a repetition time of 7.2 s and a spectral width of 57 ppm (14,006 Hz).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Arterial blood collected during intra-operative infusion was centrifuged at 2,000g and 4 °C for 20 min to collect the plasma, which was snap-frozen and stored at −80 °C.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Samples were thawed on wet ice and aliquots diluted 50-fold with cold methanol:acetonitrile:water (50:30:20) in chilled tubes, vortexed for 10 min and centrifuged at 21,100g for 10 min at 4 °C, and then the supernatants stored at −80 °C.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

On the day of analysis, supernatants were centrifuged and aliquots transferred to a 96-well plate for analysis by hydrophilic interaction liquid chromatography (HILIC) high-resolution mass spectrometry (HRMS).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The HILIC–HRMS system consisted of a Shimadzu Nexera X2 UHPLC and Sciex 6600 Triple TOF mass spectrometer, using a SeQuant ZIC-pHILIC 5 µm 150 × 2.1 mm column (with a ZIC-pHILIC 20 × 2.1 mm guard column) at 45 °C.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The liquid chromatography gradient started at 80% acetonitrile and 20% 20 mM ammonium carbonate (pH 9.4), changing to 20% acetonitrile over 17 min at 200 µl min, with a further 11.5 min of column re-equilibration.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Each sample was injected twice for analysis in both positive and negative electrospray ionization with a full-scan m/z range of 75–1,000.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Data were acquired using Sciex Analyst TF and processed using Sciex MultiQuant software.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Extracted ion chromatograms were generated from the theoretical m/z ± 20 ppm.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Peak integration was reviewed manually, and the peak area of each metabolite and isotope was exported.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Primary human cell lines were derived at Addenbrooke’s Hospital, Cambridge, UK, as described previously.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Tissue collection was approved by a Regional Ethics Committee (REC18/EE/0283) and was compliant with the UK Human Tissue Act 2004.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Resected tissue samples were washed with Hanks’ balanced salt solution and minced using sterile razor blades, followed by digestion with Accutase (Sigma-Aldrich) for 60 min at 37 °C.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Single cells were isolated by filtration through a 40 µM filter (Falcon).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Cells were centrifuged at 350g, 21 °C, and the pellet was incubated with 2–3 ml of Red Blood Cell Lysis Buffer (Sigma-Aldrich) for 5 min at room temperature (21 °C).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Following centrifugation, cells were seeded at a density of 15,000 cells per cm in serum-free Neurobasal A medium (Gibco) supplemented with B27, N2, EGF and FGF growth factors (Sigma-Aldrich).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Cells were allowed to form aggregates, and the medium was changed 3 days post derivation.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

For neurosphere formation, cells were seeded in Ultra Low Attachment 96-well plates (Corning) at a density of 10,000 cells per well.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Sphere diameter was monitored using an IncuCyte microscope (Sartorius).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Spheres were embedded in 150 μl Matrigel (Corning) domes in 24-well plates (Corning).

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The domes were covered with 2 ml of fresh Neurobasal medium and grown for 24 h. The domes were then washed three times with PBS before 2 ml of fresh glucose-free Neurobasal medium supplemented with 25 mM [U-C]glucose was added.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

Following incubation for 24 h, the medium was removed and the domes were washed three times with ice-cold PBS and then snap-frozen in liquid nitrogen.

PMC12116388

Cell-intrinsic metabolic phenotypes identified in patients with glioblastoma, using mass spectrometry imaging of 13C-labelled glucose metabolism.

The domes were sectioned to a thickness of 10 µm and analysed using DESI-MSI and MALDI-MSI, as described above.