Project description:Background In vivo stable isotope tracing is useful for natively surveying glioma metabolism but can be difficult to implement. Stable isotope tracing is tractable using in vitro glioma models, but most models lack nutrient conditions and cell populations relevant to human gliomas. This limits our ability to study glioma metabolism in the presence of an intact tumor microenvironment (TME) and immune-metabolic crosstalk. Methods We optimized an in vitro stable isotope tracing approach for human glioma explants and glioma stem-like cell (GSC) lines that integrates human plasma-like medium (HPLM). We performed 15N2-glutamine tracing in GSC monocultures and human IDH-wildtype glioblastoma explants and developed an analytical framework to evaluate microenvironment-dependent metabolic features that distinguish them. We also conducted spatial transcriptomics to assess transcriptional correlates to metabolic activities. Results Human plasma-like medium culture preserved glioma explant viability and stemness while unmasking metabolic and immune programs suppressed by conventional culture conditions. Stable isotope tracing in HPLM revealed TME-dependent and TME-independent features of tumor metabolism. Tissue explants recapitulated tumor cell-intrinsic metabolic activities, such as synthesis of immunomodulatory purines. Unlike GSC monocultures, tissue explants captured tumor cell-extrinsic activities associated with stromal cell metabolism, as exemplified by astrocytic guanosine diphosphate mannose production in heterocellular explants. Finally, glioma explants displayed tumor subtype-specific metabolic reprogramming, including robust pyrimidine degradation in mesenchymal cells. Conclusions We present a tractable approach to assess glioma metabolism in vitro under physiologic nutrient levels and in the presence of an intact TME. This platform opens new avenues to interrogate glioma metabolism and its interplay with the immune microenvironment.
Project description:This dataset is part of a study aimed at developing algorithms for the quantification of stable isotope content in microorganisms after labeling them with stable isotope-labeled substrates. For this dataset Escherichia coli cultures were labeled with 2.5 % of 15N-labeled NH4Cl and grown either anaerobically or aerobically. Cultures of E. coli were grown in M9 minimal medium in which 2.5 % of the ammonium was replaced with 15N-labeled NH4Cl for >10 generations to achieve close to complete labeling of cells. Triplicate cultures were grown. Please note that the unlabeled ammonium that was used of course had a natural content of 15N of around 0.4 %, thus the 0% added label samples have an actual 15N content of 0.4 % and all added label is on top of this value. We included a tab delimited table with this submission providing details on all raw files.
Project description:System-wide metabolic homeostasis is crucial for maintaining physiological functions of living organisms. Stable-isotope tracing metabolomics allows to unravel metabolic activity quantitatively by measuring the isotopically labeled metabolites, but has been largely restricted by coverage. Yet, delineating system-wide metabolic homeostasis at the whole-organism level remains non-trivial. Here, we develop a global isotope tracing metabolomics technology to measure labeled metabolites with a metabolome-wide coverage. Using Drosophila as an aging model organism, we probe the in vivo tracing kinetics with quantitative information on labeling patterns, extents and rates on a metabolome-wide scale. We curate a system-wide metabolic network to characterize metabolic homeostasis and disclose a system-wide loss of metabolic coordinations that impacts both intra- and inter-tissue metabolic homeostasis significantly during Drosophila aging. Importantly, we reveal an unappreciated metabolic diversion from glycolysis to serine metabolism and purine metabolism as Drosophila aging. The developed technology facilitates a system-level understanding of metabolic regulation in living organisms.
Project description:Stable isotope tracing assays track few metabolites, yet cells use many nutrients to sustain nitrogen metabolism. Here, we create a platform for tracing 30 nitrogen isotope-labeled metabolites in parallel to enable a system-level understanding of cellular nitrogen metabolism. This platform reveals that while primitive cells engage both de novo and salvage pyrimidine synthesis pathways, differentiated cells nearly exclusively salvage uridine. This link between cell state and pyrimidine synthesis pathway preference persists in murine and human tissues. Mechanistically, we find that S1900 phosphorylation of CAD, the first enzyme of the de novo pathway, is induced by uridine deprivation in differentiated cells and constitutively enriched in primitive cells. Mimicking CAD S1900 phosphorylation in differentiated cells constitutively activates de novo pyrimidine synthesis, while blocking this modification impairs the cellular response to uridine starvation. Collectively, we establish a method for nitrogen metabolism profiling and define a mechanism of cell state-specific pyrimidine synthesis pathway choice.