NMR-Based Stable Isotope Tracing of Cancer Metabolism.

NMR is widely used for metabolite profiling (metabolomics, metabonomics) particularly of various readily obtainable biofluids such as plasma and urine. It is especially valuable for stable isotope tracer studies to track metabolic pathways under control or perturbed conditions in a wide range of cell models as well as animal models and human subjects. NMR has unique properties for utilizing stable isotopes to edit or simplify otherwise complex spectra acquired in vitro and in vivo, while quantifying the level of enrichment at specific atomic positions in various metabolites (i.

Stable isotope-resolved metabolomics analyses of metabolic phenotypes reveal variable glutamine metabolism in different patient-derived models of non-small cell lung cancer from a single patient.

Introduction: Stable isotope tracers have been increasingly used in preclinical cancer model systems, including cell culture and mouse xenografts, to probe the altered metabolism of a variety of cancers, such as accelerated glycolysis and glutaminolysis and generation of oncometabolites. Comparatively little has been reported on the fidelity of the different preclinical model systems in recapitulating the aberrant metabolism of tumors.

Objectives: We have been developing several different experimental model systems for systems biochemistry analyses of non-small cell lung cancer (NSCLC) using patient-derived tissues to evaluate appropriate models for metabolic and phenotypic analyses.

Challenges of Spatially Resolved Metabolism in Cancer Research.

Stable isotope-resolved metabolomics comprises a critical set of technologies that can be applied to a wide variety of systems, from isolated cells to whole organisms, to define metabolic pathway usage and responses to perturbations such as drugs or mutations, as well as providing the basis for flux analysis. As the diversity of stable isotope-enriched compounds is very high, and with newer approaches to multiplexing, the coverage of metabolism is now very extensive. However, as the complexity of the model increases, including more kinds of interacting cell types and interorgan communication, the analytical complexity also increases.

Metabolic Reprogramming in Human Cancer Patients and Patient-Derived Models.

Stable isotope-resolved metabolomics delineates reprogrammed intersecting metabolic networks in human cancers. Knowledge gained from in vivo patient studies provides the "benchmark" for cancer models to recapitulate. It is particularly difficult to model patients' tumor microenvironment (TME) with its complex cell-cell/cell-matrix interactions, which shapes metabolic reprogramming crucial to cancer development/drug resistance.

Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via β2-adrenergic receptor (β2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism.

Portable optical spectroscopic assay for non-destructive measurement of key metabolic parameters on cancer cells and organotypic fresh tumor slices.

To enable non-destructive metabolic characterizations on cancer cells and organotypic tumor models for therapeutic studies in an easy-to-access way, we report a highly portable optical spectroscopic assay for simultaneous measurement of glucose uptake and mitochondrial function on various cancer models with high sensitivity. Well-established breast cancer cell lines (MCF-7 and MDA-MB-231) were used to validate the optical spectroscopic assay for metabolic characterizations, while fresh tumor samples harvested from both animals and human cancer patients were used to test the feasibility of our optical metabolic assay for non-destructive measurement of key metabolic parameters on organotypic tumor slices. Our optical metabolic assay captured that MCF-7 cells had higher mitochondrial metabolism, but lower glucose uptake compared to the MDA-MB-231 cells, which is consistent with our microscopy imaging and flow cytometry data, as well as the published Seahorse Assay data.

Asparagine restriction enhances CD8 T cell metabolic fitness and antitumoral functionality through an NRF2-dependent stress response.

Robust and effective T cell immune surveillance and cancer immunotherapy require proper allocation of metabolic resources to sustain energetically costly processes, including growth and cytokine production. Here, we show that asparagine (Asn) restriction on CD8 T cells exerted opposing effects during activation (early phase) and differentiation (late phase) following T cell activation. Asn restriction suppressed activation and cell cycle entry in the early phase while rapidly engaging the nuclear factor erythroid 2-related factor 2 (NRF2)-dependent stress response, conferring robust proliferation and effector function on CD8 T cells during differentiation.

Differential Inhibition of Anaplerotic Pyruvate Carboxylation and Glutaminolysis-Fueled Anabolism Underlies Distinct Toxicity of Selenium Agents in Human Lung Cancer.

Past chemopreventive human trials on dietary selenium supplements produced controversial outcomes. They largely employed selenomethionine (SeM)-based diets. SeM was less toxic than selenite or methylseleninic acid (MSeA) to lung cancer cells.

Targeting mutant p53-R248W reactivates WT p53 function and alters the onco-metabolic profile.

is the most commonly mutated gene in cancer, and gain-of-function mutations have wide-ranging effects. Efforts to reactivate wild-type p53 function and inhibit mutant functions have been complicated by the variety of mutations. Identified from a screen, the NSC59984 compound has been shown to restore activity to mutant p53 in colorectal cancer cells.

Ultrahigh resolution MS/MS-based reconstruction of metabolic networks in mammalian cells reveals changes for selenite and arsenite action.

Metabolic networks are complex, intersecting, and composed of numerous enzyme-catalyzed biochemical reactions that transfer various molecular moieties among metabolites. Thus, robust reconstruction of metabolic networks requires metabolite moieties to be tracked, which cannot be readily achieved with mass spectrometry (MS) alone. We previously developed an Ion Chromatography-ultrahigh resolution-MS/data independent-MS method to track the simultaneous incorporation of the heavy isotopes C and N into the moieties of purine/pyrimidine nucleotides in mammalian cells.

GAB functions as a bioenergetic and signalling gatekeeper to control T cell inflammation.

γ-Aminobutyrate (GAB), the biochemical form of (GABA) γ-aminobutyric acid, participates in shaping physiological processes, including the immune response. How GAB metabolism is controlled to mediate such functions remains elusive. Here we show that GAB is one of the most abundant metabolites in CD4 T helper 17 (T17) and induced T regulatory (iT) cells.

Polarization and β-Glucan Reprogram Immunomodulatory Metabolism in Human Macrophages and Ex Vivo in Human Lung Cancer Tissues.

Immunomodulatory (IM) metabolic reprogramming in macrophages (Mϕs) is fundamental to immune function. However, limited information is available for human Mϕs, particularly in response plasticity, which is critical to understanding the variable efficacy of immunotherapies in cancer patients. We carried out an in-depth analysis by combining multiplex stable isotope-resolved metabolomics with reversed phase protein array to map the dynamic changes of the IM metabolic network and key protein regulators in four human donors' Mϕs in response to differential polarization and M1 repolarizer β-glucan (whole glucan particles [WGPs]).

Resolving Enantiomers of 2-Hydroxy Acids by Nuclear Magnetic Resonance.

Biologically important 2-hydroxy carboxylates such as lactate, malate, and 2-hydroxyglutarate exist in two enantiomeric forms that cannot be distinguished under achiral conditions. The D and L (or R, S) enantiomers have different biological origins and functions, and therefore, there is a need for a simple method for resolving, identifying, and quantifying these enantiomers. We have adapted and improved a chiral derivatization technique for nuclear magnetic resonance (NMR), which needs no chromatography for enantiomer resolution, with greater than 90% overall recovery.

A Micro-Scale Analytical Method for Determining Glycogen Turnover by NMR and FTMS.

Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose anchored to the protein glycogenin. Although glycogen primarily occurs in the liver and muscle, it is found in most tissues, and its metabolism has been shown to be important in cancers and immune cells. Robust analysis of glycogen turnover requires stable isotope tracing plus a reliable means of quantifying total and labeled glycogen derived from precursors such as 13C6-glucose.

Succinate dehydrogenase/complex II is critical for metabolic and epigenetic regulation of T cell proliferation and inflammation.

Effective T cell-mediated immune responses require the proper allocation of metabolic resources to sustain growth, proliferation, and cytokine production. Epigenetic control of the genome also governs T cell transcriptome and T cell lineage commitment and maintenance. Cellular metabolic programs interact with epigenetic regulation by providing substrates for covalent modifications of chromatin.

6-Phosphogluconate dehydrogenase (6PGD), a key checkpoint in reprogramming of regulatory T cells metabolism and function.

Cellular metabolism has key roles in T cells differentiation and function. CD4 T helper-1 (Th1), Th2, and Th17 subsets are highly glycolytic while regulatory T cells (Tregs) use glucose during expansion but rely on fatty acid oxidation for function. Upon uptake, glucose can enter pentose phosphate pathway (PPP) or be used in glycolysis.

Innate immune activation by checkpoint inhibition in human patient-derived lung cancer tissues.

Although Pembrolizumab-based immunotherapy has significantly improved lung cancer patient survival, many patients show variable efficacy and resistance development. A better understanding of the drug's action is needed to improve patient outcomes. Functional heterogeneity of the tumor microenvironment (TME) is crucial to modulating drug resistance; understanding of individual patients' TME that impacts drug response is hampered by lack of appropriate models.

NMR Analysis of Carboxylate Isotopomers of C-Metabolites by Chemoselective Derivatization with N-Cholamine.

A substantial fraction of common metabolites contains carboxyl functional groups. Their C isotopomer analysis by nuclear magnetic resonance (NMR) is hampered by the low sensitivity of the C nucleus, the slow longitudinal relaxation for the lack of an attached proton, and the relatively low chemical shift dispersion of carboxylates. Chemoselective (CS) derivatization is a means of tagging compounds in a complex mixture via a specific functional group.

NMR Methods for Determining Lipid Turnover via Stable Isotope Resolved Metabolomics.

Lipids comprise diverse classes of compounds that are important for the structure and properties of membranes, as high-energy fuel sources and as signaling molecules. Therefore, the turnover rates of these varied classes of lipids are fundamental to cellular function. However, their enormous chemical diversity and dynamic range in cells makes detailed analysis very complex.

Blockade of 6-phosphogluconate dehydrogenase generates CD8 effector T cells with enhanced anti-tumor function.

Although T cell expansion depends on glycolysis, T effector cell differentiation requires signaling via the production of reactive oxygen species (ROS). Because the pentose phosphate pathway (PPP) regulates ROS by generating nicotinamide adenine dinucleotide phosphate (NADPH), we examined how PPP blockade affects T cell differentiation and function. Here, we show that genetic ablation or pharmacologic inhibition of the PPP enzyme 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP results in the generation of superior CD8 T effector cells.

An Ion Chromatography-Ultrahigh-Resolution-MS/Data-Independent High-Resolution MS Method for Stable Isotope-Resolved Metabolomics Reconstruction of Central Metabolic Networks.

The metabolome comprises a complex network of interconnecting enzyme-catalyzed reactions that involve transfers of numerous molecular subunits. Thus, the reconstruction of metabolic networks requires metabolite substructures to be tracked. Subunit tracking can be achieved by tracing stable isotopes through metabolic transformations using NMR and ultrahigh -resolution (UHR)-mass spectrometry (MS).

Fumarate hydratase-deficient renal cell carcinoma cells respond to asparagine by activation of the unfolded protein response and stimulation of the hexosamine biosynthetic pathway.

Background: The loss-of-function mutation of fumarate hydratase (FH) is a driver of hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Fumarate accumulation results in activation of stress-related mechanisms leading to upregulation of cell survival-related genes. To better understand how cells compensate for the loss of FH in HLRCC, we determined the amino acid nutrient requirements of the FH-deficient UOK262 cell line (UOK262) and its FH-repleted control (UOK262WT).

Inosine is an alternative carbon source for CD8-T-cell function under glucose restriction.

T cells undergo metabolic rewiring to meet their bioenergetic, biosynthetic and redox demands following antigen stimulation. To fulfil these needs, effector T cells must adapt to fluctuations in environmental nutrient levels at sites of infection and inflammation. Here, we show that effector T cells can utilize inosine, as an alternative substrate, to support cell growth and function in the absence of glucose in vitro.

Resolving Metabolic Heterogeneity in Experimental Models of the Tumor Microenvironment from a Stable Isotope Resolved Metabolomics Perspective.

The tumor microenvironment (TME) comprises complex interactions of multiple cell types that determines cell behavior and metabolism such as nutrient competition and immune suppression. We discuss the various types of heterogeneity that exist in solid tumors, and the complications this invokes for studies of TME. As human subjects and in vivo model systems are complex and difficult to manipulate, simpler 3D model systems that are compatible with flexible experimental control are necessary for studying metabolic regulation in TME.