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Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13 C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization. Production of inflammatory mediators by M1-polarized macrophages is thought to rely on suppression of mitochondrial metabolism in favor of glycolysis. Refining this concept, here the authors define metabolic targets of nitric oxide as responsible for the mitochondrial rewiring resulting from polarization.

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. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using β2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically. Myeloid derived suppressor cells (MDSC) are associated with tumourigenesis and therapy response. Here, the authors show that beta 2-adrenergic receptor activation in MDSC leads to metabolic rewiring which regulates chemotherapy response in preclinical models of blood cancer.

Background Metabolic perturbations arising from malignant transformation have not been systematically characterized in human lung cancers in situ . Stable isotope resolved metabolomic analysis (SIRM) enables functional analysis of gene dysregulations in lung cancer. To this purpose, metabolic changes were investigated by infusing uniformly labeled 13 C-glucose into human lung cancer patients, followed by resection and processing of paired non-cancerous lung and non small cell carcinoma tissues. NMR and GC-MS were used for 13 C-isotopomer-based metabolomic analysis of the extracts of tissues and blood plasma. Results Many primary metabolites were consistently found at higher levels in lung cancer tissues than their surrounding non-cancerous tissues. 13 C-enrichment in lactate, Ala, succinate, Glu, Asp, and citrate was also higher in the tumors, suggesting more active glycolysis and Krebs cycle in the tumor tissues. Particularly notable were the enhanced production of the Asp isotopomer with three 13 C-labeled carbons and the buildup of 13 C-2,3-Glu isotopomer in lung tumor tissues. This is consistent with the transformations of glucose into Asp or Glu via glycolysis, anaplerotic pyruvate carboxylation (PC), and the Krebs cycle. PC activation in tumor tissues was also shown by an increased level of pyruvate carboxylase mRNA and protein. Conclusion PC activation – revealed here for the first time in human subjects – may be important for replenishing the Krebs cycle intermediates which can be diverted to lipid, protein, and nucleic acid biosynthesis to fulfill the high anabolic demands for growth in lung tumor tissues. We hypothesize that this is an important event in non-small cell lung cancer and possibly in other tumor development.

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. Here, we showed that blocking 6-phosphogluconate dehydrogenase (6PGD) in the oxidative PPP resulted in substantial reduction of Tregs suppressive function and shifts toward Th1, Th2, and Th17 phenotypes which led to the development of fetal inflammatory disorder in mice model. These in turn improved anti-tumor responses and worsened the outcomes of colitis model. Metabolically, 6PGD blocked Tregs showed improved glycolysis and enhanced non-oxidative PPP to support nucleotide biosynthesis. These results uncover critical role of 6PGD in modulating Tregs plasticity and function, which qualifies it as a novel metabolic checkpoint for immunotherapy applications.

Delivering isotopic tracers for metabolic studies in rodents without overt stress is challenging. Current methods achieve low label enrichment in proteins and lipids. Here, we report noninvasive introduction of 13 C 6 -glucose via a stress-free, ad libitum liquid diet. Using NMR and ion chromatography-mass spectrometry, we quantify extensive 13 C enrichment in products of glycolysis, the Krebs cycle, the pentose phosphate pathway, nucleobases, UDP-sugars, glycogen, lipids, and proteins in mouse tissues during 12 to 48 h of 13 C 6 -glucose feeding. Applying this approach to patient-derived lung tumor xenografts (PDTX), we show that the liver supplies glucose-derived Gln via the blood to the PDTX to fuel Glu and glutathione synthesis while gluconeogenesis occurs in the PDTX. Comparison of PDTX with ex vivo tumor cultures and arsenic-transformed lung cells versus xenografts reveals differential glucose metabolism that could reflect distinct tumor microenvironment. We further found differences in glucose metabolism between the primary PDTX and distant lymph node metastases. Isotope tracer administration for probing metabolism in vivo is important to assess metabolic functions in a relevant physiological setting. Here, the authors report a non-invasive method of administering 13 C 6 - glucose to mouse models via liquid diet feeding to achieve deep metabolic network coverage.

The mechanisms underlying the metabolic adaptation of myeloid cells within the tumor microenvironment remain incompletely understood. Here, we identify 6-phosphogluconate dehydrogenase (6PGD), a rate-limiting enzyme in the pentose phosphate pathway (PPP), as an important regulator of monocytic-myeloid derived suppressor cell (M-MDSC) function. Our findings reveal that tumor M-MDSCs upregulate 6PGD expression via IL-6/STAT3 signaling. Blocking 6PGD, using either genetic or pharmacological approaches, impairs the immunosuppressive function of M-MDSCs and suppresses tumor growth. Mechanistically, 6PGD inhibition leads to the accumulation of its substrate, 6-phosphogluconate (6PG), within M-MDSCs, activates the JNK1-IRS1 and PI3K-AKT-pDRP1 signaling pathways, leading to mitochondrial fragmentation and elevated mitochondrial reactive oxygen species (ROS). This metabolic shift drives M-MDSCs toward an M1-like proinflammatory phenotype. Furthermore, 6PGD blockade synergizes with anti-PD-1 immunotherapy in a preclinical tumor model, substantially improving therapeutic outcomes. Our data reveals 6PGD as a possible therapeutic target to disrupt M-MDSC function and improve cancer immunotherapy outcomes. Myeloid derived suppressor cells (MDSC) use different metabolic mechanisms to adapt to the tumour microenvironment. Here the authors show that 6-phosphogluconate dehydrogenase (6PGD) is important for MSDC function and that blockade of 6PGD impaired MDSC function and suppresses tumour growth leading to metabolic and functional changes in the MDSC and a more pro-inflammatory phenotype.

Mitochondrial malic enzyme 2 (ME2) catalyzes the oxidative decarboxylation of malate to yield CO 2 and pyruvate, with concomitant reduction of dinucleotide cofactor NAD + or NADP + . We find that ME2 is highly expressed in many solid tumors. In the A549 non-small cell lung cancer (NSCLC) cell line, ME2 depletion inhibits cell proliferation and induces cell death and differentiation, accompanied by increased reactive oxygen species (ROS) and NADP + /NADPH ratio, a drop in ATP and increased sensitivity to cisplatin. ME2 knockdown impacts phosphoinositide-dependent protein kinase 1 (PDK1) and phosphatase and tensin homolog (PTEN) expression, leading to AKT inhibition. Depletion of ME2 leads to malate accumulation and pyruvate decrease and exogenous cell permeable dimethyl-malate (DMM) mimics the ME2 knockdown phenotype. Both ME2 knockdown and DMM treatment reduce A549 cell growth in vivo . Collectively, our data suggest that ME2 is a potential target for cancer therapy.

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. Lung organotypic tissue slice cultures (OTC) with patients’ native TME procured from primary and brain-metastasized (BM) non-small cell lung cancer (NSCLC) patients were treated with Pembrolizumab and/or beta-glucan (WGP, an innate immune activator). Metabolic tracing with 13 C 6 -Glc/ 13 C 5 , 15 N 2 -Gln, multiplex immunofluorescence, and digital spatial profiling (DSP) were employed to interrogate metabolic and functional responses to Pembrolizumab and/or WGP. Primary and BM PD-1 + lung cancer OTC responded to Pembrolizumab and Pembrolizumab + WGP treatments, respectively. Pembrolizumab activated innate immune metabolism and functions in primary OTC, which were accompanied by tissue damage. DSP analysis indicated an overall decrease in immunosuppressive macrophages and T cells but revealed microheterogeneity in immune responses and tissue damage. Two TMEs with altered cancer cell properties showed resistance. Pembrolizumab or WGP alone had negligible effects on BM-lung cancer OTC but Pembrolizumab + WGP blocked central metabolism with increased pro-inflammatory effector release and tissue damage. In-depth metabolic analysis and multiplex TME imaging of lung cancer OTC demonstrated overall innate immune activation by Pembrolizumab but heterogeneous responses in the native TME of a patient with primary NSCLC. Metabolic and functional analysis also revealed synergistic action of Pembrolizumab and WGP in OTC of metastatic NSCLC.

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. Further, as studies move further into spatially resolved biology, new technical problems have to be overcome owing to the small number of analytes present in the confines of a single cell or cell compartment. Here, we review the overall goals and solutions made possible by stable isotope tracing and their applications to models of increasing complexity. Finally, we discuss progress and outstanding difficulties in high-resolution spatially resolved tracer-based metabolic studies.

Objectives In this review we compare the advantages and disadvantages of different model biological systems for determining the metabolic functions of cells in complex environments, how they may change in different disease states, and respond to therapeutic interventions. Introduction All preclinical drug-testing models have advantages and drawbacks. We compare and contrast established cell, organoid and animal models with ex vivo organ or tissue culture and in vivo human experiments in the context of metabolic readout of drug efficacy. As metabolism reports directly on the biochemical state of cells and tissues, it can be very sensitive to drugs and/or other environmental changes. This is especially so when metabolic activities are probed by stable isotope tracing methods, which can also provide detailed mechanistic information on drug action. We have developed and been applying Stable Isotope-Resolved Metabolomics to examine metabolic reprogramming of human lung cancer cells in monoculture, in mouse xenograft/explant models, and in lung cancer patients in situ (Lane et al. in Omics 15:173–182, 2011 ; Fan et al. in Metabolomics 7(2):257–269, 2011a , in Pharmacol Ther 133:366–391, 2012a , in Metabolomics 8(3):517–527, b ; Xie et al. in Cell Metab 19:795–809, 2014 ; Ren et al. in Sci Rep 4:5414, 2014 ; Sellers et al. in J Clin Investig 125(2):687–698, 2015 ). We are able to determine the influence of the tumor microenvironment using these models. We have now extended the range of models to fresh human tissue slices, similar to those originally described by Warburg (Biochem Z 142:317–333, 1923 ), which retain the native tissue architecture and heterogeneity with a paired benign versus cancer design under defined cell culture conditions. This platform offers an unprecedented human tissue model for preclinical studies on metabolic reprogramming of human cancer cells in their tissue context, and response to drug treatment (Xie et al. 2014 ). As the microenvironment of the target human tissue is retained and individual patient’s response to drugs is obtained, this platform promises to transcend current limitations of drug selection for clinical trials or treatments Conclusions Development of ex vivo human tissue and animal models with humanized organs including bone marrow and liver show considerable promise for analyzing drug responses that are more relevant to humans. Similarly using stable isotope tracer methods with these improved models in advanced stages of the drug development pipeline, in conjunction with tissue biopsy is expected significantly to reduce the high failure rate of experimental drugs in Phase II and III clinical trials.