bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021‒05‒02
thirty-two papers selected by
Giovanny Rodriguez Blanco
University of Edinburgh

  1. Metabolites. 2021 Apr 26. pii: 270. [Epub ahead of print]11(5):
      Itaconate is derived from the tricarboxylic acid (TCA) cycle intermediate cis-aconitate and links innate immunity and metabolism. Its synthesis is altered in inflammation-related disorders and it therefore has potential as clinical biomarker. Mesaconate and citraconate are naturally occurring isomers of itaconate that have been linked to metabolic disorders, but their functional relationships with itaconate are unknown. We aimed to establish a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of itaconate, mesaconate, citraconate, the pro-drug 4-octyl-itaconate, and selected TCA intermediates. The assay was validated for itaconate, mesaconate, and citraconate for intra- and interday precision and accuracy, extended stability, recovery, freeze/thaw cycles, and carry-over. The lower limit of quantification was 0.098 µM for itaconate and mesaconate and 0.049 µM for citraconate in 50 µL samples. In spike-in experiments, itaconate remained stable in human plasma and whole blood for 24 and 8 h, respectively, whereas spiked-in citraconate and mesaconate concentrations changed during incubation. The type of anticoagulant in blood collection tubes affected measured levels of selected TCA intermediates. Human plasma may contain citraconate (0.4-0.6 µM, depending on the donor), but not itaconate or mesaconate, and lipopolysaccharide stimulation of whole blood induced only itaconate. Concentrations of the three isomers differed greatly among mouse organs: Itaconate and citraconate were most abundant in lymph nodes, mesaconate in kidneys, and only citraconate occurred in brain. This assay should prove useful to quantify itaconate isomers in biomarker and pharmacokinetic studies, while providing internal controls for their effects on metabolism by allowing quantification of TCA intermediates.
    Keywords:  CAD; Irg1; Krebs cycle; biomarker; cis-aconitate decarboxylase; citraconate; itaconate; mass spectrometry; mesaconate; metabolism
  2. Methods Mol Biol. 2021 ;2285 319-328
      Metabolomics, lipidomics, and the study of cellular metabolism are gaining increasing interest particularly in the field of immunology, since the activation and effector functions of immune cells are profoundly controlled by changes in cellular metabolic asset. Among the different techniques that can be used for the evaluation of cellular metabolism, the Seahorse Extracellular Flux Analyzer allows the real time measurement of both glycolytic and mitochondrial respiration pathways in cells of interest, through the assessment of extracellular acidification and oxygen consumption rate. Metabolomics, on the other hand, is the high-throughput analysis of metabolites, i.e., the substrates, intermediates, and products of cellular metabolism, starting from biofluids, cells or tissues. The metabolome does not include lipids as their properties are different from water-soluble metabolites and are classified under the lipidome. Lipidomics analysis allows the identification and quantification of lipid species. Metabolomics and lipidomics are currently performed with mass-spectrometry coupled with liquid or gas chromatography (LC-MS or GC-MS) and/or nuclear-magnetic resonance (NMR). Here we describe the protocol for the evaluation of metabolic rate, metabolomics, and lipidomics in T cells, examining the detailed experimental approaches.
    Keywords:  Bioenergetics; Glycolysis; Lipidomics; Metabolism; Metabolomics; Mitochondrial respiration; T cells
  3. Methods Mol Biol. 2021 ;2285 297-317
      The dynamic regulation of protein function by altered protein expression and post-translational modifications (PTMs) is essential for T cell function, but it has remained difficult to systemically quantify such events. Mass spectrometry (MS)-based proteomics has become a mainstream tool for comprehensive profiling of proteome and PTMs, especially with the development of multiplexed isobaric labeling methods, such as tandem mass tag (TMT), coupled with high-resolution two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). Here, we introduce a deep proteomics profiling protocol with an optimized 11-plex TMT-LC/LC-MS/MS platform to quantitate whole proteome, phosphoproteome, acetylome, and methylome in activated T cells. The major steps include preparation of activated T cells, protein extraction and digestion, TMT labeling, basic pH reverse phase LC, modified peptide enrichment, acidic pH reverse phase LC-MS/MS, and computational data processing. Approximately 10,000 proteins, 30,000 phosphosites, 2,000 lysine acetylated sites, and 1,000 lysine methylated sites can be identified and quantified from 1 mg of proteins per sample. Quality control steps are implemented in this protocol, and future development, such as nanoscale 16-plex TMT analysis, is discussed. This multiplexed and robust method provides a powerful tool for dissecting proteomic and PTM signatures in T cells at the systems level, and it is equally suitable for other biological samples, including effector T cell subsets.
    Keywords:  Acetylome; Isobaric labeling; Mass spectrometry; Methylome; Phosphoproteome; Post-translational modifications; Proteomics; T cell; Tandem mass tag
  4. Cell Metab. 2021 Apr 22. pii: S1550-4131(21)00168-6. [Epub ahead of print]
      Altered tissue mechanics and metabolism are defining characteristics of cancer that impact not only proliferation but also migration. While migrating through a mechanically and spatially heterogeneous microenvironment, changes in metabolism allow cells to dynamically tune energy generation and bioenergetics in response to fluctuating energy needs. Physical cues from the extracellular matrix influence mechanosignaling pathways, cell mechanics, and cytoskeletal architecture to alter presentation and function of metabolic enzymes. In cancer, altered mechanosensing and metabolic reprogramming supports metabolic plasticity and high energy production while cells migrate and metastasize. Here, we discuss the role of mechanoresponsive metabolism in regulating cell migration and supporting metastasis as well as the potential of therapeutically targeting cancer metabolism to block motility and potentially metastasis.
    Keywords:  bioenergetics; cancer invasion; energy metabolism; mechanotransduction; tumor microenvironment
  5. Int J Mol Sci. 2021 Apr 23. pii: 4419. [Epub ahead of print]22(9):
      The rewiring of lipid metabolism is a major adaptation observed in cancer, and it is generally associated with the increased aggressiveness of cancer cells. Targeting lipid metabolism is therefore an appealing therapeutic strategy, but it requires a better understanding of the specific roles played by the main enzymes involved in lipid biosynthesis. Lipin-1 is a central regulator of lipid homeostasis, acting either as an enzyme or as a co-regulator of transcription. In spite of its important functions it is only recently that several groups have highlighted its role in cancer. Here, we will review the most recent research describing the role of lipin-1 in tumor progression when expressed by cancer cells or cells of the tumor microenvironment. The interest of its inhibition as an adjuvant therapy to amplify the effects of anti-cancer therapies will be also illustrated.
    Keywords:  cancer; fatty acids; lipids; lipin-1; metabolism; phosphatidic acid phosphatase; propranolol
  6. Anal Chem. 2021 Apr 26.
      We present here a novel surface mass spectrometry strategy to perform untargeted metabolite profiling of formalin-fixed paraffin-embedded pediatric ependymoma archives. Sequential Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) and liquid extraction surface analysis-tandem mass spectrometry (LESA-MS/MS) permitted the detection of 887 metabolites (163 chemical classes) from pediatric ependymoma tumor tissue microarrays (diameter: <1 mm; thickness: 4 μm). From these 163 classes, 60 classes were detected with both techniques, whilst LESA-MS/MS and 3D OrbiSIMS individually allowed the detection of another 83 and 20 unique metabolite classes, respectively. Through data fusion and multivariate analysis, we were able to identify key metabolites and corresponding pathways predictive of tumor relapse, which were retrospectively confirmed by gene expression analysis with publicly available data. Altogether, this sequential mass spectrometry strategy has shown to be a versatile tool to perform high-throughput metabolite profiling on sample-limited tissue archives.
  7. Cell Metab. 2021 Apr 22. pii: S1550-4131(21)00169-8. [Epub ahead of print]
      NAD(H) and NADP(H) have traditionally been viewed as co-factors (or co-enzymes) involved in a myriad of oxidation-reduction reactions including the electron transport in the mitochondria. However, NAD pathway metabolites have many other important functions, including roles in signaling pathways, post-translational modifications, epigenetic changes, and regulation of RNA stability and function via NAD-capping of RNA. Non-oxidative reactions ultimately lead to the net catabolism of these nucleotides, indicating that NAD metabolism is an extremely dynamic process. In fact, recent studies have clearly demonstrated that NAD has a half-life in the order of minutes in some tissues. Several evolving concepts on the metabolism, transport, and roles of these NAD pathway metabolites in disease states such as cancer, neurodegeneration, and aging have emerged in just the last few years. In this perspective, we discuss key recent discoveries and changing concepts in NAD metabolism and biology that are reshaping the field. In addition, we will pose some open questions in NAD biology, including why NAD metabolism is so fast and dynamic in some tissues, how NAD and its precursors are transported to cells and organelles, and how NAD metabolism is integrated with inflammation and senescence. Resolving these questions will lead to significant advancements in the field.
    Keywords:  NAD pathway metabolites; NAD(+); aging; disease; humans; mitochondria; transport; vitamin B3
  8. Anal Bioanal Chem. 2021 Apr 28.
      Improving the reliability of quantification in lipidomic analyses is crucial for its successful application in the discovery of new biomarkers or in clinical practice. In this study, we propose a workflow to improve the accuracy and precision of lipidomic results issued by the laboratory. Lipid species from 11 classes were analyzed by a targeted RPLC-MRM/MS method. The peak areas of species were used to estimate concentrations by an internal standard calibration approach (IS-calibration) and by an alternative normalization signal calibration schema (NS-calibration). The latter uses a long-term reference plasma material as a matrix-matched external calibrator whose accuracy was compared to the NIST SRM-1950 mean consensus values reported by the Interlaboratory Lipidomics Comparison Exercise. The bias of lipid concentrations showed a good accuracy for 69 of 89 quantified lipids. The quantitation of species by the NS-calibration schema improved the within- and between-batch reproducibility in quality control samples, in comparison to the usual IS-calibration approach. Moreover, the NS-calibration workflow improved the robustness of the lipidomics measurements reducing the between-batch variability (relative standard deviation <10% for 95% of lipid species) in real conditions tested throughout the analysis of 120 plasma samples. In addition, we provide a free access web tool to obtain the concentration of lipid species by the two previously mentioned quantitative approaches, providing an easy follow-up of quality control tasks related to lipidomics.
    Keywords:  Lipidomics; Mass spectrometry; Molar concentration; NIST SRM-1950; Quality assurance; Software
  9. Cancers (Basel). 2021 Apr 06. pii: 1727. [Epub ahead of print]13(7):
      Tumor cells display metabolic alterations when compared to non-transformed cells. These characteristics are crucial for tumor development, maintenance and survival providing energy supplies and molecular precursors. Anaplerosis is the property of replenishing the TCA cycle, the hub of carbon metabolism, participating in the biosynthesis of precursors for building blocks or signaling molecules. In advanced prostate cancer, an upshift of succinate-driven oxidative phosphorylation via mitochondrial Complex II was reported. Here, using untargeted metabolomics, we found succinate accumulation mainly in malignant cells and an anaplerotic effect contributing to biosynthesis, amino acid, and carbon metabolism. Succinate also stimulated oxygen consumption. Malignant prostate cells displayed higher mitochondrial affinity for succinate when compared to non-malignant prostate cells and the succinate-driven accumulation of metabolites induced expression of mitochondrial complex subunits and their activities. Moreover, extracellular succinate stimulated migration, invasion, and colony formation. Several enzymes linked to accumulated metabolites in the malignant cells were found upregulated in tumor tissue datasets, particularly NME1 and SHMT2 mRNA expression. High expression of the two genes was associated with shorter disease-free survival in prostate cancer cohorts. Moreover, in-vitro expression of both genes was enhanced in prostate cancer cells upon succinate stimulation. In conclusion, the data indicate that uptake of succinate from the tumor environment has an anaplerotic effect that enhances the malignant potential of prostate cancer cells.
    Keywords:  anaplerosis; cancer metabolism; mitochondria; prostate cancer; succinate
  10. Sci Data. 2021 Apr 26. 8(1): 118
      Murine models are amongst the most widely used systems to study biology and pathology. Targeted quantitative proteomic analysis is a relatively new tool to interrogate such systems. Recently the need for relative quantification on hundreds to thousands of samples has driven the development of Data Independent Acquisition methods. One such technique is SWATH-MS, which in the main requires prior acquisition of mass spectra to generate an assay reference library. In stem cell research, it has been shown pluripotency can be induced starting with a fibroblast population. In so doing major changes in expressed proteins is inevitable. Here we have created a reference library to underpin such studies. This is inclusive of an extensively documented script to enable replication of library generation from the raw data. The documented script facilitates reuse of data and adaptation of the library to novel applications. The resulting library provides deep coverage of the mouse proteome. The library covers 29519 proteins (53% of the proteome) of which 7435 (13%) are supported by a proteotypic peptide.
  11. J Clin Invest. 2021 Apr 29. pii: 143691. [Epub ahead of print]
      Hypoxia is a hallmark of solid tumors that promotes cell growth, survival, metastasis and confers resistance to chemo and radiotherapies. Hypoxic responses are largely mediated by the transcription factor hypoxia-inducible factor (HIF)-1α and HIF-2α. Our work demonstrates that HIF-2α is essential for colorectal cancer (CRC) progression. However, targeting hypoxic cells is difficult and tumors rapidly acquire resistance to recently developed inhibitors of HIF-2α. To overcome this limitation, we performed a small molecule screen to identify HIF-2α dependent vulnerabilities. Several known ferroptosis activators and dimethyl fumarate (DMF), a cell permeable mitochondrial metabolite derivative, led to selective synthetic lethality in HIF-2α expressing tumor enteroids. Our work demonstrates that HIF-2α integrates two independent forms of cell death via regulation of cellular iron and oxidation. First, activation of HIF-2α upreguated lipid and iron regulatory genes in colon cancer cells and colon tumors in mice and led to a ferroptosis-susceptible cell state. Secondly, via an iron dependent, lipid peroxidation-independent pathway, HIF-2α activation potentiated ROS, via irreversible cysteine oxidation and enhanced cell death. Inhibition or knockdown of HIF-2α decreased ROS and resistance to oxidative cell death in vitro and in vivo. Our results demonstrate a mechanistic vulnerability in cancer cells that were the dependent on HIF-2α that can be leveraged for colon cancer treatment.
    Keywords:  Cancer; Cell stress; Hypoxia; Metabolism; Oncology
  12. Metabolites. 2021 Apr 26. pii: 269. [Epub ahead of print]11(5):
      Metabolome profiling is becoming more commonly used in the study of complex microbial communities and microbiomes; however, to date, little information is available concerning appropriate extraction procedures. We studied the influence of different extraction solvent mixtures on untargeted metabolomics analysis of two continuous culture enrichment communities performing enhanced biological phosphate removal (EBPR), with each enrichment targeting distinct populations of polyphosphate-accumulating organisms (PAOs). We employed one non-polar solvent and up to four polar solvents for extracting metabolites from biomass. In one of the reactor microbial communities, we surveyed both intracellular and extracellular metabolites using the same set of solvents. All samples were analysed using ultra-performance liquid chromatography mass spectrometry (UPLC-MS). UPLC-MS data obtained from polar and non-polar solvents were analysed separately and evaluated using extent of repeatability, overall extraction capacity and the extent of differential abundance between physiological states. Despite both reactors demonstrating the same bioprocess phenotype, the most appropriate extraction method was biomass specific, with methanol: water (50:50 v/v) and methanol: chloroform: water (40:40:20 v/v) being chosen as the most appropriate for each of the two different bioreactors, respectively. Our approach provides new data on the influence of solvent choice on the untargeted surveys of the metabolome of PAO enriched EBPR communities and suggests that metabolome extraction methods need to be carefully tailored to the specific complex microbial community under study.
    Keywords:  activated sludge; enhanced biological phosphorus removal; extraction; mass spectrometry; microbial ecology; polyphosphate-accumulating organisms; ultra-high performance liquid chromatography; untargeted metabolomics
  13. Endocr Relat Cancer. 2021 Apr 01. pii: ERC-21-0092. [Epub ahead of print]
      The existence of molecular links that facilitate colorectal cancer (CRC) development in the population with type 2 diabetes (T2D) is supported by substantial epidemiological evidence. This review summarizes how the systemic metabolic and hormonal imbalances from T2D alter CRC cell metabolism, signaling and gene expression as well as their reciprocal meshing, with an overview of CRC molecular subtypes and animal models to study the diabetes-CRC cancer links. Metabolic and growth factor checkpoints ensure a physiological cell proliferation rate compatible with limited nutrient supply. Hyperinsulinemia and hyperleptinemia in prediabetes and excess circulating glucose and lipids in T2D, overcome formidable barriers for tumor development. Increased nutrient availability favours metabolic reprogramming, alters signaling and generate mutations and epigenetic modifications, through increased reactive oxygen species and oncometabolites. The reciprocal control between metabolism and hormone signaling is lost in diabetes. Excess adipose tissue at the origin of T2D, unbalances adipokine (leptin / adiponectin) secretion ratios and function and disrupts the Insulin/IGF axes. Leptin/adiponectin imbalances in T2D are believed to promote proliferation and invasion of CRC cancer cells and contribute to inflammation, an important component of CRC tumorigenesis. Disruption of the Insulin/IGF axes in T2D targets systemic and CRC cell metabolic reprogramming, survival and proliferation. Future research to clarify the molecular diabetes-CRC links will help to prevent CRC and reduce its incidence in the diabetic population and must guide therapeutic decisions.
  14. Cancer Metab. 2021 Apr 30. 9(1): 20
      Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate (OAA), serving to replenish the tricarboxylic acid (TCA) cycle. In nonmalignant tissue, PC plays an essential role in controlling whole-body energetics through regulation of gluconeogenesis in the liver, synthesis of fatty acids in adipocytes, and insulin secretion in pancreatic β cells. In breast cancer, PC activity is linked to pulmonary metastasis, potentially by providing the ability to utilize glucose, fatty acids, and glutamine metabolism as needed under varying conditions as cells metastasize. PC enzymatic activity appears to be of particular importance in cancer cells that are unable to utilize glutamine for anaplerosis. Moreover, PC activity also plays a role in lipid metabolism and protection from oxidative stress in cancer cells. Thus, PC activity may be essential to link energy substrate utilization with cancer progression and to enable the metabolic flexibility necessary for cell resilience to changing and adverse conditions during the metastatic process.
    Keywords:  Energy metabolism; Metastasis; Pyruvate carboxylase
  15. Methods Mol Biol. 2021 ;2271 205-219
      Analysis of N-glycans are commonly conducted via enzymatic release, labeling, and liquid chromatography (LC) separation and fluorescent detection. Mass spectrometry (MS) has been increasingly used as an orthogonal detection method to provide additional structural information and increase the confidence of N-glycan analysis. In this chapter, we describe a method to perform routine analysis of N-glycans including the sample preparation with a signal-enhancement label, LC-MS data generation, and data analysis. Using this method, up to 24 N-glycan samples can be prepared at one time and analyzed by LC-MS. With the addition of automation platform, up to 96 N-glycan samples can be prepared and analyzed in a high-throughput manner.
    Keywords:  Analytical characterization; Automation; Cell line development; Critical quality attribute; High-throughput analysis; LC-MS; N-glycan analysis; Process development; Routine mass detection
  16. Nat Rev Endocrinol. 2021 Apr 29.
      This Review focuses on the mechanistic evidence for a link between obesity, dysregulated cellular metabolism and breast cancer. Strong evidence now links obesity with the development of 13 different types of cancer, including oestrogen receptor-positive breast cancer in postmenopausal women. A number of local and systemic changes are hypothesized to support this relationship, including increased circulating levels of insulin and glucose as well as adipose tissue-derived oestrogens, adipokines and inflammatory mediators. Metabolic pathways of energy production and utilization are dysregulated in tumour cells and this dysregulation is a newly accepted hallmark of cancer. Dysregulated metabolism is also hypothesized to be a feature of non-neoplastic cells in the tumour microenvironment. Obesity-associated factors regulate metabolic pathways in both breast cancer cells and cells in the breast microenvironment, which provides a molecular link between obesity and breast cancer. Consequently, interventions that target these pathways might provide a benefit in postmenopausal women and individuals with obesity, a population at high risk of breast cancer.
  17. Metabolites. 2021 Apr 07. pii: 226. [Epub ahead of print]11(4):
      Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.
    Keywords:  aldose reductase inhibitors; cardiovascular disease; endothelial metabolism; fluxomics; hexosamine biosynthetic pathway; metabolic flux analysis; pentose phosphate pathway; polyol pathway
  18. Molecules. 2021 Apr 11. pii: 2194. [Epub ahead of print]26(8):
      Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study's results may support a better understanding of bladder cancer development and progression mechanisms.
    Keywords:  bladder cancer (BC); liquid chromatography; mass spectrometry; metabolomics; solid phase microextraction (SPME); urine
  19. Methods Mol Biol. 2021 ;2271 281-301
      The existence of glycans in isomeric forms is responsible for the multifariousness of their properties and biological functions. Their altered expression has been associated with various diseases and cancers. Analysis of native glycans is not very sensitive due to the low ionization efficiency of glycans. These facts necessitate their comprehensive structural studies and establishes a high demand for sensitive and reliable techniques. In this chapter, we discuss the strategies for effective separation and identification of permethylated isomeric glycans. The sample preparation for permethylated glycans derived from model glycoproteins and complex biological samples, analyzed using LC-MS/MS, is delineated. We introduce protein extraction and release of glycans, followed by strategies to purify the released glycans, which are reduced and permethylated to improve ionization efficiency and stabilize sialic acid residues. High-temperature LC-based separation on PGC (porous graphitized carbon) column is conducive to isomeric separation of glycans and allows their sensitive identification and quantification using MS/MS.
    Keywords:  Glycomics; Isomeric separation; LC-MS; Permethylation; Porous graphitized carbon
  20. Biochim Biophys Acta Rev Cancer. 2021 Apr 26. pii: S0304-419X(21)00050-0. [Epub ahead of print] 188553
      The commencement of cancer is attributed to one or a few cells that become rogue and attain the property of immortality. The inception of distinct cancer cell clones during the hyperplastic and dysplastic stages of cancer progression is the utimate consequence of the dysregulated cellular pathways and the proliferative potential itself. Furthermore, a critical factor that adds a layer of complexity to this pre-existent intra-tumoral heterogeneity (ITH) is the foundation of an oxygen gradient, that is established due to the improper architecture of the tumor vasculature. Therefore, as a resultant effect, the poorly oxygenated regions thus formed and characterized as hypoxic, promote the emergence of aggressive and treatment-resistant cancer cell clones. The extraordinary property of the hypoxic cancer cells to exist harmoniously with cancerous and non-cancerous cells in the tumor microenvironment (TME) further increases the intricacies of ITH. Here in this review, the pivotal influence of differential oxygen concentrations in shaping the ITH is thoroughly discussed. We also emphasize on the vitality of the interacting networks that govern the overall fate of oxygen gradient-dependent origin of tumor heterogeneity. Additionally, the implications of less-appreciated reverse Warburg effect, a symbiotic metabolic coupling, and the associated epigenetic regulation of rewiring of cancer metabolism in response to oxygen gradients, have been highlighted as critical influencers of ITH.
    Keywords:  Altered metabolism; Cancer epigenetics; Hypoxia; Inter-cellular networks; Oxygen-gradient; Tumor heterogeneity; Tumor microenvironment
  21. Metabolites. 2021 Apr 23. pii: 264. [Epub ahead of print]11(5):
      A comprehensive view of cell metabolism provides a new vision of cancer, conceptualized as tissue with cellular-altered metabolism and energetic dysfunction, which can shed light on pathophysiological mechanisms. Cancer is now considered a heterogeneous ecosystem, formed by tumor cells and the microenvironment, which is molecularly, phenotypically, and metabolically reprogrammable. A wealth of evidence confirms metabolic reprogramming activity as the minimum common denominator of cancer, grouping together a wide variety of aberrations that can affect any of the different metabolic pathways involved in cell physiology. This forms the basis for a new proposed classification of cancer according to the altered metabolic pathway(s) and degree of energy dysfunction. Enhanced understanding of the metabolic reprogramming pathways of fatty acids, amino acids, carbohydrates, hypoxia, and acidosis can bring about new therapeutic intervention possibilities from a metabolic perspective of cancer.
    Keywords:  tumor ecosystem; tumor microenvironment; tumor reprogramming
  22. Annu Rev Immunol. 2021 Apr 26. 39 395-416
      Recent evidence supports the notion that mitochondrial metabolism is necessary for T cell activation, proliferation, and function. Mitochondrial metabolism supports T cell anabolism by providing key metabolites for macromolecule synthesis and generating metabolites for T cell function. In this review, we focus on how mitochondrial metabolism controls conventional and regulatory T cell fates and function.
    Keywords:  ROS; TCA cycle; acetyl-CoA; epigenetics; inflammation; l-2-hydroxyglutarate; l-2HG
  23. Oncogene. 2021 Apr 29.
      Ferroptosis, a form of regulated cell death triggered by lipid peroxidation, was recently identified as an important mechanism in radiotherapy (RT)-mediated tumor suppression and radioresistance, although the exact genetic contexts in which to target ferroptosis in RT remains to be defined. p53 is the most commonly mutated gene in human cancers and a major effector to RT. Here, we identify ferroptosis as a critical mechanism to mediate p53 function in tumor radiosensitivity. Mechanistically, RT-mediated p53 activation antagonizes RT-induced SLC7A11 expression and represses glutathione synthesis, thereby promoting RT-induced lipid peroxidation and ferroptosis. p53 deficiency promotes radioresistance in cancer cells or tumors at least partly through SLC7A11-mediated ferroptosis inhibition. Ferroptosis inducers (FINs) that inhibit SLC7A11 exert significant radiosensitizing effects in tumor organoids and patient-derived xenografts with p53 mutation or deficiency. Finally, we show that RT-induced ferroptosis correlates with p53 activation and better clinical outcomes to RT in cancer patients. Together, our study uncovers a previously unappreciated role of ferroptosis in p53-mediated radiosensitization and suggest using FINs in combination with RT to treat p53-mutant cancers.
  24. Metabolites. 2021 Apr 27. pii: 277. [Epub ahead of print]11(5):
      Lipids are a ubiquitous class of structurally complex molecules involved in various biological processes. In the fast-growing field of lipidomics, preanalytical issues are frequently neglected. Here, we investigated the stability of lipid profiles of murine liver, brain, lung, heart, and spleen homogenates by quantitative flow injection analysis using tandem mass spectrometry and high-resolution mass spectrometry. Storage of tissue homogenates at room temperature showed substantial alterations of the lipid profiles reflecting lipolytic action. Therefore, ratios of ceramide to sphingomyelin, lysophosphatidylethanolamine to phosphatidylethanolamine, lysophosphatidylcholine to phosphatidylcholine, and diglyceride to triglyceride were applied to monitor sample stability and the effect of sodium dodecyl sulfate (SDS) as a potential stabilizing agent. The addition of SDS led to a concentration-dependent stabilization of lipid profiles in liver, brain, and heart homogenates, while in lung and spleen homogenates, in particular, the lysophosphatidylethanolamine to phosphatidylethanolamine ratio increased upon addition of SDS. In conclusion, we demonstrated that lipid class ratios reflecting lipolytic activity could be applied to evaluate both the stability of samples and the influence of stabilizers.
    Keywords:  lipid class ratio; lipidomics; lipolytic ratios; mass spectrometry; sodium dodecyl sulfate; stabilization; tissue
  25. J Proteome Res. 2021 Apr 28.
      Mass spectrometry data sets from omics studies are an optimal information source for discriminating patients with disease and identifying biomarkers. Thousands of proteins or endogenous metabolites can be queried in each analysis, spanning several orders of magnitude in abundance. Machine learning tools that effectively leverage these data to accurately identify disease states are in high demand. While mass spectrometry data sets are rich with potentially useful information, using the data effectively can be challenging because of missing entries in the data sets and because the number of samples is typically much smaller than the number of features, two challenges that make machine learning difficult. To address this problem, we have modified a new supervised classification tool, the Aristotle Classifier, so that omics data sets can be better leveraged for identifying disease states. The optimized classifier, AC.2021, is benchmarked on multiple data sets against its predecessor and two leading supervised classification tools, Support Vector Machine (SVM) and XGBoost. The new classifier, AC.2021, outperformed existing tools on multiple tests using proteomics data. The underlying code for the classifier, provided herein, would be useful for researchers who desire improved classification accuracy when using their omics data sets to identify disease states.
    Keywords:  Alzheimer’s disease; Aristotle Classifier; ROC; SVM; XGBoost; machine learning; mass spectrometry; proteomics
  26. Curr Rheumatol Rep. 2021 Apr 28. 23(6): 42
      PURPOSE OF REVIEW: Rheumatoid arthritis (RA) is a chronic autoimmune, inflammatory disease of the synovium that affects the movable joints. It develops due to the infiltration and invasion of the synovial joints by immune cells. Metabolism is anabolic or catabolic chemical reactions occurring in a cell. The biochemical pathways in synovial and immune cells are altered affecting the downstream metabolite formation. Changes in the metabolite levels alter signaling cascades which further intensify the disease. Despite current knowledge of metabolomics, there remain certain features that need to be elucidated to correlate the differential metabolite levels with RA.RECENT FINDINGS: Metabolite profiling can be used to find altered patterns of metabolites in RA. Glucose, lipid, amino acid, and estrogen metabolism are the key pathways that are altered and contribute to the aggravation of RA. The altered metabolic pathways involved in different cells in RA results in complex interactions between metabolites and biomacromolecules; thus, it generates autoantigens. Moreover, understanding the correlation between differential metabolites and disease severity might help reveal potential new biomarkers and therapeutic targets for RA pathogenesis. So, considering the multi-faceted role of altered metabolites in the pathogenesis of RA, metabolic pathways of different cells are needed to be studied for a better understanding of their functions in the disease and thus, improving the present therapeutic strategies.
    Keywords:  Anti-citrullinated peptide antibodies (ACPAs); Interleukin (IL); Reactive oxygen species (ROS); Rheumatoid arthritis (RA); Rheumatoid factors (RF)
  27. Int J Mol Sci. 2021 Apr 22. pii: 4367. [Epub ahead of print]22(9):
      Early detection of prostate cancer (PC) is largely carried out using assessment of prostate-specific antigen (PSA) level; yet it cannot reliably discriminate between benign pathologies and clinically significant forms of PC. To overcome the current limitations of PSA, new urinary and serum biomarkers have been developed in recent years. Although several biomarkers have been explored in various scenarios and patient settings, to date, specific guidelines with a high level of evidence on the use of these markers are lacking. Recent advances in metabolomic, genomics, and proteomics have made new potential biomarkers available. A number of studies focused on the characterization of the specific PC metabolic phenotype using different experimental approaches has been recently reported; yet, to date, research on metabolomic application for PC has focused on a small group of metabolites that have been known to be related to the prostate gland. Exosomes are extracellular vesicles that are secreted from all mammalian cells and virtually detected in all bio-fluids, thus allowing their use as tumor biomarkers. Thanks to a general improvement of the technical equipment to analyze exosomes, we are able to obtain reliable quantitative and qualitative information useful for clinical application. Although some pilot clinical investigations have proposed potential PC biomarkers, data are still preliminary and non-conclusive.
    Keywords:  biomarkers; early diagnosis; exosomes; metabolomics; prostate cancer
  28. Cancers (Basel). 2021 Apr 20. pii: 1980. [Epub ahead of print]13(8):
      Hepatocellular carcinoma (HCC) is a common malignancy with poor prognosis, high morbidity and mortality concerning with lack of effective diagnosis and high postoperative recurrence. Similar with other cancers, HCC cancer cells have to alter their metabolism to adapt to the changing requirements imposed by the environment of the growing tumor. In less vascularized regions of tumor, cancer cells experience hypoxia and nutrient starvation. Here, we show that HCC undergoes a global metabolic reprogramming during tumor growth. A combined proteomics and metabolomics analysis of paired peritumoral and tumor tissues from 200 HCC patients revealed liver-specific metabolic reprogramming and metabolic alterations with increasing tumor sizes. Several proteins and metabolites associated with glycolysis, the tricarboxylic acid cycle and pyrimidine synthesis were found to be differentially regulated in serum, tumor and peritumoral tissue with increased tumor sizes. Several prognostic metabolite biomarkers involved in HCC metabolic reprogramming were identified and integrated with clinical and pathological data. We built and validated this combined model to discriminate against patients with different recurrence risks. An integrated and comprehensive metabolomic analysis of HCC is provided by our present work. Metabolomic alterations associated with the advanced stage of the disease and poor clinical outcomes, were revealed. Targeting cancer metabolism may deliver effective therapies for HCC.
    Keywords:  NMR spectroscopy; hepatocellular carcinoma; metabolomics; predictive model; proteomics
  29. Horm Metab Res. 2021 Apr 26.
      The adrenal gland is important for many physiological and pathophysiological processes, but studies are often restricted by limited availability of sample material. Improved methods for sample preparation are needed to facilitate analyses of multiple classes of adrenal metabolites and macromolecules in a single sample. A procedure was developed for preparation of chromaffin cells, mouse adrenals, and human chromaffin tumors that allows for multi-omics analyses of different metabolites and preservation of native proteins. To evaluate the new procedure, aliquots of samples were also prepared using conventional procedures. Metabolites were analyzed by liquid-chromatography with mass spectrometry or electrochemical detection. Metabolite contents of chromaffin cells and tissues analyzed with the new procedure were similar or even higher than with conventional methods. Catecholamine contents were comparable between both procedures. The TCA cycle metabolites, cis-aconitate, isocitate, and α-ketoglutarate were detected at higher concentrations in cells, while in tumor tissue only isocitrate and potentially fumarate were measured at higher contents. In contrast, in a broad untargeted metabolomics approach, a methanol-based preparation procedure of adrenals led to a 1.3-fold higher number of detected metabolites. The established procedure also allows for simultaneous investigation of adrenal hormones and related enzyme activities as well as proteins within a single sample. This novel multi-omics approach not only minimizes the amount of sample required and overcomes problems associated with tissue heterogeneity, but also provides a more complete picture of adrenal function and intra-adrenal interactions than previously possible.
  30. J Am Chem Soc. 2021 Apr 29.
      Nicotinamide adenine dinucleotide (NAD+) is a multifunctional molecule. Beyond redox metabolism, NAD+ has an equally important function as a substrate for post-translational modification enzymes, the largest family being the poly-ADP-ribose polymerases (PARPs, 17 family members in humans). The recent surprising discoveries of noncanonical NAD (NAD+/NADH)-binding proteins suggests that the NAD interactome is likely larger than previously thought; yet, broadly useful chemical tools for profiling and discovering NAD-binding proteins do not exist. Here, we describe the design, synthesis, and validation of clickable, photoaffinity labeling (PAL) probes, 2- and 6-ad-BAD, for interrogating the NAD interactome. We found that 2-ad-BAD efficiently labels PARPs in a UV-dependent manner. Chemical proteomics experiments with 2- and 6-ad-BAD identified known and unknown NAD+/NADH-binding proteins. Together, our study shows the utility of 2- and 6-ad-BAD as clickable PAL NAD probes.
  31. Curr Rheumatol Rep. 2021 Apr 28. 23(6): 41
      PURPOSE OF REVIEW: Osteoarthritis (OA) and rheumatoid arthritis (RA) are characterized by abnormal lipid metabolism manifested as altered fatty acid (FA) profiles of synovial fluid and tissues and in the way dietary FA supplements can influence the symptoms of especially RA. In addition to classic eicosanoids, the potential roles of polyunsaturated FA (PUFA)-derived specialized pro-resolving lipid mediators (SPM) have become the focus of intensive research. Here, we summarize the current state of knowledge of the roles of FA and oxylipins in the degradation or protection of synovial joints.RECENT FINDINGS: There exists discordance between the large body of literature from cell culture and animal experiments on the adverse and beneficial effects of individual FA and the lack of effective treatments for joint destruction in OA and RA patients. Saturated 16:0 and 18:0 induce mostly deleterious effects, while long-chain n-3 PUFA, especially 20:5n-3, have positive influence on joint health. The situation can be more complex for n-6 PUFA, such as 18:2n-6, 20:4n-6, and its derivative prostaglandin E2, with a combination of potentially adverse and beneficial effects. SPM analogs have future potential as analgesics for arthritic pain. Alterations in FA profiles and their potential implications in SPM production may affect joint lubrication, synovial inflammation, pannus formation, as well as cartilage and bone degradation and contribute to the pathogeneses of inflammatory joint diseases. Further research directions include high-quality randomized controlled trials on dietary FA supplements and investigations on the significance of lipid composition of microvesicle membrane and cargo in joint diseases.
    Keywords:  Fatty acid; Osteoarthritis; Oxylipins; Polyunsaturated fatty acids; Rheumatoid arthritis; Specialized pro-resolving lipid mediators
  32. Biochem J. 2021 Apr 26. pii: BCJ20210155. [Epub ahead of print]
      Fatty acid transport protein 4 (FATP4) belongs to a family of acyl-CoA synthetases which activate long-chain fatty acids into acyl-CoAs subsequently used in specific metabolic pathways. Patients with FATP4 mutations and Fatp4-null mice show thick desquamating skin and other complications, however, FATP4 role on macrophage functions has not been studied. We here determined whether the levels of macrophage glycerophospholipids, sphingolipids including ceramides, triacylglycerides, and cytokine release could be altered by FATP4 inactivation. Two in vitro experimental systems were studied: FATP4-knockdown in THP-1-derived macrophages undergoing M1 (LPS+IFNγ) or M2 (IL-4) activation and bone marrow-derived macrophages (BMDMs) from macrophage-specific Fatp4-knockout (Fatp4M-/-) mice undergoing tunicamycin (TM)-induced ER stress. FATP4-deficient macrophages showed a metabolic shift towards triacylglycerides and were protected from M1- or TM-induced release of pro-inflammatory cytokines and cellular injury. Fatp4M-/- BMDMs showed specificity in attenuating TM-induced activation of inositol-requiring enzyme1α, but not other unfolded protein response pathways. Under basal conditions, FATP4/Fatp4 deficiency decreased the levels of ceramides and induced an upregulation of mannose receptor CD206 expression. The deficiency led to an attenuation of IL-8 release in THP-1 cells as well as TNF-α and IL-12 release in BMDMs. Thus, FATP4 functions as an acyl-CoA synthetase in macrophages and its inactivation suppresses the release of pro-inflammatory cytokines by shifting fatty acids towards the synthesis of specific lipids.
    Keywords:  ER stress; ceramides; fatty acid transport protein 4; lipidomics; polarized macrophages; triacylglycerides