bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2019–12–22
sixteen papers selected by
Giovanny Rodriguez Blanco, The Beatson Institute for Cancer Research



  1. Metabolites. 2019 Dec 17. pii: E308. [Epub ahead of print]9(12):
      Untargeted metabolomics (including lipidomics) is a holistic approach to biomarker discovery and mechanistic insights into disease onset and progression, and response to intervention. Each step of the analytical and statistical pipeline is crucial for the generation of high-quality, robust data. Metabolite identification remains the bottleneck in these studies; therefore, confidence in the data produced is paramount in order to maximize the biological output. Here, we outline the key steps of the metabolomics workflow and provide details on important parameters and considerations. Studies should be designed carefully to ensure appropriate statistical power and adequate controls. Subsequent sample handling and preparation should avoid the introduction of bias, which can significantly affect downstream data interpretation. It is not possible to cover the entire metabolome with a single platform; therefore, the analytical platform should reflect the biological sample under investigation and the question(s) under consideration. The large, complex datasets produced need to be pre-processed in order to extract meaningful information. Finally, the most time-consuming steps are metabolite identification, as well as metabolic pathway and network analysis. Here we discuss some widely used tools and the pitfalls of each step of the workflow, with the ultimate aim of guiding the reader towards the most efficient pipeline for their metabolomics studies.
    Keywords:  data processing; experimental design; liquid chromatography–mass spectrometry (LC-MS); metabolic pathway and network analysis; metabolism; metabolite identification; sample preparation; univariate and multivariate statistics; untargeted metabolomics
    DOI:  https://doi.org/10.3390/metabo9120308
  2. Cell Rep. 2019 Dec 17. pii: S2211-1247(19)31570-0. [Epub ahead of print]29(12): 4086-4098.e6
      The tumor microenvironment (TME) plays a pivotal role in cancer progression, and, in ovarian cancer (OvCa), the primary TME is the omentum. Here, we show that the diabetes drug metformin alters mesothelial cells in the omental microenvironment. Metformin interrupts bidirectional signaling between tumor and mesothelial cells by blocking OvCa cell TGF-β signaling and mesothelial cell production of CCL2 and IL-8. Inhibition of tumor-stromal crosstalk by metformin is caused by the reduced expression of the tricarboxylic acid (TCA) enzyme succinyl CoA ligase (SUCLG2). Through repressing this TCA enzyme and its metabolite, succinate, metformin activated prolyl hydroxylases (PHDs), resulting in the degradation of hypoxia-inducible factor 1α (HIF1α) in mesothelial cells. Disruption of HIF1α-driven IL-8 signaling in mesothelial cells by metformin results in reduced OvCa invasion in an organotypic 3D model. These findings indicate that tumor-promoting signaling between mesothelial and OvCa cells in the TME can be targeted using metformin.
    Keywords:  HIF1; IL-8; SUCLG2; TGF; mesothelial cells; metformin; omental metastasis; ovarian cancer; succinate; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.celrep.2019.11.079
  3. Metabolites. 2019 Dec 13. pii: E301. [Epub ahead of print]9(12):
      Metabolomics is the latest 'omics' technology and systems biology science that allows for comprehensive profiling of small-molecule metabolites in biological systems at a specific time and condition. Metabolites are cellular intermediate products of metabolic reactions, which reflect the ultimate response to genomic, transcriptomic, proteomic, or environmental changes in a biological system. Aging is a complex biological process that is characterized by a gradual and progressive decline in molecular, cellular, tissue, organ, and organismal functions, and it is influenced by a combination of genetic, environmental, diet, and lifestyle factors. The precise biological mechanisms of aging remain unknown. Metabolomics has emerged as a powerful tool to characterize the organism phenotypes, identify altered metabolites, pathways, novel biomarkers in aging and disease, and offers wide clinical applications. Here, I will provide a comprehensive overview of our current knowledge on metabolomics led studies in aging with particular emphasis on studies leading to biomarker discovery. Based on the data obtained from model organisms and humans, it is evident that metabolites associated with amino acids, lipids, carbohydrate, and redox metabolism may serve as biomarkers of aging and/or longevity. Current challenges and key questions that should be addressed in the future to advance our understanding of the biological mechanisms of aging are discussed.
    Keywords:  MS; NMR; aging; biomarker; human longitudinal studies; metabolism; metabolites; metabolomics; model organisms
    DOI:  https://doi.org/10.3390/metabo9120301
  4. Anal Chem. 2019 Dec 17.
      Although capillary liquid chromatography married with tandem mass spectrometry (cLC-MS/MS) has become a powerful technique for proteomics and metabolomics research, it is still a great challenge to fabricate durable capillary-based analytical columns coupling continuous nanoflow (<1,000 nL/min) electrospray ionization (ESI) with MS, owing to the issue of clogging and fragile of emitters. Here, we proposed a simple approach to integrate microstructured photonic fibers (MPFs) into wide bore capillaries with 150 μm I.D., serving as integral bifunctional frit or/and ESI emitter of packed column. Two kinds of MPFs containing 126 homogeneous microchannels with different inner diameter, 3.2 μm for MPF-1 and 2.6 μm for MPF-2, were explored for preparation. The octadecylsilicate (ODS) silica-packed column using MPF-1 as frit exhibited the lowest plate heights of 14.2-19.7 μm for five alkylbenzenes at the velocity of 1.5 mm/s, which were slightly lower than those of packed column with porous polymer monolith (PPM)-based frit by cLC coupling with ultraviolet (UV) detection. Additionally, the packed columns with integral MPFs frit-emitters were further applied in analysis of a complex biological sample of digest of Hela cells by cLC-MS. An average of 7109 unique peptides could be identified in a single analysis by using MPF-1 emitter, and 7110 unique peptides were identified by using MPF-2 emitter, which were superior to that of packed column with an integral tapered tip emitter (6894 peptides). It is obvious that this novel integral MPF-based frit-emitter do not easily suffer from the issues of cracking owing to the silica cladding around independent microchannels (>100), which always encumber both independent and integral tapered tip emitters for cLC-MS.
    DOI:  https://doi.org/10.1021/acs.analchem.9b04997
  5. Metabolites. 2019 Dec 18. pii: E4. [Epub ahead of print]10(1):
      Cardiovascular diseases (CVDs) represent a major concern in today's society, with more than 17.5 million deaths reported annually worldwide. Recently, five metabolites related to the gut metabolism of phospholipids were identified as promising predictive biomarker candidates for CVD. Validation of those biomarker candidates is crucial for applications to the clinic, showing the need for high-throughput analysis of large numbers of samples. These five compounds, trimethylamine N-oxide (TMAO), choline, betaine, l-carnitine, and deoxy-l-carnitine (4-trimethylammoniobutanoic acid), are highly polar compounds and show poor retention on conventional reversed phase chromatography, which can lead to strong matrix effects when using mass spectrometry detection, especially when high-throughput analysis approaches are used with limited separation of analytes from interferences. In order to reduce the potential matrix effects, we propose a novel fast parallel electromembrane extraction (Pa-EME) method for the analysis of these metabolites in plasma samples. The evaluation of Pa-EME parameters was performed using multi segment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Recoveries up to 100% were achieved, with variability as low as 2%. Overall, this study highlights the necessity of protein precipitation prior to EME for the extraction of highly polar compounds. The developed Pa-EME method was evaluated in terms of concentration range and response function, as well as matrix effects using fast-LC-MS/MS. Finally, the developed workflow was compared to conventional sample pre-treatment, i.e., protein precipitation using methanol, and fast-LC-MS/MS. Data show very strong correlations between both workflows, highlighting the great potential of Pa-EME for high-throughput biological applications.
    Keywords:  capillary electrophoresis–mass spectrometry; cardiovascular disease; electromembrane extraction; liquid chromatography–mass spectrometry; multi-segment injection
    DOI:  https://doi.org/10.3390/metabo10010004
  6. Bioanalysis. 2019 Dec;11(24): 2297-2318
      Metabolomics is the comprehensive study of small-molecule metabolites. Obtaining a wide coverage of the metabolome is challenging because of the broad range of physicochemical properties of the small molecules. To study the compounds of interest spectroscopic (NMR), spectrometric (MS) and separation techniques (LC, GC, supercritical fluid chromatography, CE) are used. The choice for a given technique is influenced by the sample matrix, the concentration and properties of the metabolites, and the amount of sample. This review discusses the most commonly used analytical techniques for metabolomic studies, including their advantages, drawbacks and some applications.
    Keywords:  multiplatform approaches; separation techniques; spectrometric techniques; spectroscopic techniques; targeted metabolomics; untargeted metabolomics
    DOI:  https://doi.org/10.4155/bio-2019-0014
  7. Antioxid Redox Signal. 2019 Dec 16.
      Significance: Cancer cells exhibit altered metabolic pathways to keep up with biosynthetic and reduction-oxidation needs during tumor proliferation and metastasis. The common induction of metabolic pathways during cancer progression, regardless of cancer histio- or genotype, makes cancer metabolism an attractive target for therapeutic exploitation. Recent Advances: Emerging data suggest that these altered pathways may even result in resistance to anticancer therapies. Identifying specific metabolic dependencies that are unique to cancer cells has proved challenging in this field, limiting the therapeutic window for many candidate drug approaches. Critical Issues: Cancer cells display significant metabolic flexibility in nutrient-limited environments, hampering the longevity of suppressing cancer metabolism through any singular approach. Combinatorial "synthetic lethal" approaches may have a better chance for success and promising strategies are reviewed here. The dynamism of the immune system adds a level of complexity, as various immune populations in the tumor microenvironment often share metabolic pathways with cancer, with successive alterations during immune activation and quiescence. Decoding the reprogramming of metabolic pathways within cancer cells and stem cells, as well as examining metabolic symbiosis between components of the tumor microenvironment, would be essential to further meaningful drug development within the tumor's metabolic ecosystem. Future Directions: In this article, we examine evidence for the therapeutic potential of targeting metabolic alterations in cancer, and we discuss the drawbacks and successes that have stimulated this field. Antioxid. Redox Signal. 00, 000-000.
    Keywords:  cancer therapy; cell metabolism; metabolic vulnerability
    DOI:  https://doi.org/10.1089/ars.2019.7947
  8. J Proteomics. 2019 Dec 11. pii: S1874-3919(19)30377-X. [Epub ahead of print]213 103605
      We investigated the alterations in the plasma concentrations of energy-balance-related metabolites in patients with lung (LC) or head & neck (HNC) cancer and the changes on these parameters induced by radiotherapy. The study was conducted in 33 patients with non-small cell LC and 28 patients with HNC. We analyzed the concentrations of 17 metabolites involved in glycolysis, citric acid cycle and amino acid metabolism using targeted gas chromatography coupled to quadrupole time-of-flight mass spectrometry. For comparison, a control group of 50 healthy individuals was included in the present study. Patients with LC or HNC had significant alterations in the plasma levels of several energy-balance-related metabolites. Radiotherapy partially normalized these alterations in patients with LC, but not in those with HNC. The measurement of plasma glutamate concentration was an excellent predictor of the presence of LC or HNC, with sensitivity >90% and specificity >80%. Also, associations with disease prognosis were observed with plasma glutamate, amino acids and β-hydroxybutyrate concentrations. SIGNIFICANCE: This study analyzed the changes produced in the plasma concentrations of energy-balance-related metabolites in patients with lung cancer or head and neck cancer. The results obtained identified glutamate as the parameter with the highest discrimination capacity between patients and the control group. The relationships between various metabolites and clinical outcomes were also analyzed. These results extend the knowledge of metabolic alterations in cancer, thus facilitating the search for biomarkers and therapeutic targets.
    Keywords:  Cancer metabolism; Glutamate; Metabolomics; Radiotherapy; β-Hydroxybutyrate
    DOI:  https://doi.org/10.1016/j.jprot.2019.103605
  9. Acta Diabetol. 2019 Dec 20.
       AIMS: The population of immigrants from the Middle East in Sweden show a higher prevalence of type 2 diabetes (T2D) compared to native Swedes. The exact reason for this is unknown. Here, we have performed metabolite profiling to investigate these differences.
    METHODS: Metabolite profiling was conducted in Iraqi immigrants (n = 93) and native Swedes (n = 77) using two complementary mass spectrometry-based platforms. Differences in metabolite levels were compared after adjustment for confounding anthropometric, diet and clinical variables.
    RESULTS: The Iraqi immigrant population were more obese (44.1 vs 24.7%, p < 0.05), but had a lower prevalence of hypertension (32.3 vs 54.8%, p < 0.01) than the native Swedish population. We detected 140 metabolites, 26 of which showed different levels between populations (q < 0.05,) after adjustment for age, sex, BMI, T2D and use of metformin. Twenty-two metabolites remained significant after further adjustment for HOMA-IR, HOMA-beta or insulin sensitivity index. Levels of polyunsaturated acylcarnitines (14:2 and 18:2) and fatty acid (18:2) were higher, whereas those of saturated and monounsaturated acylcarnitines (14:0, 18:1, and 8:1), fatty acids (12:0, 14:0, 16:0, and 18:1), uremic solutes (urate and quinate) and ketone bodies (beta-hydroxybutyrate) were lower in Iraqi immigrants. Further, levels of phospholipids were generally lower in the Iraqi immigrant population.
    CONCLUSIONS: Our result suggests an overall beneficial lipid profile in Iraqi immigrants, despite a higher risk to develop T2D. Higher levels of polyunsaturated fatty acids may suggest differences in dietary pattern, which in turn may reduce the risk of hypertension.
    Keywords:  Acylcarnitines; Fatty acids; Hypertension; Ketone bodies; Metabolomics; Middle East; Migration; Obesity; Type 2 diabetes; Uremic solutes
    DOI:  https://doi.org/10.1007/s00592-019-01464-w
  10. Int J Mol Sci. 2019 Dec 19. pii: E40. [Epub ahead of print]21(1):
      Non-alcoholic fatty liver disease (NAFLD) has emerged as one of the main causes of chronic liver disease worldwide. NAFLD comprises a group of conditions characterized by the accumulation of hepatic lipids that can eventually lead to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), the fifth most common cancer type with a poor survival rate. In this context, several works have pointed out perturbations in lipid metabolism and, particularly, changes in bioactive sphingolipids, as a hallmark of NAFLD and derived HCC. In the present work, we have reviewed existing literature about sphingolipids and the development of NAFLD and NAFLD-derived HCC. During metabolic syndrome, considered a risk factor for steatosis development, an increase in ceramide and sphigosine-1-phosphate (S1P) have been reported. Likewise, other reports have highlighted that increased sphingomyelin and ceramide content is observed during steatosis and NASH. Ceramide also plays a role in liver fibrosis and cirrhosis, acting synergistically with S1P. Finally, during HCC, metabolic fluxes are redirected to reduce cellular ceramide levels whilst increasing S1P to support tumor growth.
    Keywords:  HCC; Metabolic syndrome; NAFLD; NASH; S1P; ceramide; cirrhosis; lipidomics; metabolomics; sphingolipids; sphingomyelin
    DOI:  https://doi.org/10.3390/ijms21010040
  11. Sci Rep. 2019 Dec 20. 9(1): 19593
      Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor in adults. Despite the multimodal standard treatments for GBM, the median survival is still about one year. Analysis of brain tissues from GBM patients shows that lipid droplets are highly enriched in tumor tissues while undetectable in normal brain tissues, yet the identity and functions of lipid species in GBM are not well understood. The aims of the present work are to determine how GBM utilizes fatty acids, and assess their roles in GBM proliferation. Treatment of U138 GBM cells with a monounsaturated fatty acid, oleic acid, induces accumulation of perilipin 2-coated lipid droplets containing triglycerides enriched in C18:1 fatty acid, and increases fatty acid oxidation. Interestingly, oleic acid also increases glucose utilization and proliferation of GBM cells. In contrast, pharmacologic inhibition of monoacylglycerol lipase attenuates GBM proliferation. Our findings demonstrate that monounsaturated fatty acids promote GBM proliferation via triglyceride metabolism, suggesting a novel lipid droplet-mediated pathway which may be targeted for GBM treatment.
    DOI:  https://doi.org/10.1038/s41598-019-55985-z
  12. Metabolites. 2019 Dec 18. pii: E2. [Epub ahead of print]10(1):
      A gas chromatography mass spectrometry (GC-MS) metabolomics protocol was modified for quenching, harvesting, and extraction of metabolites from adherent cells grown under high (20%) fetal calf serum conditions. The reproducibility of using either 50% or 80% methanol for quenching of cells was compared for sample harvest. To investigate the efficiency and reproducibility of intracellular metabolite extraction, different volumes and ratios of chloroform were tested. Additionally, we compared the use of total protein amount versus cell mass as normalization parameters. We demonstrate that the method involving 50% methanol as quenching buffer followed by an extraction step using an equal ratio of methanol:chloroform:water (1:1:1, v/v/v) followed by the collection of 6 mL polar phase for GC-MS measurement was superior to the other methods tested. Especially for large sample sets, its comparative ease of measurement leads us to recommend normalization to protein amount for the investigation of intracellular metabolites of adherent human cells grown under high (or standard) fetal calf serum conditions. To avoid bias, care should be taken beforehand to ensure that the ratio of total protein to cell number are consistent among the groups tested. For this reason, it may not be suitable where culture conditions or cell types have very different protein outputs (e.g., hypoxia vs. normoxia). The full modified protocol is available in the Supplementary Materials.
    Keywords:  20% FCS; GC-MS; extraction; harvesting; metabolites; normalization
    DOI:  https://doi.org/10.3390/metabo10010002
  13. Nature. 2019 Dec 18.
      Metastasis requires cancer cells to undergo metabolic changes that are poorly understood1-3. Here we show that metabolic differences among melanoma cells confer differences in metastatic potential as a result of differences in the function of the MCT1 transporter. In vivo isotope tracing analysis in patient-derived xenografts revealed differences in nutrient handling between efficiently and inefficiently metastasizing melanomas, with circulating lactate being a more prominent source of tumour lactate in efficient metastasizers. Efficient metastasizers had higher levels of MCT1, and inhibition of MCT1 reduced lactate uptake. MCT1 inhibition had little effect on the growth of primary subcutaneous tumours, but resulted in depletion of circulating melanoma cells and reduced the metastatic disease burden in patient-derived xenografts and in mouse melanomas. In addition, inhibition of MCT1 suppressed the oxidative pentose phosphate pathway and increased levels of reactive oxygen species. Antioxidants blocked the effects of MCT1 inhibition on metastasis. MCT1high and MCT1-/low cells from the same melanomas had similar capacities to form subcutaneous tumours, but MCT1high cells formed more metastases after intravenous injection. Metabolic differences among cancer cells thus confer differences in metastatic potential as metastasizing cells depend on MCT1 to manage oxidative stress.
    DOI:  https://doi.org/10.1038/s41586-019-1847-2
  14. Wiley Interdiscip Rev Syst Biol Med. 2019 Dec 16. e1474
      It is becoming increasingly appreciated that intermediates of metabolic pathways, besides their anabolic and catabolic functions, can act as signaling molecules and influence the outcome of immune responses. Although lactate was previously considered as a waste product of glucose metabolism, accumulating evidence has highlighted its pivotal role in regulating diverse biological processes, including immune cell polarization, differentiation and effector functions. In addition, lactate is a key player in modulating tumor immune surveillance. Hence, targeting lactate-induced signaling pathways is a promising tool to reduce inflammation, to prevent autoimmunity and to restore anti-tumor immune response. This article is characterized under: Biological Mechanisms > Metabolism.
    Keywords:  immunity; inflammation; lactate; metabolism; tumor
    DOI:  https://doi.org/10.1002/wsbm.1474
  15. Drug Resist Updat. 2019 Nov 29. pii: S1368-7646(19)30067-6. [Epub ahead of print]49 100670
      Lipids, phospholipids and cholesterol in particular, are the predominant components of the plasma membrane, wherein multidrug efflux transporters of the ATP-binding cassette (ABC) superfamily reside as integral pump proteins. In the current review, we discuss how lipids potently modulate the expression and activity of these multidrug efflux pumps, contributing to the development of the multidrug resistance (MDR) phenotype in cancer. The molecular mechanisms underlying this modulation of the MDR phenotype are pleiotropic. First, notwithstanding the high intra-and inter-tumor variability, MDR cells display an altered composition of plasma membrane phospholipids and glycosphingolipids, and are enriched with very long saturated fatty acid chains. This feature, along with the increased levels of cholesterol, decrease membrane fluidity, alter the spatial organization of membrane nano- and micro-domains, interact with transmembrane helices of ABC transporters, hence favoring drug binding and release. Second, MDR cells exhibit a peculiar membrane lipid composition of intracellular organelles including mitochondria and endoplasmic reticulum (ER). In this respect, they contain a lower amount of oxidizable fatty acids, hence being more resistant to oxidative stress and chemotherapy-induced apoptosis. Third, drug resistant cancer cells have a higher ratio of monosatured/polyunsatured fatty acids: this lipid signature reduces the production of reactive aldehydes with cytotoxic and pro-inflammatory activity and, together with the increased activity of anti-oxidant enzymes, limits the cellular damage induced by lipid peroxidation. Finally, specific precursors of phospholipids and cholesterol including ceramides and isoprenoids, are highly produced in MDR cells; by acting as second messengers, they trigger multiple signaling cascades that induce the transcription of drug efflux transporter genes and/or promote a metabolic reprogramming which supports the MDR phenotype. High-throughput lipidomics and computational biology technologies are a great tool in analyzing the tumor lipid signature in a personalized manner and in identifying novel biomarkers of drug resistance. Moreover, beyond the induction of MDR, lipid metabolism offers a remarkable opportunity to reverse MDR by using lipid analogues and repurposing lipid-targeting drugs (e.g. statins and aminobisphosphonates) that reprogram the lipid composition of drug resistant cells, hence rendering them drug sensitive.
    Keywords:  Cholesterol; Drug resistance; Isoprenoids; Lipid peroxidation; MDR reversal; Membrane fluidity; Membranes; Phospholipids
    DOI:  https://doi.org/10.1016/j.drup.2019.100670
  16. Antioxid Redox Signal. 2019 Dec 18.
       SIGNIFICANCE: Cancer cells are stabilized in an undifferentiated state similar to stem cells. This leads to profound modifications of their metabolism, which further modifies their genetics and epigenetics as malignancy progresses. Specific metabolites and enzymes may serve as clinical markers of cancer progression. Recent Advances: Both 2-hydroxyglutarate (2HG) enantiomers are associated with reprogrammed metabolism, in grade III/IV glioma, glioblastoma and acute myeloid leukemia (AML) cells, and numerous other cancer types, while acting also in the crosstalk of tumors with immune cells. 2HG contributes to specific alternations in cancer metabolism and developed oxidative stress, while also inducing decisions on the differentiation of naïve T lymphocytes, and serves as a signal messenger in immune cells. Moreover, 2HG inhibits chromatin-modifying enzymes, namely 2-oxoglutarate-dependent dioxygenases, interferes with hypoxia-induced factor (HIF) transcriptome reprogramming and mTOR pathway, thus dysregulating gene expression and further promoting cancerogenesis.
    CRITICAL ISSUES: Typically heterozygous mutations within the active sites of isocitrate dehydrogenase IDH1R132H and mitochondrial IDH2R140Q provide cells with millimolar R-2HG concentrations, while side-activities of lactate and malate dehydrogenase form submillimolar S-2HG. However, even wild-type IDH1 and IDH2, notably under shifts toward reductive carboxylation glutaminolysis or changes in other enzymes, lead to "intermediate" 0.01-0.1 mM 2HG levels, e.g. in breast carcinoma, compared to 10-8 M in non-cancer cells.
    FUTURE DIRECTIONS: Uncovering further molecular metabolism details specific for given cancer cell types and sequence-specific epigenetic alternations will lead to the design of diagnostic approaches, not only predicting patients' prognosis or uncovering metastases and tumor remissions, but also for early diagnostics.
    DOI:  https://doi.org/10.1089/ars.2019.7902