218 research outputs found
Stable Isotope Resolved Metabolomics Studies \u3cem\u3ein ex vivo\u3c/em\u3e Tissue Slices
An important component of this methodology is to assess the role of the tumor microenvironment on tumor growth and survival. To tackle this problem, we have adapted the original approach of Warburg (Warburg, 1923), by combining thin tissue slices with Stable Isotope Resolved Metabolomics (SIRM) to determine detailed metabolic activity of human tissues. SIRM enables the tracing of metabolic transformations of source molecules such as glucose or glutamine over defined time periods, and is a requirement for detailed pathway tracing and flux analysis. In our approach, we maintain freshly resected tissue slices (both cancerous and non- cancerous from the same organ of the same subject) in cell culture media, and treat with appropriate stable isotope-enriched nutrients, e.g. 13C6-glucose or 13C5, 15N2 -glutamine. These slices are viable for at least 24 h, and make it possible to eliminate systemic influence on the target tissue metabolism while maintaining the original 3D cellular architecture. It is therefore an excellent pre-clinical platform for assessing the effect of therapeutic agents on target tissue metabolism and their therapeutic efficacy on individual patients (Xie et al., 2014; Sellers et al., 2015)
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. Stable Isotope Resolved Metabolomics (SIRM) is a valuable tool for tracing metabolic networks in complex systems, but at present does not directly address heterogeneous metabolism at the individual cell level. We compare the advantages and disadvantages of different model systems for SIRM experiments, with a focus on lung cancer cells, their interactions with macrophages and T cells, and their response to modulators in the immune microenvironment. We describe the experimental set up, illustrate results from 3D cultures and co-cultures of lung cancer cells with human macrophages, and outline strategies to address the heterogeneous TME
Chloroformate Derivatization for Tracing the Fate of Amino Acids in Cells and Tissues by Multiple Stable Isotope Resolved Metabolomics (mSIRM)
Amino acids have crucial roles in central metabolism, both anabolic and catabolic. To elucidate these roles, steady-state concentrations of amino acids alone are insufficient, as each amino acid participates in multiple pathways and functions in a complex network, which can also be compartmentalized. Stable Isotope-Resolved Metabolomics (SIRM) is an approach that uses atom-resolved tracking of metabolites through biochemical transformations in cells, tissues, or whole organisms. Using different elemental stable isotopes to label multiple metabolite precursors makes it possible to resolve simultaneously the utilization of these precursors in a single experiment. Conversely, a single precursor labeled with two (or more) different elemental isotopes can trace the allocation of e.g. C and N atoms through the network.
Such dual-label experiments however challenge the resolution of conventional mass spectrometers, which must distinguish the neutron mass differences among different elemental isotopes. This requires ultrahigh resolution Fourier transform mass spectrometry (UHR-FTMS). When combined with direct infusion nano-electrospray ion source (nano-ESI), UHR-FTMS can provide rapid, global, and quantitative analysis of all possible mass isotopologues of metabolites. Unfortunately, very low mass polar metabolites such as amino acids can be difficult to analyze by current models of UHR-FTMS, plus the high salt content present in typical cell or tissue polar extracts may cause unacceptable ion suppression for sources such as nano-ESI.
Here we describe a modified method of ethyl chloroformate (ECF) derivatization of amino acids to enable rapid quantitative analysis of stable isotope labeled amino acids using nano-ESI UHR-FTMS. This method showed excellent linearity with quantifiable limits in the low nanomolar range represented in microgram quantities of biological specimens, which results in extracts with total analyte abundances in the low to sub-femtomole range. We have applied this method to profile amino acids and their labeling patterns in 13C and 2H doubly labeled PC9 cell extracts, cancerous and non-cancerous tissue extracts from a lung cancer patient and their protein hydrolysates as well as plasma extracts from mice fed with a liquid diet containing 13C6-glucose.
The multi-element isotopologue distributions provided key insights into amino acid metabolism and intracellular pools in human lung cancer tissues in high detail. The 13C labeling of Asp and Glu revealed de novo synthesis of these amino acids from 13C6-glucose via the Krebs cycle, specifically the elevated level of 13C3-labeled Asp and Glu in cancerous versus non-cancerous lung tissues was consistent with enhanced pyruvate carboxylation. In addition, tracking the fate of double tracers, (13C6-Glc + 2H2-Gly or 13C6-Glc + 2H3-Ser) in PC9 cells clearly resolved pools of Ser and Gly synthesized de novo from 13C6-Glc (13C3-Ser and 13C2-Gly) versus Ser and Gly derived from external sources (2H3-Ser, 2H2-Gly). Moreover the complex 2H labeling patterns of the latter were results of Ser and Gly exchange through active Ser-Gly one-carbon metabolic pathway in PC9 cells
Exploring Cancer Metabolism Using Stable Isotope-Resolved Metabolomics (SIRM)
Metabolic reprogramming is a hallmark of cancer. The changes in metabolism are adaptive to permit proliferation, survival, and eventually metastasis in a harsh environment. Stable isotope-resolved metabolomics (SIRM) is an approach that uses advanced approaches of NMR and mass spectrometry to analyze the fate of individual atoms from stable isotope-enriched precursors to products to deduce metabolic pathways and networks. The approach can be applied to a wide range of biological systems, including human subjects. This review focuses on the applications of SIRM to cancer metabolism and its use in understanding drug actions
A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions
<p>Abstract</p> <p>Background</p> <p>Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of <sup>13</sup>C isotopologue data for UDP-<it>N</it>-acetyl-<smcaps>D</smcaps>-glucosamine (UDP-GlcNAc) synthesized from [U-<sup>13</sup>C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes.</p> <p>Results</p> <p>We utilized a stable isotope resolved metabolomics (SIRM) approach to determine the timecourse of <sup>13</sup>C incorporation from [U-<sup>13</sup>C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. <sup>13</sup>C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS) was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional <sup>13</sup>C enrichment in these subunits against experimental data. The reproducibility and robustness of the deconvolution were verified by replicate experiments, extensive statistical analyses, and cross-validation against NMR data.</p> <p>Conclusions</p> <p>This computational approach revealed the relative fluxes through the different biosynthetic pathways of UDP-GlcNAc, which comprises simultaneous sequential and parallel reactions, providing new insight into the regulation of UDP-GlcNAc levels and <it>O</it>-linked protein glycosylation. This is the first such analysis of UDP-GlcNAc dynamics, and the approach is generally applicable to other complex metabolites comprising distinct metabolic subunits, where sufficient numbers of isotopologues can be unambiguously resolved and accurately measured.</p
Effects of the pathogenic water mold Saprolegnia ferax on survival of amphibian larvae
Infectious diseases are a significant threat to worldwide biodiversity. Amphibian declines, a significant part of current biodiversity losses, are in many cases associated with infectious disease. Water molds are one group of pathogens affecting amphibians on a worldwide basis. Although water molds have been studied extensively for their effects on host embryos, little information is available about how they affect post-embryonic amphibians. We tested the effects of one species of water mold, Saprolegnia ferax, in a comparative study of larvae of 4 amphibian species: Pseudacris regilla (Pacific treefrog), Rana cascadae (Cascades frog), Ambystoma macrodactylum (long-toed salamander), and R. aurora (red-legged frog). S. ferax can kill amphibians at the embryonic and juvenile life history stages, depending on the amphibian species. In the present study, a 1 wk exposure to S. ferax killed P. regilla larvae and a 2 wk exposure killed R. aurora larvae. Larvae of the other host species were unaffected after 1 wk of exposure to S. ferax. Our results suggest that S. ferax can kill amphibian larvae and further suggest that evaluation of how pathogens affect amphibians at the population level requires investigation at various life stages
Probing the Metabolic Phenotype of Breast Cancer Cells by Multiple Tracer Stable Isotope Resolved Metabolomics
Breast cancers vary by their origin and specific set of genetic lesions, which gives rise to distinct phenotypes and differential response to targeted and untargeted chemotherapies. To explore the functional differences of different breast cell types, we performed Stable Isotope Resolved Metabolomics (SIRM) studies of one primary breast (HMEC) and three breast cancer cells (MCF-7, MDAMB-231, and ZR75-1) having distinct genotypes and growth characteristics, using 13C6-glucose, 13C-1+2-glucose, 13C5,15N2-Gln, 13C3-glycerol, and 13C8-octanoate as tracers. These tracers were designed to probe the central energy producing and anabolic pathways (glycolysis, pentose phosphate pathway, Krebs Cycle, glutaminolysis, nucleotide synthesis and lipid turnover). We found that glycolysis was not associated with the rate of breast cancer cell proliferation, glutaminolysis did not support lipid synthesis in primary breast or breast cancer cells, but was a major contributor to pyrimidine ring synthesis in all cell types; anaplerotic pyruvate carboxylation was activated in breast cancer versus primary cells. We also found that glucose metabolism in individual breast cancer cell lines differed between in vitro cultures and tumor xenografts, but not the metabolic distinctions between cell lines, which may reflect the influence of tumor architecture/microenvironment
Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes
<p>Abstract</p> <p>Background</p> <p>The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30) and human primary myocytes in culture. Uniformly <sup>13</sup>C-labeled glucose was used as a source molecule to follow the incorporation of <sup>13</sup>C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis.</p> <p>Results</p> <p>The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by <sup>13</sup>C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.</p> <p>Conclusion</p> <p>The specific <sup>13</sup>C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells. The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis. This may be a common adaptive strategy in cancer cells.</p
Altered regulation of metabolic pathways in human lung cancer discerned by 13C stable isotope-resolved metabolomics (SIRM)
<p>Abstract</p> <p>Background</p> <p>Metabolic perturbations arising from malignant transformation have not been systematically characterized in human lung cancers <it>in situ</it>. 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 <sup>13</sup>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 <sup>13</sup>C-isotopomer-based metabolomic analysis of the extracts of tissues and blood plasma.</p> <p>Results</p> <p>Many primary metabolites were consistently found at higher levels in lung cancer tissues than their surrounding non-cancerous tissues. <sup>13</sup>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 <sup>13</sup>C-labeled carbons and the buildup of <sup>13</sup>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.</p> <p>Conclusion</p> <p>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.</p
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