Analyzing Melanoma Cell Oxygen Consumption and Extracellular Acidification Rates Using Seahorse Technology.

Cancer cells have deregulated metabolism that can contribute to the unique metabolic makeup of the tumor microenvironment. This can be variable between patients, and it is important to understand these differences since they potentially can affect therapy response. Here we discuss a method of processing and assaying metabolism from direct ex vivo murine and human tumor samples using seahorse extracellular flux analysis. This provides real-time profiling of oxidative versus glycolytic metabolism and can help infer the metabolic status of the tumor microenvironment.

Studying the Metabolism of Epithelial-Mesenchymal Plasticity Using the Seahorse XFe96 Extracellular Flux Analyzer.

The critical role of metabolism in facilitating cancer cell growth and survival has been demonstrated by a combination of methods including, but not limited to, genomic sequencing, transcriptomic and proteomic analyses, measurements of radio-labelled substrate flux and the high throughput measurement of oxidative metabolism in unlabelled live cells using the Seahorse Extracellular Flux (XF) technology. These studies have revealed that tumour cells exhibit a dynamic metabolic plasticity, using numerous pathways including both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to support cell proliferation, energy production and the synthesis of biomass. These advanced technologies have also demonstrated metabolic differences between cancer cell types, between molecular subtypes within cancers and between cell states. This has been exemplified by examining the transitions of cancer cells between epithelial and mesenchymal phenotypes, referred to as epithelial-mesenchymal plasticity (EMP). A growing number of studies are demonstrating significant metabolic alterations associated with these transitions, such as increased use of glycolysis by triple negative breast cancers (TNBC) or glutamine addiction in lung cancer. Models of EMP, including invasive cell lines and xenografts, isolated circulating tumour cells and metastatic tissue have been used to examine EMP metabolism. Understanding the metabolism supporting molecular and cellular plasticity and increased metastatic capacity may reveal metabolic vulnerabilities that can be therapeutically exploited. This chapter describes protocols for using the Seahorse Extracellular Flux Analyzer (XFe96), which simultaneously performs real-time monitoring of oxidative phosphorylation and glycolysis in living cells. As an example, we compare the metabolic profiles generated from two breast cancer sublines that reflect epithelial and mesenchymal phenotypes, respectively. We use this example to show how the methodology described can generate bioenergetic results that in turn can be correlated to EMP phenotypes. Normalisation of bioenergetic studies should be considered with respect to cell number, and to potential differences in mitochondrial mass, itself being an important bioenergetics endpoint.

Analyzing Intracellular Gradients in Pollen Tubes.

Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca2+ probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.

Direct Estimation of Metabolic Flux by Heavy Isotope Labeling Simultaneous with Pathway Inhibition: Metabolic Flux Inhibition Assay.

Heavy isotope labeled metabolites are readily detected by mass spectrometry and are commonly used to analyze the rates of metabolic reactions in cultured cells. The ability to detect labeled metabolites-and infer fluxes-is influenced by a number of factors that can confound simplistic comparative assays. The accumulation of labeled metabolites is strongly influenced by the pool size of the metabolite of interest and also by changes in downstream reactions, which are not always fully perceived. Here, we describe a method that overcomes some of these limitations and allows simple calculation of reaction rates under low nutrient, rapid reaction rate conditions. Acutely increasing the pool of the metabolite of interest (by adding a pulse of excess unlabeled nutrient to the cells) rapidly increases accumulation of labeled metabolite, facilitating a more accurate assessment of reaction rate.

Measuring Glycolytic and Mitochondrial Fluxes in Endothelial Cells Using Radioactive Tracers.

Endothelial cells (ECs) form the inner lining of the vascular network. Although they can remain quiescent for years, ECs exhibit high plasticity in both physiological and pathological conditions, when they need to rapidly form new blood vessels in a process called angiogenesis. EC metabolism recently emerged as an important driver of this angiogenic switch. The use of radioactive tracer substrates to assess metabolic flux rates in ECs has been essential for the discovery that fatty acid, glucose, and glutamine metabolism critically contribute to vessel sprouting. In the future, these assays will be useful as a tool for the characterization of pathological conditions in which deregulation of EC metabolism underlies and/or precedes the disease, but also for the identification of anti-angiogenic metabolic targets. This chapter describes in detail the radioactive tracer substrate assays that have been used for the determination of EC metabolic flux in vitro.

Determining Compartment-Specific Metabolic Fluxes.

In this chapter, we present an experimental protocol for the targeted metabolic profiling of full cells and mitochondria in selectively permeabilized cells. Mitochondria of adherent cell cultures are made accessible by the addition of digitonin-a compound that selectively permeabilizes the cytosolic membrane without affecting mitochondrial integrity. The generated in situ mitochondria are subsequently used in a stable isotope labeling assay in which their metabolic fluxes can be analyzed without any interfering influence originating from cytosolic components. The protocol is complemented by oxygen consumption measurements of permeabilized cells on a Seahorse XF instrument. The additional data on mitochondrial respiration can be used to validate the functionality of mitochondria in the applied setup but are also a valuable add-on to the stable isotope labeling data.

Determining Macrophage Polarization upon Metabolic Perturbation.

Metabolic reprograming controlling macrophage activation and function is emerging as new regulatory circuit on shaping immune responses. Generally, lipopolysaccharides (LPS)-induced pro-inflammatory activated macrophages, known as M1 macrophages, display higher glycolysis. In contrast, interleukin-4 (IL-4)-skewed anti-inflammatory activated macrophages, known as M2 macrophages, mainly rely on oxidative phosphorylation for their bioenergetic demands. Emerging evidence reveals that these metabolic preferences further fine-tune macrophage polarization process, including signaling cascades and epigenetic reprogramming. Thus, specific nutrient microenvironments may affect inflammatory responses of macrophages by intervening these metabolic machineries. How to measure the metabolic switch of macrophages both in vitro and in vivo is an important issue for understanding immunometabolic regulations in macrophages. Here, we describe a basic protocol for examining how glutamine metabolism affects macrophage polarization by using the Extracellular Flux (XF(e)96) Analyzer (Seahorse Bioscience), which takes real-time measurements of oxidative phosphorylation and glycolysis. We also present a detailed procedure for detecting the expression of inflammatory genes in polarized macrophages under glutamine-replete or -deprived conditions.

Extracellular Flux Assays to Determine Oxidative Phosphorylation and Glycolysis in Chronic Lymphocytic Leukemia Cells.

Extracellular flux assays are conducted using seahorse XF96 analyzer. They are used to calculate oxygen consumption rate which is to determine mitochondrial oxidative phosphorylation and extracellular acidification rate which is a measure of glycolysis. Collectively, these assays are used to assess the metabolic phenotype of a cell. Up to four drugs can be loaded and tested in the XF cartridges used in the assay and their effect on cells could be determined. While adherent cell lines are easy to use for this assay, suspension cultures or primary cells are difficult to use. In the following sections, we describe the methodology for this assay for CLL cells in suspension cultures and CLL-stroma cocultures.

Dynamic 13C Labeling of Fast Turnover Metabolites for Analysis of Metabolic Fluxes and Metabolite Channeling.

Dynamic or isotopically nonstationary 13C labeling experiments are a powerful tool not only for precise carbon flux quantification (e.g., metabolic flux analysis of photoautotrophic organisms) but also for the investigation of pathway bottlenecks, a cell's phenotype, and metabolite channeling. In general, isotopically nonstationary metabolic flux analysis requires three main components: (1) transient isotopic labeling experiments; (2) metabolite quenching and isotopomer analysis using LC-MS; (3) metabolic network construction and flux quantification. Labeling dynamics of key metabolites from 13C-pulse experiments allow flux estimation of key central pathways by solving ordinary differential equations to fit time-dependent isotopomer distribution data. Additionally, it is important to provide biomass requirements, carbon uptake rates, specific growth rates, and carbon excretion rates to properly and precisely balance the metabolic network. Labeling dynamics through cascade metabolites may also identify channeling phenomena in which metabolites are passed between enzymes without mixing with the bulk phase. In this chapter, we outline experimental protocols to probe metabolic pathways through dynamic labeling. We describe protocols for labeling experiments, metabolite quenching and extraction, LC-MS analysis, computational flux quantification, and metabolite channeling observations.

Microphysiological flux balance platform unravels the dynamics of drug induced steatosis.

Drug development is currently hampered by the inability of animal experiments to accurately predict human response. While emerging organ on chip technology offers to reduce risk using microfluidic models of human tissues, the technology still mostly relies on end-point assays and biomarker measurements to assess tissue damage resulting in limited mechanistic information and difficulties to detect adverse effects occurring below the threshold of cellular damage. Here we present a sensor-integrated liver on chip array in which oxygen is monitored using two-frequency phase modulation of tissue-embedded microprobes, while glucose, lactate and temperature are measured in real time using microfluidic electrochemical sensors. Our microphysiological platform permits the calculation of dynamic changes in metabolic fluxes around central carbon metabolism, producing a unique metabolic fingerprint of the liver's response to stimuli. Using our platform, we studied the dynamics of human liver response to the epilepsy drug Valproate (Depakine™) and the antiretroviral medication Stavudine (Zerit™). Using E6/E7LOW hepatocytes, we show TC50 of 2.5 and 0.8 mM, respectively, coupled with a significant induction of steatosis in 2D and 3D cultures. Time to onset analysis showed slow progressive damage starting only 15-20 hours post-exposure. However, flux analysis showed a rapid disruption of metabolic homeostasis occurring below the threshold of cellular damage. While Valproate exposure led to a sustained 15% increase in lipogenesis followed by mitochondrial stress, Stavudine exposure showed only a transient increase in lipogenesis suggesting disruption of ß-oxidation. Our data demonstrates the importance of tracking metabolic stress as a predictor of clinical outcome.

Metabolic Measurements of Nonpermeating Compounds in Live Cells Using Hyperpolarized NMR.

Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) has emerged as a technique for enhancing NMR signals by several orders of magnitude, thereby facilitating the characterization of metabolic pathways both in vivo and in vitro. Following the introduction of an externally hyperpolarized compound, real-time NMR enables the measurement of metabolic flux in the corresponding pathway. Spin relaxation however limits the maximum experimental time and prevents the use of this method with compounds exhibiting slow membrane transport rates. Here, we demonstrate that on-line electroporation can serve as a method for membrane permeabilization for use with D-DNP in cell cultures. An electroporation apparatus hyphenated with stopped-flow sample injection permits the introduction of the hyperpolarized metabolite within 3 s after the electrical pulse. In yeast cells that do not readily take up pyruvate, the addition of the electroporation pulse to the D-DNP experiment increases the signals of the downstream metabolic products CO2 and HCO3-, which otherwise are near the detection limit, by 8.2- and 8.6-fold. Modeling of the time dependence of these signals then permits the determination of the respective kinetic rate constants. The observed conversion rate from pyruvate to CO2 normalized for cell density was found to increase by a factor of 12 due to the alleviation of the membrane transport limitation. The use of electroporation therefore extends the applicability of D-DNP to in vitro studies with a wider range of metabolites and at the same time reduces the influence of membrane transport on the observed conversion rates.

Assessment of metabolism-dependent drug efficacy and toxicity on a multilayer organs-on-a-chip.

Pharmaceutical development is greatly hindered by the poor predictive power of existing in vitro models for drug efficacy and toxicity testing. In this work, we present a new and multilayer organs-on-a-chip device that allows for the assessment of drug metabolism, and its resultant drug efficacy and cytotoxicity in different organ-specific cells simultaneously. Four cell lines representing the liver, tumor (breast cancer and lung cancer), and normal tissue (gastric cells) were cultured in the compartmentalized micro-chambers of the multilayer microdevice. We adopted the prodrug capecitabine (CAP) as a model drug. The intermediate metabolites 5'-deoxy-5-fluorocytidine (DFUR) of CAP that were metabolized from liver and its active metabolite 5-fluorouracil (5-FU) from the targeted cancer cells and normal tissue cells were identified using mass spectrometry. CAP exhibited strong cytoxicity on breast cancer and lung cancer cells, but not in normal gastric cells. Moreover, the drug-induced cytotoxicity on cells varied in various target tissues, suggesting the metabolism-dependent drug efficacy in different tissues as exisits in vivo. This in vitro model can not only allow for characterizing the dynamic metabolism of anti-cancer drugs in different tissues simultaneously, but also facilitate the assessment of drug bioactivity on various target tissues in a simple way, indicating the utility of this organs-on-chip for applications in pharmacodynamics/pharmacokinetics studies, drug efficacy and toxicity testing.

Simultaneous recording of brain extracellular glucose, spike and local field potential in real time using an implantable microelectrode array with nano-materials.

Glucose is the main substrate for neurons in the central nervous system. In order to efficiently characterize the brain glucose mechanism, it is desirable to determine the extracellular glucose dynamics as well as the corresponding neuroelectrical activity in vivo. In the present study, we fabricated an implantable microelectrode array (MEA) probe composed of platinum electrochemical and electrophysiology microelectrodes by standard micro electromechanical system (MEMS) processes. The MEA probe was modified with nano-materials and implanted in a urethane-anesthetized rat for simultaneous recording of striatal extracellular glucose, local field potential (LFP) and spike on the same spatiotemporal scale when the rat was in normoglycemia, hypoglycemia and hyperglycemia. During these dual-mode recordings, we observed that increase of extracellular glucose enhanced the LFP power and spike firing rate, while decrease of glucose had an opposite effect. This dual mode MEA probe is capable of examining specific spatiotemporal relationships between electrical and chemical signaling in the brain, which will contribute significantly to improve our understanding of the neuron physiology.

BIOENERGETIC CHARACTERIZATION OF H9C2 CELLS USING THE EXTRACELLULAR FLUX ANALYZER.

UNLABELLED: The aim of the present work was to standardize the working methodology for assessing the bioenergetic profile of H9c2 cardiomyoblasts cells, with reference to the optimization of cell seeding number and the establishment of favorable concentrations for the classic modulators of mitochondrial respiratory function, in particular the one of a classical uncoupler, FCCP. MATERIAL AND METHODS: The extracellular flux analyzer (XF, Seahorse Bioscience) is a novel high-throughput instrument able to monitor the metabolism of living cells by simultaneously measuring mitochondrial respiration and glycolysis. The in vitro platform will be further used to better understand the pathophysiology and the unrecognized side effects of drugs currently used in the therapy of major cardiovascular diseases. CONCLUSIONS: In the long run, characterization of novel pharmacological agents' effects on other cell lines, including tumoral ones, will be also considered.

GC-MS-based ¹³C metabolic flux analysis.

The in vivo analysis of metabolic fluxes has become a valuable method for the investigation of microorganisms. It turned out especially useful in industrial biotechnology for the prediction of beneficial genetic targets for rational strain optimization. Here, we describe in detail the procedure for the state-of-the-art approach of (13)C metabolic flux analysis comprising steady-state cultivations in a mineral salt medium with (13)C-labeled substrates, GC-MS measurement for labeling analysis, as well as metabolic modeling using the open-source software OpenFlux.

A novel platform for automated high-throughput fluxome profiling of metabolic variants.

Advances in metabolic engineering are enabling the creation of a large number of cell factories. However, high-throughput platforms do not yet exist for rapidly analyzing the metabolic network of the engineered cells. To fill the gap, we developed an integrated solution for fluxome profiling of large sets of biological systems and conditions. This platform combines a robotic system for (13)C-labelling experiments and sampling of labelled material with NMR-based isotopic fingerprinting and automated data interpretation. As a proof-of-concept, this workflow was applied to discriminate between Escherichia coli mutants with gradual expression of the glucose-6-phosphate dehydrogenase. Metabolic variants were clearly discriminated while pathways that support metabolic flexibility towards modulation of a single enzyme were elucidating. By directly connecting the data flow between cell cultivation and flux quantification, considerable advances in throughput, robustness, release of resources and screening capacity were achieved. This will undoubtedly facilitate the development of efficient cell factories.

Bioenergetic analysis of intact mammalian cells using the Seahorse XF24 Extracellular Flux analyzer and a luciferase ATP assay.

Metabolic pathways and bioenergetics were described in great detail over half a century ago, and during the past decade there has been a resurgence in integrating these cellular processes with other biological properties of the cell, including growth control, protein kinase cascade signaling, cell cycle division, and autophagy. Since many disease conditions are associated with altered metabolism and production of energy, it is important to develop new approaches to measure these cellular parameters. This chapter summarizes a new and exciting approach based on the Seahorse XF24 Extracelluar Flux analyzer, which takes real time measurements of oxidative phosphorylation and glycolysis in living cells. These bioenergetic profiles are then compared with steady-state levels of cellular ATP as measured by a luciferase assay.

A high sensitivity MEA probe for measuring real time rat brain glucose flux.

The mammalian central nervous system (CNS) relies on a constant supply of external glucose for its undisturbed operation. This article presents an implantable Multi-Electrode Array (MEA) probe for brain glucose measurement. The MEA was implemented on Silicon-On-Insulator (SOI) wafer using Micro-Electro-Mechanical-Systems (MEMS) methods. There were 16 platinum recording sites on the probe and enzyme glucose oxidase (GOx) was immobilized on them. The glucose sensitivity of the MEA probe was as high as 489 µA mM(-1) cm(-2). 1,3-Phenylenediamine (mPD) was electropolymerized onto the Pt recording surfaces to prevent larger molecules such as ascorbic acid (AA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), and dopamine (DA) from reaching the recording sites surface. The MEA probe was implanted in the anesthetized rat striatum and responded to glucose levels which were altered by intraperitoneal injection of glucose and insulin. After the in vivo experiment, the MEA probe still kept sensitivity to glucose, these suggested that the MEA probe was reliable for glucose monitoring in brain extracellular fluid (ECF).