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.

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.

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.

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.

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).