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Metabolism is a compartmentalized process, and it is apparent in studying cancer that tumors, like normal tissues, demonstrate metabolic cooperation between different cell types. Metabolic profiling of cells in 2D culture systems often fails to reflect the metabolism occurring within tissues in vivo due to lack of other cell types and 3D interaction. We designed a tooling and methodology to metabolically profile and compare 2D cultures with cancer cell spheroids, and microtissue slices from tumors, and normal organs. We observed differences in the basal metabolism of 2D and 3D cell cultures in response to metabolic inhibitors, and chemotherapeutics. The metabolic profiles of microtissues derived from normal organs (heart, kidney) were relatively consistent when comparing microtissues derived from the same organ. Treatment of heart and kidney microtissues with cardio- or nephro-toxins had early and marked effects on tissue metabolism. In contrast, microtissues derived from different regions of the same tumors exhibited significant metabolic heterogeneity, which correlated to histology. Hence, metabolic profiling of complex microtissues is necessary to understand the effects of metabolic co-operation and how this interaction, not only can be targeted for treatment, but this method can be used as a reproducible, early and sensitive measure of drug toxicity.

Triclosan (TCS) is a synthetic antimicrobial agent used in many consumer goods at millimolar concentrations. As a result of exposure, TCS has been detected widely in humans. We have recently discovered that TCS is a proton ionophore mitochondrial uncoupler in multiple types of living cells. Here, we present novel data indicating that TCS is also a mitochondrial uncoupler in a living organism: 24-hour post-fertilization (hpf) zebrafish embryos. These experiments were conducted using a Seahorse Bioscience XFe 96 Extracellular Flux Analyzer modified for bidirectional temperature control, using the XF96 spheroid plate to position and measure one zebrafish embryo per well. Using this method, after acute exposure to TCS, the basal oxygen consumption rate (OCR) increases, without a decrease in survival or heartbeat rate. TCS also decreases ATP-linked respiration and spare respiratory capacity and increases proton leak: all indicators of mitochondrial uncoupling. Our data indicate, that TCS is a mitochondrial uncoupler in vivo, which should be taken into consideration when assessing the toxicity and/or pharmaceutical uses of TCS. This is the first example of usage of a Seahorse Extracellular Flux Analyzer to measure bioenergetic flux of a single zebrafish embryo per well in a 96-well assay format. The method developed in this study provides a high-throughput tool to identify previously unknown mitochondrial uncouplers in a living organism. Copyright © 2016 John Wiley & Sons, Ltd.

Cell-based assays have become a favored format for drug discovery because living cells have relevant biological complexity and can be highly multiplexed to screen for drugs and their mechanisms. In response to a changing extracellular environment, disease and/or drug exposure, cells remodel bioenergetic pathways in a matter of minutes to drive phenotypic changes associated with these perturbations. By measuring the extracellular flux (XF), that is the changes in oxygen and proton concentrations in the media surrounding cells, one can simultaneously determine their relative state of aerobic and glycolytic metabolism, respectively. In addition, XF is time-resolved and non-invasive, making it an attractive format for studying drug effects in vitro.

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells.

Triclosan (TCS) is a synthetic antimicrobial agent used in many consumer goods at millimolar concentrations. As a result of exposure, TCS has been detected widely in humans. We have recently discovered that TCS is a proton ionophore mitochondrial uncoupler in multiple types of living cells. Here we present novel data indicating that TCS is also a mitochondrial uncoupler in a living organism: 24 hour post fertilization zebrafish embryos. These experiments were conducted using a Seahorse Bioscience XFe 96 Extracellular Flux Analyzer modified for bidirectional temperature control, using the XF96 spheroid plate to position and measure one zebrafish embryo per well. Using this method, following acute exposure to TCS, basal oxygen consumption rate (OCR) increases, without a decrease in survival or heartbeat rate. TCS also decreases ATP-linked respiration and spare respiratory capacity and increases proton leak: all indicators of mitochondrial uncoupling. Our data indicate, that TCS is a mitochondrial uncoupler in vivo, which should be taken into consideration when assessing the toxicity and/or pharmaceutical uses of TCS. This is the first example of usage of a Seahorse Extracellular Flux Analyzer to measure bioenergetic flux of a single zebrafish embryo per well in a 96 well assay format. The method developed in this study provides a high-throughput tool to identify previously-unknown mitochondrial uncouplers in a living organism.

Cellular metabolism and bioenergetics processes are increasingly recognized as critical regulators in the pathogenesis of cancers. Metabolic activity and bioenergetic processes are often characterized using typical 2 dimensional (2D) adherent cell culture systems; however, this approach lacks the 3 dimensional (3D) context and heterogeneity of tumor tissue in vivo, which may have significant effects on metabolic and bioenergetic behavior. A novel and more physiologically relevant model is the use of microtissues (also known as spheroids). Metabolic activity and bioenergetic function were accessed in individual cancer cell line microtissues using a newly designed 96-well plate for the Seahorse Bioscience XFe96 Extracellular Flux Analyzer. Mitochondrial and glycolytic functions were determined simultaneously using the Cell Mito Stress and Glycolytic Stress Tests, respectively. Several parameters, including: seeding density, microtissue size, and substrate identity and availability were investigated. Further...

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Background The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. Methodology/Principal Findings The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. Conclusions/Significance The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

Respirometry using modified cell culture microplates offers an increase in throughput and a decrease in biological material required for each assay. Plate based respirometers are susceptible to a range of diffusion phenomena; as O(2) is consumed by the specimen, atmospheric O(2) leaks into the measurement volume. Oxygen also dissolves in and diffuses passively through the polystyrene commonly used as a microplate material. Consequently the walls of such respirometer chambers are not just permeable to O(2) but also store substantial amounts of gas. O(2) flux between the walls and the measurement volume biases the measured oxygen consumption rate depending on the actual [O(2)] gradient. We describe a compartment model-based correction algorithm to deconvolute the biological oxygen consumption rate from the measured [O(2)]. We optimize the algorithm to work with the Seahorse XF24 extracellular flux analyzer. The correction algorithm is biologically validated using mouse cortical synaptosomes and liver mitochondria attached to XF24 V7 cell culture microplates, and by comparison to classical Clark electrode oxygraph measurements. The algorithm increases the useful range of oxygen consumption rates, the temporal resolution, and durations of measurements. The algorithm is presented in a general format and is therefore applicable to other respirometer systems.

Perfluorocyclobutyl (PFCB) polymers exhibit a superior combination of optical and structural properties including: low transmission loss, unprecedented processability, tailorable refractive index and thermoptic coefficients compared to other fluoropolymers. An overview of PFCB synthesis, fabrication, properties, and commercial opportunities will be presented.

The search for metabolic liabilities in tumor cells is typically performed in standard 2D cultures. This approach lacks the 3D context and heterogeneity of tumor tissue in vivo, which has significant impact on their metabolic behavior. One solution to modeling complex tumor cell interactions is to use cultured tumor spheroids. To assess bioenergetic function in individual spheroids, a modified, 96-well plate for the Seahorse Bioscience XFe96 Extracellular Flux Analyzer was created. Here, we demonstrate the capability of measuring mitochondrial oxygen consumption – a measure or mitochondrial respiration – and extracellular acidification – a measure of glycolytic flux – in individual HCT116 colon carcinoma spheroids. Spheroids were cultured using the hanging drop method for 4 days. Mitochondrial function was determined using the Cell Mito Stress Test which profiles 6 specific indices of mitochondrial activity. Cells grown in spheroids had a markedly increased spare respiratory capacity compared to cells in 2D culture. These data are consistent with a shift from the highly proliferative nature of 2D culture to a more physiological model where proliferation and differentiation is more balanced. We hypothesize that viewing metabolism holistically allows for a more complete view of cellular bioenergetic function and potential liabilities. To understand the metabolic potential of cells in the context of their basal metabolism, two new metrics for describing mitochondrial function were established. By stimulating maximal oxygen consumption by combined exposure to oligomycin and FCCP, and maximal extracellular acidification rate (ECAR) using rotenone, antimycin A, and oligomycin, the potential to switch metabolic pathways was determined. Metabolic Capacity Index was identified using the maximal responses in each direction, and Metabolic Flexibility Index was calculated as the angle of the line to identify cellular preference for OXPHOS or glycolysis. Interestingly, plastic-adherent HCT116 cells only exhibited potential to increase glycolysis, while HCT116 spheroids had a more balanced response. These data are consistent with the finding that spheroids have increased spare capacity. Implications of this work for future studies in cancer biology will be discussed. Citation Format: Brian P. Dranka, Pamela Swain, George W. Rogers, Ajit S. Divakaruni, Andy Neilson, David A. Ferrick. Metabolic liabilities of human colon carcinoma spheroids are different compared to standard 2D cultures. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B07. doi:10.1158/1538-7445.CHTME14-B07