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Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate.

ABSTRACT: Cancer cells exhibit remarkable alterations in cellular metabolism, particularly in their nutrient substrate preference. We have devised several experimental methods that rapidly analyze the metabolic substrate flux in cancer cells: glycolysis and the oxidation of major fuel substrates glucose, glutamine, and fatty acids. Using the XF Extracellular Flux analyzer, these methods measure, in real-time, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in a microplate as they respond to substrates and metabolic perturbation agents. In proof-of-principle experiments, we analyzed substrate flux and mitochondrial bioenergetics of two human glioblastoma cell lines, SF188s and SF188f, which were derived from the same parental cell line but proliferate at slow and fast rates, respectively. These analyses led to three interesting observations: 1) both cell lines respired effectively with substantial endogenous substrate respiration; 2) SF188f cells underwent a significant shift from glycolytic to oxidative metabolism, along with a high rate of glutamine oxidation relative to SF188s cells; and 3) the mitochondrial proton leak-linked respiration of SF188f cells increased significantly compared to SF188s cells. It is plausible that the proton leak of SF188f cells may play a role in allowing continuous glutamine-fueled anaplerotic TCA cycle flux by partially uncoupling the TCA cycle from oxidative phosphorylation. Taken together, these rapid, sensitive and high-throughput substrate flux analysis methods introduce highly valuable approaches for developing a greater understanding of genetic and epigenetic pathways that regulate cellular metabolism, and the development of therapies that target cancer metabolism.

SUBMITTER: Pike Winer LS

PROVIDER: S-EPMC4215881 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate.

Pike Winer Lisa S LS Wu Min M

PloS one 20141031 10

Cancer cells exhibit remarkable alterations in cellular metabolism, particularly in their nutrient substrate preference. We have devised several experimental methods that rapidly analyze the metabolic substrate flux in cancer cells: glycolysis and the oxidation of major fuel substrates glucose, glutamine, and fatty acids. Using the XF Extracellular Flux analyzer, these methods measure, in real-time, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of living cells in ...[more]

PMID: 25360519

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Project description:Following UV irradiation of skin, dendritic cells (DCs) differentiating from the bone marrow (BM) of mice have a reduced ability to prime new immune responses; their reduced immunogenicity is maintained for at least 16 weeks in UV-chimeric mice. We hypothesized that different metabolic states underpin changes in DC function. Compared with DCs from the BM of non-irradiated mice, DCs from the BM of UV-irradiated mice produced more lactate and utilized greater amounts of glucose, a profile that was supported by greater glycolytic flux when incubated in low-serum-containing medium. Responses to a mitochondrial stress test were similar suggesting that the DCs from the BM of UV-irradiated mice had not switched from a profile of oxidative phosphorylation, but were imprinted for greater glycolytic responses. After microarray profiling, RT-qPCR confirmation and Ingenuity pathway analysis, greater expression of the enzyme, 3-hydroxyanthranilate 3,4-dioxygenase, was identified as a potential contributor to increased glycolysis by BM-differentiated DCs. This enzyme provides the final step of the biosynthetic pathway from tryptophan to quinolinate, the universal de novo precursor to the pyridine ring of nicotinamide adenine dinucleotide (NAD), and may provide a mechanism to ensure sufficient NAD is available to support enhanced glycolysis. Increased lactate production was also measured for DCs from the BM of 16-week engrafted UV-chimeric mice and suggests long-lasting imprinting of progenitor cells for altered immunometabolism in their progeny cells. This study provides evidence of changes to metabolic states that associate with altered DC function. Female C57BL/6J (CD45.2 alloantigen) and B6.SJL-Ptprca (CD45.1 alloantigen) mice were obtained from the Animal Resources Centre (Murdoch, Western Australia). A bank of TL40W/12RS lamps (Philips, Amsterdam, The Netherlands) emitting broadband UVR with 65% UVB (280-320 nm) and peak emission at 313 nm was used. Twenty-four h prior to irradiation, a uniform area of dorsal skin of mice was shaved (8 cm2). To administer UVR, mice were held in perspex compartments which were covered with 0.2 mm polyvinyl chloride plastic to eliminate wavelengths <290 nm. The compartments were placed 20 cm beneath the UV lamps and up to 8 kJ/m2 UVR was delivered. Eight kJ/m2 UVR is equivalent to 3-4 minimal erythemal doses . The UVB output by the lamps was measured using a UVX radiometer (Ultraviolet Products Inc., Upland, CA). Mice were 6-10 wk old at the time of irradiation unless otherwise stated. Mice were subcutaneously implanted with two PGE2 pellets, each containing 0.1 mg with a constant release of 4.76 Âµg/day over 21 days (total 9.52 Âµg/day, Innovative Research of America, Sarasota, FL), three days prior to isolation of BM cells. The pellets were inserted into the loose skin at the top of the back. Freshly-isolated BM cells were cultured for 5 days in RPMI-1640 (HyClone, GE Health Care Life Sciences, Logan, UT) supplemented with 10% FCS (RPMI-10), 10 ng/ml GM-CSF and 10 ng/ml IL-4 (Peprotech Inc, Rocky Hill, NJ) at a density of 8 x 105 cells/ml in 24 well plates to promote CD11c+ cell differentiation (medium replaced on days 2 and 4). Non-adherent cells were enriched to >95% CD11c+ cells (confirmed by flow cytometry) using anti-CD11c magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and autoMACS-Pro (Miltenyi Biotec) separation. Total RNA was extracted from triplicate preparations of BM-differentiated CD11c+ cells. Two sample groups; BM-differentiated DC sample from C57BL/6J mice injected with 2 x 10^6 BM cells from a naÃ¯ve congenic B6.SJL-Ptprca mouse BM-differentiated DC sample from C57BL/6J mice injected with 2 x 10^6 BM cells from a UV irradiated congenic B6.SJL-Ptprca mouse

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Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate.

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Rapid analysis of glycolytic and oxidative substrate flux of cancer cells in a microplate.

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