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

Pike Winer LS, Wu M - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Seahorse Bioscience Inc., North Billerica, Massachusetts, United States of America.

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.

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Lowered basal glycolytic flux but acquired glycolytic capacity in SF188f cells compared with SF188s cells.A. ECAR response of SF188s and SF188f cells to glucose (10 mM), oligomycin (1 µM), and 2-DG (100 mM). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 2 mM glutamine. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. ECAR values were normalized to mpH/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B. Calculated glycolytic flux and glycolytic capacity of SF188s and SF188f cells normalized to mpH/min/10,000 cells. * p<0.05.
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pone-0109916-g007: Lowered basal glycolytic flux but acquired glycolytic capacity in SF188f cells compared with SF188s cells.A. ECAR response of SF188s and SF188f cells to glucose (10 mM), oligomycin (1 µM), and 2-DG (100 mM). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 2 mM glutamine. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. ECAR values were normalized to mpH/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B. Calculated glycolytic flux and glycolytic capacity of SF188s and SF188f cells normalized to mpH/min/10,000 cells. * p<0.05.

Mentions: Having determined the basal OCR and ECAR, we investigated glycolytic and oxidative substrate flux in these cells. First, we examined the glycolytic arm of metabolism. The normalized basal glycolytic flux was much lower in SF188f cells, at 5.6±0.8 mpH/104 cells, compared with SF188s cells, at 14.4±0.8 mpH/104 cells (Figure7A). Interestingly, oligomycin stimulated a large ECAR increase over basal glycolysis in SF188f cells (Figure 7A), but failed to evoke a significant increase in SF188s cells. Thus, under basal conditions, glycolysis in SF188s cells occurs at full capacity while SF188f cells possess a substantial unused glycolytic capacity. The respective glycolysis flux and glycolytic capacity of the pair are shown in Figure 7B.


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

Pike Winer LS, Wu M - PLoS ONE (2014)

Lowered basal glycolytic flux but acquired glycolytic capacity in SF188f cells compared with SF188s cells.A. ECAR response of SF188s and SF188f cells to glucose (10 mM), oligomycin (1 µM), and 2-DG (100 mM). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 2 mM glutamine. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. ECAR values were normalized to mpH/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B. Calculated glycolytic flux and glycolytic capacity of SF188s and SF188f cells normalized to mpH/min/10,000 cells. * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215881&req=5

pone-0109916-g007: Lowered basal glycolytic flux but acquired glycolytic capacity in SF188f cells compared with SF188s cells.A. ECAR response of SF188s and SF188f cells to glucose (10 mM), oligomycin (1 µM), and 2-DG (100 mM). SF188s and SF188f cells were plated at 30,000 and 20,000 cells/well, respectively, in XF24 V7 cell culture plates 24–28 hours prior to the assays. The assay medium was the substrate-free base medium supplemented with 2 mM glutamine. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization. ECAR values were normalized to mpH/104 cells. A representative experiment out of three is shown here. Each data point represents mean ± SD, n = 4. B. Calculated glycolytic flux and glycolytic capacity of SF188s and SF188f cells normalized to mpH/min/10,000 cells. * p<0.05.
Mentions: Having determined the basal OCR and ECAR, we investigated glycolytic and oxidative substrate flux in these cells. First, we examined the glycolytic arm of metabolism. The normalized basal glycolytic flux was much lower in SF188f cells, at 5.6±0.8 mpH/104 cells, compared with SF188s cells, at 14.4±0.8 mpH/104 cells (Figure7A). Interestingly, oligomycin stimulated a large ECAR increase over basal glycolysis in SF188f cells (Figure 7A), but failed to evoke a significant increase in SF188s cells. Thus, under basal conditions, glycolysis in SF188s cells occurs at full capacity while SF188f cells possess a substantial unused glycolytic capacity. The respective glycolysis flux and glycolytic capacity of the pair are shown in Figure 7B.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Seahorse Bioscience Inc., North Billerica, Massachusetts, United States of America.

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.

Show MeSH
Related in: MedlinePlus