Limits...
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.

Show MeSH

Related in: MedlinePlus

High proliferation rates of SF188f cells and associated shift in their basal OCR and ECAR compared with SF188s cells.A. Number of SF188s and SF188f cells at 24, 48 and 72 hours in their respective culture medium. B. Basal OCR and ECAR of SF188s and SF188f cells. 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. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization; Assay medium was the substrate-free base medium supplemented with 25 mM glucose, 6 mM Glutamine and 1 mM pyruvate. A representative experiment out of three is shown here. The OCR and ECAR values were normalized to pmoles/min/104 cells or mpH/min/104cells. Each data point represents mean ± SD, n = 3.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215881&req=5

pone-0109916-g006: High proliferation rates of SF188f cells and associated shift in their basal OCR and ECAR compared with SF188s cells.A. Number of SF188s and SF188f cells at 24, 48 and 72 hours in their respective culture medium. B. Basal OCR and ECAR of SF188s and SF188f cells. 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. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization; Assay medium was the substrate-free base medium supplemented with 25 mM glucose, 6 mM Glutamine and 1 mM pyruvate. A representative experiment out of three is shown here. The OCR and ECAR values were normalized to pmoles/min/104 cells or mpH/min/104cells. Each data point represents mean ± SD, n = 3.

Mentions: Having established the methods to analyze glycolysis and oxidation of exogenous substrates, we performed proof-of-principle experiments in SF188s and SF188f cells (see Material and Methods). These two cell lines were derived from the same parental cell line SF188 (which harbor c-MYC amplification) [35], but proliferated at very different rates, with the former much slower than the latter (Figure 6A). The fast-growing behavior of SF188 cells occurred only after, and not within, the initial four weeks' culture in DMEM containing 25 mM glucose and 6 mM glutamine, whereas SF188 cells cultured in DMEM containing 5.5 mM glucose and 2 mM glutamine maintained the same growth rate as the parental cells, suggesting an intrinsic change in SF188f cells' growth program as they acquired the fast growing behavior. We were curious as to whether there were any metabolic alterations associated with SF188f cells' fast growing behavior. Upon examining their basal OCR and ECAR in assay medium containing 25 mM glucose, 6 mM glutamine and 1 mM pyruvate, we found that SF188f cells displayed a lower ECAR and a higher OCR compared with SF188s cells (after adjusting for the number of cells being measured) (Figure 6B). This suggested that the fast growing SF188f glioblastoma cells adopted a more oxidative metabolism (perhaps reprogramming their metabolic network via certain epigenetic events), shifting away from glycolytic metabolism.


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

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

High proliferation rates of SF188f cells and associated shift in their basal OCR and ECAR compared with SF188s cells.A. Number of SF188s and SF188f cells at 24, 48 and 72 hours in their respective culture medium. B. Basal OCR and ECAR of SF188s and SF188f cells. 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. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization; Assay medium was the substrate-free base medium supplemented with 25 mM glucose, 6 mM Glutamine and 1 mM pyruvate. A representative experiment out of three is shown here. The OCR and ECAR values were normalized to pmoles/min/104 cells or mpH/min/104cells. Each data point represents mean ± SD, n = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109916-g006: High proliferation rates of SF188f cells and associated shift in their basal OCR and ECAR compared with SF188s cells.A. Number of SF188s and SF188f cells at 24, 48 and 72 hours in their respective culture medium. B. Basal OCR and ECAR of SF188s and SF188f cells. 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. Upon completion of an assay, cells were treated with trypsin and counted for the purpose of normalization; Assay medium was the substrate-free base medium supplemented with 25 mM glucose, 6 mM Glutamine and 1 mM pyruvate. A representative experiment out of three is shown here. The OCR and ECAR values were normalized to pmoles/min/104 cells or mpH/min/104cells. Each data point represents mean ± SD, n = 3.
Mentions: Having established the methods to analyze glycolysis and oxidation of exogenous substrates, we performed proof-of-principle experiments in SF188s and SF188f cells (see Material and Methods). These two cell lines were derived from the same parental cell line SF188 (which harbor c-MYC amplification) [35], but proliferated at very different rates, with the former much slower than the latter (Figure 6A). The fast-growing behavior of SF188 cells occurred only after, and not within, the initial four weeks' culture in DMEM containing 25 mM glucose and 6 mM glutamine, whereas SF188 cells cultured in DMEM containing 5.5 mM glucose and 2 mM glutamine maintained the same growth rate as the parental cells, suggesting an intrinsic change in SF188f cells' growth program as they acquired the fast growing behavior. We were curious as to whether there were any metabolic alterations associated with SF188f cells' fast growing behavior. Upon examining their basal OCR and ECAR in assay medium containing 25 mM glucose, 6 mM glutamine and 1 mM pyruvate, we found that SF188f cells displayed a lower ECAR and a higher OCR compared with SF188s cells (after adjusting for the number of cells being measured) (Figure 6B). This suggested that the fast growing SF188f glioblastoma cells adopted a more oxidative metabolism (perhaps reprogramming their metabolic network via certain epigenetic events), shifting away from glycolytic metabolism.

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