Seahorse Metabolic Analyzers Enable Rapid, Quantitative Measurement of Cellular Bioenergetics

David Ferrick, PhD Chief Scientific Officer

Changing the Way Metabolism is Measured Mitochondria

Energy Map

Glycolysis

Fatty Acids

Glucose Glutamine

O 2 O 2

O 2

O 2

O 2

O 2

+ H + H

+ H

+ H + H H +

O 2 O 2

O 2

Seahorse Stressor Reagent (oligomycin & FCCP)

Let’s start with the most rapid, user friendly measure of bioenergetic behavior: The XF Cell Energy Phenotype Test

Sensor Cartridge and miniPlate with your cells

Your XFp

Measure any metabolic change, in any cell, for any cause, with the XF Energy Map

Glycolysis

Mito

chon

dria

l Res

pira

tion

Aerobic

Glycolytic

Energetic

Oxy

gen

Cons

umpt

ion

Rate

(OCR

)

Extracellular Acidification Rate (ECAR)

XF Energy Map

Quiescent

Define metabolic phenotypes

Glycolysis

Mito

chon

dria

l Res

pira

tion

Aerobic

Glycolytic

Energetic

Oxy

gen

Cons

umpt

ion

Rate

(OCR

)

Extracellular Acidification Rate (ECAR)

XF Energy Map

Quiescent

Neurons

Activated T cells

Memory T Cells Tumor Cells

Measure metabolic potential

Glycolysis

Mito

chon

dria

l Res

pira

tion

Aerobic

Glycolytic

Energetic

Oxy

gen

Cons

umpt

ion

Rate

(OCR

)

Extracellular Acidification Rate (ECAR)

XF Energy Map

Neurons

Stressed Neurons

Quiescent

Stressed

Impaired Neurons

Stressed

At risk Neurons

Watch cells switch metabolic pathways

Glycolysis

Mito

chon

dria

l Res

pira

tion

Aerobic

Glycolytic

Energetic

Oxy

gen

Cons

umpt

ion

Rate

(OCR

)

Extracellular Acidification Rate (ECAR)

XF Energy Map

Tumor Cells

Quiescent

Stressed

p53 Arrested tumor Cell

Stressed

Energy map reveals three metabolic phenotypes regulating Mycobacterium tuberculosis drug susceptibility

Drug Sensitive

Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium tuberculosis. Saini et al., 2016, Cell Reports 14, 1–14, January 26, 2016

Metabolic reprogramming regulates stem cell differentiation

Reprogramming of Stem Cell Epigenetics. Ryall JG, Cliff T, Dalton S, Sartorelli V Cell Stem Cell. 2015 Dec;3;17(6):651-62.

Changes in metabolism regulate many of the important cell fate decisions made by stem cells

Seahorse standardized assays measure the key metabolic pathways

XF Mito Stress Test XF Glycolysis Stress Test

XF Cell Energy Phenotype Test

Metabolic Engines: XF Stress Test Kits

Measure the key parameters of glycolytic function:

• Glycolysis • Glycolytic Capacity • Glycolytic Reserve • Non-glycolytic Acidification

Extr

acel

lula

r Aci

dific

atio

n

EC

AR

(mpH

/min

)

Non-glycolytic Acidification

Glycolytic Capacity

Glycolysis

Glycolytic Reserve Capacity

Measure the key parameters of mitochondrial function:

• Basal respiration • ATP coupled respiration • Maximal Respiration • Spare Respiratory Capacity • Non-mitochondrial Oxygen Consumption

XF Mito Stress Test XF Glycolysis Stress Test A B C A B C

Gold Standard XF Assays

Mitochondrial Function Across Multiple Therapeutic Areas

Cell. 2014 Sep 11;158(6):1254-69. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Takashima Y, Guo G, Loos R, Nichols J, Ficz G, Krueger F, Oxley D, Santos F, Clarke J, Mansfield W, Reik W, Bertone P, Smith A.

Cancer Cell. 2014 Dec 8;26(6):840-50 ABL1-mediated oxidative stress adaptation in fumarate hydratase-deficient cancer Sourbier C, Ricketts CJ, Matsumoto S, Crooks DR, Liao PJ, Mannes PZ, Yang Y, Wei MH, Srivastava G, Ghosh S, Chen V, Vocke CD, Merino M, Srinivasan R, Krishna MC, Mitchell JB, Pendergast AM, Rouault TA, Neckers L, Linehan WM

Cancer Cancer Cell, 2014

Immunology Science Translational Medicine, 2015

Sci Transl Med. 2015 Feb 11;7(274):274ra18 Normalization of CD4+ T cell metabolism reverses lupus. Yin Y, Choi SC, Xu Z, Perry DJ, Seay H, Croker BP, Sobel ES, Brusko TM, Morel L.

Obesity/Diabetes Nature Medicine, 2014

Cardiovascular Disease Nature Medicine, 2014

Nat Med. 2014 Jun;20(6):616-23 Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies Wang G, McCain ML, Yang L, He A, Pasqualini FS, Agarwal A, Yuan H, et al.

Stem Cell Biology Cell, 2014

Nat Neurosci. 2014 Dec;17(12):1710-9. An α2-Na/K ATPase/α-adducin complex in astrocytes triggers non-cell autonomous neurodegeneration. Gallardo G, Barowski J, Ravits J, Siddique T, Lingrel JB, Robertson J, Steen H, Bonni A.

Neurodegeneration Nature Neuroscience, 2014

2014 Dec;20(12):1427-35. Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity Arruda AP, Pers BM, Parlakgül G, Güney E, Inouye K, Hotamisligil GS

.

Glycolytic Function Across Multiple Therapeutic Areas

Immunology Journal of Immunology, 2014

Autoimmunity Nature Medicine, 2015

Nat Med. 2015 Jan;21(1):55-61. Notch controls the survival of memory CD4+ T cells by regulating glucose uptake. Maekawa Y, Ishifune C, Tsukumo S, Hozumi K, Yagita H, Yasutomo K.

Toxicology Journal of Applied Toxicology, 2015

Cell Physiology Mitochondrion, 2015

Mitochondrion. 2015 Mar;21:19-26. Polymerase gamma mutator mice rely on increased glycolytic flux for energy production. Saleem A, Safdar A, Kitaoka Y, Ma X, Marquez OS, Akhtar M, Nazli A, Suri R, Turnbull J, Tarnopolsky MA

Stem Cell Biology Cell Reports 2015

Cell Rep. 2015 Jun 16;11(10):1614-24. mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling. Hämäläinen RH, Ahlqvist KJ, Ellonen P, Lepistö M, Logan A, Otonkoski T, Murphy MP, Suomalainen A.

Cancer Nature, 2014

J Appl Toxicol. 2015 Aug;35(8):945-51. Culture conditions profoundly impact phenotype in BEAS-2B, a human pulmonary epithelial model. Zhao F, Klimecki WT.

.

Control Activated

Cancer Cell. 2014 Dec 8;26(6):840-50 ABL1-mediated oxidative stress adaptation in fumarate hydratase-deficient cancer Sourbier C, Ricketts CJ, Matsumoto S, Crooks DR, Liao PJ, Mannes PZ, Yang Y, Wei MH, Srivastava G, Ghosh S, Chen V, Vocke CD, Merino M, Srinivasan R, Krishna MC, Mitchell JB, Pendergast AM, Rouault TA, Neckers L, Linehan WM

Nature 514, no. 7524 (2014): 628-32 Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function Viale A, Pettazzoni P, Lyssiotis CA, Ying H, Sánchez N, Marchesini M, Carugo A, Green T, Seth S, Giuliani V, Kost-Alimova M, Muller F, Colla S, Nezi L, Genovese G, Deem AK, Kapoor A, Yao W, Brunetto E, Kang Y, Yuan M, Asara JM, Wang YA, Heffernan TP, Kimmelman AC, et al

Seahorse mito stress test reveals requirement of fatty acid oxidation to develop immune memory

Key discovery in immune metabolism may improve immunotherapy

+ FA

Immunity 2011 December 28 Washington University School of Medicine, St. Louis

Durability of engineered patient T cells may depend on triggering the appropriate metabolic pathways

“The implications of Prof. Pearce’s findings may help explain the dramatic clinical successes of cancer

immunotherapies”, Dr. Carl June

- FA

Memory T Cell

Memory failure

Naive T cell Activate T cell

Effector T cell

Memory T cell

Metabolic programs control immune cell differentiation and function

Aerobic Energetic

Glycolytic Quiescent

Aerobic Energetic

Glycolytic Quiescent

Aerobic Energetic

Glycolytic Quiescent

GSEA (Gene set enrichment analysis) identifies metabolic program for Dendritic Cell maturation

Direct type I IFN but not MDA5/TLR3 activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly IC stimulation. Pantel A et al. PLoS Biol. 2014 Jan;12(1):e1001759.

Monocyte / resident macrophage M1 macrophage M2 macrophage

Metabolic programs control immune cell differentiation and function

Aerobic Energetic

Glycolytic Quiescent

Aerobic Energetic

Glycolytic Quiescent

Aerobic Energetic

Glycolytic Quiescent

Seahorse standardized assays measure the key metabolic pathways

XF Mito Stress Test XF Glycolysis Stress Test

XF Cell Energy Phenotype Test

XF Mito Fuel Flex Test

Metabolic Fuels: XF Mito Fuel Flex Test

Measure the key parameters of mitochondrial fuel use:

• Dependency

• Flexibility

• Capacity

Glucose Pathway

Flexibility

Dependency

Capacity

100

80

60

40

20

0 Glutamine Pathway

80

60

40

20

0

Flexibility

Dependency

Capacity

100

Fatty Acid Pathway

Flexibility

80

60

40

20

0

Dependency

Capacity

100

- The Newest Standard for Live Cell Metabolism

This unique substrate assay is highly complimentary to other substrate utilization assays

2-NBDG

+ Live cell rate + Real-time + Fuel Dependency + Fuel Flexibility + Fuel Capacity

= XF Mito Fuel Flex Test

13C Glucose Flux Analysis 2-NBDG Glucose uptake

[14C] 2-DG uptake Glucose consumption

XF Mito Fuel Flex Test

The Agilent-Seahorse Advantage

Metabolic signatures uncover distinct targets in molecular subsets of diffuse large B cell lymphoma Caro P, et al, Cancer Cell; 2012 Oct 16. 22 (4):547-560.

- Seahorse Assays inform 13C isotopomer analyses

How do cells survive without a functional TCA cycle in HLRCC (Hereditary leiomyomatosis and renal-cell cancer)

Aerobic Glycolysis

Lactate

Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Frezza C et al. Nature. 2011 Aug 17;477(7363):225-8.

Computational model predicts novel pathway that is synthetic lethal for Fh1-/-

fumarate

succinate

Haem

18/24 reactions predicted to be synthetic lethal belonged to haem metabolism

Confirmed by metabolomics

Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Frezza C et al. Nature. 2011 Aug 17;477(7363):225-8.

Potential new approach for treating HLRCC patients

Synthetic lethal (in silico) Upregulated in Fh-/-

Substrates induced in Fh-/-

bilirubin

bilirubin-UDP

fumarate

succinate

glutamine

Lactate

Haem

Bilirubin-UDP

bilirubin

Haem oxygenase is synthetically lethal with the tumour suppressor fumarate hydratase. Frezza C et al. Nature. 2011 Aug 17;477(7363):225-8.

Seahorse provides metabolic information on key pathways in living cells

Mitochondrial function

Glycolytic function

Metabolic phenotype

Glucose

Glutamine

Fatty acids

ENGINES

FUELS

Next Generation Metabolism

Questions?

THANK YOU!