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THE CANCER-METABOLISM LINK
MEASURING THE KEY PARAMETERS
OF CANCER METABOLISM
METABOLIC PHENOTYPES OF CANCER CELLS
Cancer cells exhibit a phenotype that reflects their metabolic needs. Researchers are using Seahorse XF technology
and XF stress tests to explore these metabolic changes, and the effect of metabolic therapies to increase their
understanding of cancer. The Seahorse XF Cell Mito Stress Test measures the key parameters of respiration:
basal respiration, proton leak, ATP-linked respiration, maximal respiration, and spare respiratory capacity.
The Seahorse XF Glycolysis Stress Test measures the key parameters of glycolytic function: glycolysis, glycolytic
capacity, and glycolytic reserve.
METABOLIC PROFILES
Cancer cells have a metabolic profile which reflects their altered bioenergetic requirements to support proliferation.
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Glucose
25 μM
2DG
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0
0
20
40
Time (min)
Control
Oligomycin
30
ECAR (mpH/min/mg protein)
OCR (pmol/min/ug/protein)
Oxygen Consumption Rate (OCR)
antimycin A
Extracellular Acidification Rate (ECAR)
2DG Oligomycin FCCP
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Time (min)
50 μM
75 μM
100 μM
Prostate tumor cells
Mouradian M., et al., (2014) Mol Carcinog.
Prostate epithelial cells
Ibrahim-Hashim A, et al., (2011) J Cancer Sci Ther.
Seahorse XF Cell Mito Stress Test reveals the dose-dependent
susceptibility of breast cancer cells to polyunsaturated fatty
acids as shown by a depression in all parameters of
mitochondrial respiration.
Seahorse XF Glycolysis Stress Test identifies prostate tumor
cell susceptibility to buffer therapy illustrated by an increased
glycolytic capacity over normal prostate epithelial cells.
METABOLIC SWITCHING
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- 2DG
e -sitiv - sen - - IL - - - T-R-A- - -
+ Glucose
<
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+ 2DG
0 40
<-- Glucose
- -TRA
- - IL - - res
- - ista
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--
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(mpH/min/106 cells)
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OCR (pmo/min/10K cells)
1000
Oxygen Consumption Rate (OCR)
BASAL OCR (pmo/min/106 cells)
Oxygen Consumption Rate (OCR)
Cancer cells are known to switch to a metabolic phenotype that drives proliferation, such as shifting towards glycolysis
(known as the Warburg effect), as illustrated by these Seahorse XF phenograms.
High OXPHOS
High Energetic
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OVCAR3
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ree
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eg
ed
OVCA420
SKOV3
v
asi
inv
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SKOV3 Ip
Low Energetic
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High Glycolysis
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Extracellular Acidification Rate (ECAR)
Extracellular Acidification Rate (ECAR)
Robinson GL., et al., (2012) Oncogene
Yang L., et al., (2014) Mol Syst Biol.
Seahorse XF Metabolic Switch Assay illustrates a Reverse Warburg
phenotype in mantle cell lymphomas sensitive to TRAIL induced
by 2DG inhibition, unlike the prototypic Warburg switch to aerobic
glycolysis in the presence of glucose (TRAIL-resistant).
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(mpH/10K cells)
Seahorse XF Metabolic Switch Assay identifies highly invasive
ovarian cancer cells which have decreased energetics.
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THE WORLD’S MOST ADVANCED
METABOLIC ANALYZERS
XF DATA IN PUBLICATIONS
There are over 2,000 references
utilizing Seahorse XF technology
published in leading journals such
as Nature and Cell. Scientists are
embracing Seahorse XF technology
to identify metabolic phenotypes and
reprogramming to target metabolic
pathways for therapeutic purposes.
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Glucose
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Pyruvate
Lactate
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Mitochondrial
Respiration
Glycolysis
ECAR (Extracellular
Acidification Rate)
0
OCR (Oxygen
Consumption Rate)
2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
PATHWAYS AND MECHANISM OF ACTION IN CANCER CELLS
Cancer therapies have exploited rapid proliferation as a treatment option. These treatment options can result in
unwanted and unacceptable side effects. Using Seahorse XF technology to focus on understanding cell
metabolism, more selective therapeutic agents can be studied and explored, not only for the effect on cancer
cells, but for their systemic effects as well.
Rotenone/
antimycin A
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Cell# (X106)
Oligomycin FCCP
Oxygen Consumption Rate (OCR) (%)
Permeabilized Cells
Extracelluar Acidification Rate (ECAR)
% of baseline
Glucose
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2.0
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1.0
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Time (hrs)
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1.0 mM
Colorectal cancer cells +Metformin
Time (min)
Wild type squamous carcinoma cell
p53 knockdown
Zhou G., et al., (2014) Mol Cell.
Seahorse XF assay reveals p53 pathway is critical for reversing
Warburg metabolism illustrated by reduced glycolytic activity in
p53 knockdown squamous carcinoma cells.
0mM Metformin
0.5mM Metformin
1mM Metformin
Wheaton W., et al., (2014) eLife
Seahorse XF assay reveals an unexpected dose-dependent
metformin inhibition of complex I correlating to proliferation
in colorectal cancer cells.
XF ASSAYS FOR THE
METABOLIC HALLMARKS OF CANCER
TUMOR MICROENVIRONMENT
To mimic a tumors’ in vivo environment, researchers employ methods such as culturing cells under
hypoxia or modeling tumors as multicellular spheroids. Seahorse XF technology is capable of adapting to
a variety of culturing conditions to provide precise, in vivo-like, physiologically relevant metabolic data.
HYPOXIA AND SPHEROIDS
Tumors are heterogeneous and exist in a complex, 3D environment defined by nutrient and chemical gradients
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Spare
Respiratory
Capacity
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Rotenone/
FCCP antimycin A
Oligomycin
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Oxygen Consumption Rate (OCR)
(μmo/e/h/μl cells)
Rotenone/
Oligomycin FCCP antimycin A
Oxygen Consunption Rate (OCR) (pmol/min)
Oxygen Consunption Rate (OCR) (pmol/min)
(O2, pH, etc.).
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Spare
Respiratory
Capacity
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Murine Breast Tumor Cells
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Time (min)
2D cultures
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Human Pancreatic Cancer Cells
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DMEM
Time (min)
3D cultures
No glucose
No glutamine
Fan J., et al., (2013) Mol Syst Biol.
Seahorse XF technology enables precise metabolic measurements in 3D cultures
as illustrated by an increase in spare respiratory capacity in 3D cultures of colorectal
cancer cells.
Seahorse XF technology reveals glutamine
oxidation requirement for hypoxia survival
in both murine breast cancer and human
pancreatic cells.
SUBSTRATE PREFERENCE
Cancer cells alter their substrate preference to maintain their rapid proliferation. Seahorse XF technology
provides the necessary tools that facilitate the exploration of substrate preferences,enabling a greater
understanding of cancer cell progression.
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Control
Palmitate
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Glucose
Glutamine
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CELLS GENE-ENRICHED
FOR OXPHOS
NON-ACTIVATED
B CELLS
Caro P., et al., (2012) Cancer Cell.
Seahorse XF assay identifies substrate preference of
lymphoma subsets.
Oxygen Consumption Rate (OCR) (%)
Oxygen Consumption Rate (OCR)
Basal OCR (pmoles O/min)
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CTL
FA-dependent
RAS
FA-independent
Quijano C, et al., (2012) Cell Cycle
Seahorse XF assay reveals the critical role of fatty acid
oxidation in Ras-mediated senescence of fibroblasts.
THE CANCER-METABOLISM LINK
XF assays measure the hallmarks of cancer: oncogene reprogramming of metabolism, substrate preference
of tumor cells, and metabolic phenotypes.
Proliferation, associated with carcinogenesis, involves oncogenes, proto-oncogenes, and mutated
tumor-suppressor genes. Rapid proliferation correlates to the cells’ metabolic phenotype. To maintain
rapid growth cancer cells will reprogram their metabolic phenotype,switching between glycolytic and
aerobic phenotypes.
PHENOTYPES & METABOLISM LINK
GLUCOSE
LACTATE
GLYCOLYSIS
GLUCOSE
GLYCOLYSIS
Cancer cells change their substrate preference
as they alter their metabolic phenotypes. For
example, cancer cells may increase glutamine
metabolism, alter lipid metabolism, or shift the
balance between anabolic and catabolic
processes. There is increasing evidence of the
interactions amongst genes, substrates, and
phenotypes. Seahorse XF technology and assays
are bringing unique value to investigate the
mechanisms behind the hallmarks of cancer
and altered cell metabolism.
RIBOSE
LACTATE
RIBOSE
GLUTAMINE
GLUTAMINE
TCA CYCLE
O2
TCA CYCLE
ATP
OXPHOS
O2
ATP
OXPHOS
OXPHOS
GENES & METABOLISM LINK
Glycolysis
SUBSTRATES & METABOLISM LINK
GLUCOSE
GLUCOSE
p53
PI3/AKT
FATTY ACID
GLYCOLYSIS
MYC
HIF1
FATTY ACID
LACTATE
HIF1
MYC
CITRATE
PYRUVATE
Acetyl-CoA
A
HIF1
HIF
1
PYRUVATE
LIPID SYNTHESIS
NADH
P /AKT
PI3
GLUTAMATE
TCA
ETC
MYC
YC
GLU
LUTAMINOLYSIS
GLU
UTAM
TAMINE
IN
XF METABOLIC ASSAYS
MEASURING THE KEY PARAMETERS OF CELL METABOLISM
Seahorse XF Cell Mito Stress Test Profile
Seahorse XF Glycolysis Stress Test Profile
Mitochondrial Respiration
Glycolytic Function
Oligomycin
Rotenone &
antimycin A
FCCP
Glucose
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280
Spare
Capacity
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200
Maximal
Respiration
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120
ATP
Production
Basal
Respiration
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Proton Leak
Non-mitochondrial Oxygen Consumption
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0
Oligomycin
2-DG
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0
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Time (minutes)
Extracellular Acidification Rate (ECAR)
(mpH/min)
Oxygen Consumption Rate (OCR)
(pmol/min)
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40
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25
Glycolytic
Capacity
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15
Glycolysis
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5
0
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Glycolytic
Reserve
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Non-glycolytic Acidification
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Time (minutes)
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90 100
Seahorse XF Mito Fuel Flex Test Profile
Seahorse XF Cell Energy Phenotype Test
Metabolic Phenotype & Potential
Mitochondrial Function
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Stressed
Phenotype
Baseline
Phenotype
Quiescent
Glycolytic
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60
Flexibility
40
Flexibility
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Dependency
Glycolysis
Extracellular Acidification Rate (ECAR)
For Research Use Only. Not for use in diagnostic procedures.
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Glucose Pathway
Dependency
Glutamine Pathway
Dependency
Capacity
t
l
Capacity
b
Po
ia
Capacity
Mitochondrial Respiration
Oxygen Consumption Rate (OCR)
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a
et
ic
ol
t
en
% GLC+GLN+FA Fuel Oxidation
Energetic
Aerobic
Fatty Acid Pathway
This information is subject to change without notice.
© Agilent Technologies, Inc., 2016
Published in the USA, August 1, 2016
5991-7107EN