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THE CANCER-METABOLISM LINK
MEASURING THE KEY PARAMETERS OF CANCER METABOLISM
METABOLIC SWITCHINGCancer 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.
Seahorse XF Metabolic Switch Assay identifi es highly invasive ovarian cancer cells which have decreased energetics.
10100
150
200
250
300
350
400
450
(mpH/10K cells)
OCR
(pm
o/m
in/1
0K c
ells
)
20 30 40 50 60 70 80
High OXPHOS
Low Energetic
High Energetic
High Glycolysis
SKOV3
SKOV3 Ip
OVCA420
OVCAR3
invasive degree
Oxy
gen
Con
sum
ptio
n R
ate
(OC
R)
Extracellular Acidifi cation Rate (ECAR)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).
(mpH/min/106 cells)
BASA
L O
CR (p
mo/
min
/106 c
ells
)
00
200
400
600
800
1000
40 120 200 280
- 2DG
+ 2DG
00
200
400
600
800
1000
40 80 120 160
- Glucose
+ Glucose<- - - -
- - - - - - -
- - - -
TRAIL - sensitive
<- - - - - - - - - - - - - -
TRAIL - resistant
Oxy
gen
Con
sum
ptio
n R
ate
(OC
R)
Extracellular Acidifi cation Rate (ECAR)Robinson GL., et al., (2012) Oncogene
METABOLIC PROFILESCancer cells have a metabolic profi le which refl ects their altered bioenergetic requirements to support proliferation.
Seahorse XF Glycolysis Stress Test identifi es prostate tumor cell susceptibility to buffer therapy illustrated by an increased glycolytic capacity over normal prostate epithelial cells.
00
5
10
15
20
25
30
Time (min)
ECA
R (m
pH/m
in/m
g pr
otei
n)
20 40 60 80 100
Prostate tumor cells Prostate epithelial cells
Glucose Oligomycin 2DG
Ibrahim-Hashim A, et al., (2011) J Cancer Sci Ther.
Extr
acel
lula
r Aci
difi c
atio
n R
ate
(EC
AR
)
00
10
20
30
40
50
60
Time (min)
OCR
(pm
ol/m
in/u
g/pr
otei
n)
25 50 75 100 125 150 175 200
Control 25 μM 50 μM 75 μM 100 μM
2DG Oligomycin FCCP antimycin A
Mouradian M., et al., (2014) Mol Carcinog.
Oxy
gen
Con
sum
ptio
n R
ate
(OC
R)
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.
METABOLIC PHENOTYPES OF CANCER CELLSCancer cells exhibit a phenotype that refl ects 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, glycolyticcapacity, and glycolytic reserve.
PATHWAYS AND MECHANISM OF ACTION IN CANCER CELLSCancer 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.
THE WORLD’S MOST ADVANCED METABOLIC ANALYZERS
Seahorse XF assay reveals p53 pathway is critical for reversing Warburg metabolism illustrated by reduced glycolytic activity in p53 knockdown squamous carcinoma cells.
0-162
32
226421615809
100311971392
15861780
Time (min)
Extr
acel
luar
Aci
dific
atio
n Ra
te (E
CA
R)
% o
f bas
elin
e
3317 50 83 116 14966 100 133 166
Glucose Oligomycin FCCPRotenone/
antimycin A
Wild type squamous carcinoma cell p53 knockdown
Zhou G., et al., (2014) Mol Cell. 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.
0 .25 .50 .75 1.0 mM
25
50
0
75
100
125
Colorectal cancer cells +Metformin
Oxy
gen
Cons
umpt
ion
Rate
(OCR
) (%
)P
erm
eabi
lized
Cel
ls
Cell#
(X10
6 )
0 24 48 72
0.5
1.0
0.0
1.5
2.0
2.5
Time (hrs)
0mM Metformin 0.5mM Metformin 1mM Metformin
XF DATA IN PUBLICATIONSThere 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.
Lactate
Pyruvate
Glucose
Glycolysis
ECAR (Extracellular
Acidification Rate)
Mitochondrial
Respiration OCR (Oxygen
Consumption Rate) 20060
100
200
300
400
500
600
700
800
900
2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
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.
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.
Seahorse XF assay reveals the critical role of fatty acid oxidation in Ras-mediated senescence of fi broblasts.
Quijano C, et al., (2012) Cell Cycle
0
50
100
150
200
250
300
Oxy
gen
Cons
umpt
ion
Rate
(OCR
) (%
)
FA-dependent FA-independent
RASCTL0
100
200
300
400
500
600
700
800
900
1000
1100
Oxy
gen
Cons
umpt
ion
Rate
(OCR
)Ba
sal O
CR (p
mol
es O
/min
)
Control Palmitate
Glucose Glutamine
CELLS GENE-ENRICHED FOR OXPHOS
NON-ACTIVATEDB CELLS
Seahorse XF assay identifi es substrate preference of lymphoma subsets.
Caro P., et al., (2012) Cancer Cell.
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.
0.00
0.20
0.40
0.60
0.80
0.10
0.12
0.14
0.16
0.18
Oxy
gen
Cons
umpt
ion
Rate
(OCR
)(μ
mo/
e/h/
μl c
ells
)
Murine Breast Tumor Cells
DMEM
Human Pancreatic Cancer Cells
No glucose No glutamine
Fan J., et al., (2013) Mol Syst Biol. 2D cultures 3D cultures
0
10
20
30
40
50
60
70
Time (min)
Oxy
gen
Cons
unpt
ion
Rate
(OCR
) (pm
ol/m
in)
25 50 75 100 125 150 175 200
Oligomycin FCCPRotenone/
antimycin A
SpareRespiratoryCapacity
0
5
10
15
20
25
30
35
40
Time (min)
30 60 90 120 150 180 210
Oligomycin FCCPRotenone/
antimycin A
SpareRespiratoryCapacity
Oxy
gen
Cons
unpt
ion
Rate
(OCR
) (pm
ol/m
in)
HYPOXIA AND SPHEROIDSTumors are heterogeneous and exist in a complex, 3D environment defi ned by nutrient and chemical gradients (O2, pH, etc.).
XF ASSAYS FOR THE METABOLIC HALLMARKS OF CANCER
THE CANCER-METABOLISM LINKXF 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.
PI3/AKT
GLULUTAMINOLYSIS
PI3P /AKT
HIF1
HIFHIF11 LIPID SYNTHESIS
HIF1
p53
MYC
MYCYC
MYC
PYRUVATE
TCA
GLUCOSE
GLYCOLYSIS
GENES & METABOLISM LINK SUBSTRATES & METABOLISM LINKSUBSTRATES & METABOLISM LINK
GLUCOSE FATTY ACID
FATTY ACID
PYRUVATE
LACTATE CITRATE
Acetyl-CoA
NADH
GLUUTAMTAMINEIN
GLUTAMATE
A
ETC
GLUCOSE
RIBOSE
GLUTAMINE
LACTATE
TCA CYCLE
ATPOXPHOSO2
GLY
COLY
SIS
GLUCOSE
RIBOSE
GLUTAMINE
LACTATE
TCA CYCLE
ATPOXPHOSO2
GLY
COLY
SIS
OXPHOS Glycolysis
PHENOTYPES & METABOLISM LINKCancer 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.
XF METABOLIC ASSAYSMEASURING THE KEY PARAMETERS OF CELL METABOLISM
Seahorse XF Mito Fuel Flex Test Profi le
Mitochondrial Function
Seahorse XF Cell Energy Phenotype Test
Metabolic Phenotype & Potential
Oxy
gen
Con
sum
ptio
n R
ate
(OC
R)
Extracellular Acidifi cation Rate (ECAR)
Glycolysis
Mito
chon
dria
l Res
pira
tion
Aerobic
Quiescent
Energetic
Glycolytic
BaselinePhenotype
StressedPhenotype
Metabolic Potentia
l
Glucose Pathway
% G
LC+
GLN
+FA
Fue
l Oxi
datio
n
Glutamine Pathway Fatty Acid Pathway
100
80
60
40
20
0
Cap
acity Cap
acity
Cap
acity
Flexibility
DependencyDependency
Dependency
Flexibility
0
5
10
15
20
25
30
35
40
45
Extr
acel
lula
r A
cidi
fi cat
ion
Rat
e (E
CA
R)
(m
pH/m
in)
Glycolysis
Glucose Oligomycin 2-DG
GlycolyticCapacity
GlycolyticReserve
0 10 20 30 40 50 60 70 80 90 100
Non-glycolytic Acidifi cation
Time (minutes)
BasalRespiration
FCCPRotenone &antimycin A
00
40
80
120
160
200
240280
320360
10 20 30 40 50 60 70 80 90 100 110
Non-mitochondrial Oxygen Consumption
Time (minutes)
Oxy
gen
Con
sum
ptio
n R
ate
(OC
R)
(pm
ol/m
in)
SpareCapacity
ATPProduction
Oligomycin
MaximalRespiration
Proton Leak
Seahorse XF Glycolysis Stress Test Profi le
Glycolytic Function
Seahorse XF Cell Mito Stress Test Profi le
Mitochondrial Respiration
This information is subject to change without notice.
© Agilent Technologies, Inc., 2016Published in the USA, August 1, 20165991-7107EN
For Research Use Only. Not for use in diagnostic procedures.