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.