Aerobic Energetic - agilent.com · Dr. Nika Danial. Department of Cell Biology Dana Farber Cancer...

For Research Use Only. Not for diagnostic purposes.

Daniel Disher

Manager, Seahorse XF Instrument Sales

Midwest and West Coast

Baseline

Stressed

Glycolytic

Aerobic Energetic

Quiescent

For Research Use Only. Not for diagnostic purposes.

Metabolic effects can be seen everywhere

July 11, 2017

Confidentiality Label

2

Seahorse is the most used, cited measure of functional metabolism

>1,300 instruments >10,000 users

>2,400 publications (Jan 2016)

232

409

600

783

1 7 16 41 103

240

472

881

1,481

2,264

0

500

1000

1500

2000

2500

Publications Cumulative

Energy metabolism is a leader not a follower: therefore reprograming metabolism may alter outcomes

Proliferation

Normal

Tumor cells

Novel cancer

therapies

Death

Healthy

neurons

Dying

neurons

Neurodegeneration

Activation

Naive

immune cells

Effector & memory

immune cells

Vaccines &

Immunotherapy

Differentiation

Stem Cells Differentiated

cardiomyocytes

Stem cell

therapies

Aerobic Glycolysis Required for Optimal IFN-γ Production

Cell 153, 1239–1251, June 6, 2013 ª2013

Elsevier Inc. Erika Pearce et. al

For Research Use Only. Not for diagnostic purposes

The synergy of using Mass Spec with XF technology

0

50

100

150

200

250

300

350

400

Cumulative XF + MS Publication Count thru End

2015 ~2400

Total number of publications citing

XF data ~ 400

publications citing XF data and

Mass Spec data

For Research Use Only. Not for diagnostic purposes

Seahorse Cellular Metabolism and Agilent Molecular Metabolism

Video of inputs

and outputs

No zoom

Snap shots,

Fully zoomed

10,000x

Cellular Metabolism

Live cell rates

Molecular Metabolism

Pathway roadmaps

For Research Use Only. Not for diagnostic purposes

‘Omics provide a parts list

For Research Use Only. Not for diagnostic purposes

Multiplex iTRAQ Proteome Analysis

Caro, P. et. Al. Cancer cell. 16 Oct. 2012. 22(4) 547-560.

Oxygen

Consu

mption R

ate

(O

CR

)

Basal O

CR

(pm

ole

s/m

in)

Extracellular Flux (XF) analysis

MS+XF = Better Together!

DLBCL subsets

Oxidative subsets in DLBCL

For Research Use Only. Not for diagnostic purposes

Seahorse closes the loop in validation proteomic and genomic data Peroccchi et al1 leveraged data from comparative physiology, including OXPHOS studies, evolutionary genomics, organelle proteomics, and RNA interference studies to identify a product of CBARA1 gene as the founding member of a set of proteins required for high-capacity mitochondrial calcium uptake. Named MICU1 by the investigators, this gene product is associated with the mitochondrial inner membrane, and has several characteristics required for Ca2+ transport, including the two canonical EF hands essential for activity.

The investigators concluded that these

data show that OXPHOS remains intact in

MICU1-silenced cells, and that

mitochondrial Ca2+ uptake phenotype

does not occur as a secondary

consequence of generalized mitochondrial

dysfunction.

Dr. Nika Danial. Department of

Cell Biology Dana Farber

Cancer Institute

XF assay narrows search

Norberg E & Caro P et al. Caner Cell.

2012 Oct 16;22(4):547-60.

Targeted 13C flux provides answer

U13C-Palmitate

For Research Use Only. Not for diagnostic purposes.

XF Analyzers Measure the Two Major Energy Pathways of the Cell

Mitochondrial Respiration

……………… Measured by the

Oxygen Consumption Rate

OCR

Glycolysis ………………

Measured by the Extracellular

Acidification Rate

ECAR

For Research Use Only. Not for diagnostic purposes

For Research Use Only. Not for diagnostic purposes.

The transient micro-chamber enables rapid measurement of mitochondrial respiration and glycolysis in minutes

Well 1 Well 2

The bio-cartridge is

raised, bringing the system back

to baseline

The sensor cartridge is

raised, bringing the system

back to baseline

A transient micro-chamber

is formed

Measuring Cellular Bioenergetic Processes in Real Time

For Research Use Only. Not for diagnostic purposes.

Drug injection ports – the experimental possibilities are inifinite!

•4 drug injection ports per well

•Add inhibitors, stimulants, substrates,

and compounds

• Injections defined by the user [ NOTE: Video Requires QuickTime ]

For Research Use Only. Not for diagnostic purposes.

XFe Analyzers, FluxPaks, Stress Test Kits & Reagents

XF Stress Test Kits, XF phenotype

kit and reagents (PMP, BSA-PAL) XFe96, XFe24 and XFp

FluxPaks

XFe96 Analyzer XFe24 Analyzer XFp Analyzer

Mitochondrial Respiration

Oxidation of:

• Glucose

• Fatty Acids

• Amino Acids

Which Engine

(Pathway)?

Which Fuel

Source?

What questions can I answer?

Glycolysis

Fermentation of Glucose

For Research Use Only. Not for diagnostic purposes

For Research Use Only. Not for diagnostic purposes.

What experiment should I try first?

Profiling Metabolic Engines: The XF Cell Energy Phenotype Test

Glycolysis

Mitochondria

l R

espiration

Aerobic

Glycolytic

Energetic

Oxyg

en

Co

nsu

mp

tio

n R

ate

(O

CR

)

Extracellular Acidification Rate (ECAR)

XF Energy Map

Memory T Cells

Activated T cells

Baseline

Neurons

Lymphoma Cells

Stressed

Neurons

Quiescent

Pairwise comparisons

Same? Different?

Comprehensive understanding of

your metabolic phenotype(s)

Baseline metabolisms

Stressed metabolisms

Metabolic Potential

For Research Use Only. Not for diagnostic purposes

XF Assays Measure Metabolic Switching

+Glc

-Glc

Substrate Induced Signaling Pathway Induced Gene Induced

0 0 40 60 20 80

100

300

200

400

OC

R (

pm

ol m

in-1

)

ECAR (mpH per min)

Fh1fl/fl

Fh1-/-CL1

Fh1-/-CL19

Hedgehog partial agonism drives Warburg-like

metabolism in muscle and brown fat.

Teperino R, et al., Cell. 2012 Oct 12;151(2):414-26.

Switching from aerobic glycolysis to oxidative

phosphorylation modulates the sensitivity of mantle cell

lymphoma cells to TRAIL.

Robinson et al. Oncogene. 2012 Nov 29;31(48):4996-5006.

Haem oxygenase is synthetically lethal with the tumour

suppressor fumarate hydratase.

Frezza et al. Nature. 2011 Aug 17;477(7363):225-8.

Adipocytes +/- Hedgehog ligand Mantle cell lymphoma cells +/- glucose Kidney epithelial cells +/- fumarate hydratase

Aerobic More

Metabolic

Less

Metabolic Glycolytic

For Research Use Only. Not for diagnostic purposes

Cell Energy Phenotype Test

Analyzing metabolic functionality – Broad to Specific

Aerobic? Glycolytic?

Mito Stress Test

FAO

Glycolysis Stress

Test

Substrates

Isolated Mitochondria

Substrates

For Research Use Only. Not for diagnostic purposes

For Research Use Only. Not for diagnostic purposes.

van der Windt, JW, … and Pearce, EL, Washington School of Medicine. Immunity 2011 December 28

Naïve T cells

Effector T cells

Memory T cells

Mouse Lymphocytes

BASELINE 1 2 3

Mouse CD-1 Synaptosomes

Why the Mitochondrial Stress Test?

.Flynn J M, Choi S W, Day N U, Gerencser A A, Hubbard A and Melov S., Free Radic Biol Med. 2011 Apr 1; 50(7):866-873

BASELINE 1 2 3

Mouse CD-1 Synaptosomes

For Research Use Only. Not for diagnostic purposes.

XF Cell Mito Stress Test Profile

Measure the key parameters

of mitochondrial function:

• Basal Respiration

• ATP Production

• Maximal Respiration

and Spare Capacity

• Proton Leak

Basal

Respiration

ATP

Production

Proton Leak

Spare

Capacity

Maximal

Respiration

Non-mitochondrial Respiration

• Non-mitochondrial Respiration

XF Cell Mito Stress Test

For Research Use Only. Not for diagnostic purposes.

Non-mitochondrial

Basal

Resp.

ATP

turnover

Maximal

Respiration

H+ Leak

Oligomycin FCCP Rotenone +

Antimycin A

C2C12

The Mitochondrial Stress Test

I II III IV

V

H+

H+ H+ H+

O2 H20

ADP ATP

X

X X

Assessing electron flow through the ETC

OC

R (

pM

ole

s/m

in)

FCCP

Pyruvate

Malate

BASELINE 1 2 3

Mouse CD-1 Synaptosomes

For Research Use Only. Not for diagnostic purposes

For Research Use Only. Not for diagnostic purposes.

XF Glycolysis Stress Test Profile

Glycolytic

Capacity

Non-glycolytic Acidification

Glycolytic

Reserve

Capacity

Glycolysis Glycolysis

EC

AR

(m

pH

/min

)

Extr

acell

ula

r A

cid

ific

ati

on

E

CA

R (

mpH

/min

)

10mM

XF Glycolysis Stress Test

For Research Use Only. Not for diagnostic purposes.

Free Base Lysine Increases Survival and Reduces Metastasis in Prostate Cancer Model

Ibrahim-Hashim, et al., J Cancer Sci Ther 2011, S1

PC-3M

PCS

Prostate Tumor Cells

Primary Prostate Epithelial Cells

Why the Glycolysis Stress Test?

XF Mito Fuel Flex Test

Glucose Glutamine Fatty Acids

nucleus

mitochondria

O2

Physiological

For Research Use Only. Not for diagnostic purposes

For Research Use Only. Not for diagnostic purposes

First demonstration of Seahorse to detect T cell activation opens up early activation window

July 11, 2017

Real time analysis of T cell activation

30

Gubser 2013 Nat Immunol

For Research Use Only. Not for diagnostic purposes

You don’t just get a more sensitive and faster assay, there’s more!

July 11, 2017

Real time analysis of T cell activation

31

Metabolic Requirements

Exogenous glucose

Pathway Requirements

mTOR

Signaling Requirements

CD28

Gubser, PM et al. 2013. Rapid effector function of memory CD8 T cells

requires an immediate-early glycolytic switch. Nature 14(10):1064-72.

For Research Use Only. Not for diagnostic purposes.

Glycolytic PER = Total PPR – CO2-dependent PPR

ECAR (mpH/min) * BF (mmol/L) * Vol ch (µL)

mit OCR (pmol/min)* CF

(pmol/min)

* Kvol

Improving proton flux as a measure of glycolysis

Functional metabolic data compliments your current data set

Mass Spectometry

End-point Assays

Flow Cytometry

qPCR

Only one company can deliver on both cellular and molecular metabolism – The unique differentiator

Cellular Metabolism

Molecular Metabolism

For Research Use Only. Not for diagnostic purposes

Questions?

dan.disher@agilent.com

Manager Seahorse XF Instruments

Midwest & West Coast

312.519.4352

For Research Use Only. Not for diagnostic purposes