Cyto gi bioke4

Analysis of Cellular Signaling using Activation-State Specific Antibodies

Greg Innocenti

Cell Signaling Technology

December 19, 2006

Outline

What are activation state-specific antibodies?

Imaging Cytometry

• immunofluorescence & antibody validation

• antibody conjugation

• high content analysis (HCA)

Flow Cytometry

• protocols

• clinical assays

Total (bind regardless of activation state)

Phospho-specific (eg. phospho-EGFR)

Cleavage-specific (eg. cleaved Caspase-3)

Acetylation-specific (eg. acetyl-Stat3)

Methylation-specific (eg. methyl-Histone H3)

Substrate-specific (eg. phospho-Akt Substrate)

Motif-specific (eg. phospho-Tyrosine)

Activation State-Specific Antibodies

+ - + -

p38

phos

pho-

p38

Wnt 5’

β-Catenin (C-term)

Insulin

Akt (pan) (11E7)

Total Antibodies

Untreated

LY294002

C2

C1

2 C

ells

He

La

Ce

lls

CD4S

ide

Sca

tter

CD13

CD

5

Whole Blood

Untreated EGF-Treated

Phospho-specific Antibodies

Green = Phospho-EGF Receptor (Tyr1068)Blue = DRAQ5

EGF Receptor (FITC)

Pho

spho

-EG

FR

(P

E)

Untreated Staurosporine

Caspase-3 Cleavage in Apoptosis

Green = Cleaved Caspase-3 (Asp175) (5A1) RmAbRed = F-Actin (Phalloidin)Blue = DRAQ5

Cleaved-Caspase 3

TU

NE

L

Antibody Validation & Applications

CST’s activation-specific antibodies are purified to distinguish between only one or two posttranslationally modified amino acid residues

Most antibodies raised against such modified sites have a lower affinity when compared with antibodies recognizing whole proteins

Thus, all product development follows rigorous in-house testing on a wide range of assay applications by our clinical applications specialists

Stringent validation requirements Protocol optimization Cross platform functionality

Outline


Imaging Cytometry




Flow Cytometry

• protocols

• clinical assays

Antibody Validation for Immunofluorescence/HCA

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20000.00

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30000.00

35000.00

40000.00

45000.00

0 5 10 15 20 25 30 35 40 45

Antibody Dilution (ug/ml)

Mean

Cha

nnel

Fluo

resc

ence

0.00

1.00

2.00

3.00

4.00

5.00

6.00

Sign

al to

Noi

se R

atio 2966

Control

S/N Ratio

Titration Curve

Confocal Imaging on Chamber Slides

Part II - Specificity Testing• treat cells with specific

ligands, drugs, inhibitors, etc.

• test antibody on cells that do and do not express target

• verify expression or treatment efficacy with another antibody (same target, or different target in same pathway)

Part I - Titration to determine optimal dilution/concentration

Antibody Validation for Immunofluorescence/HCA

Ant

ibod

yIs

otyp

e

Antibody

Isotype

[Ab]

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2000.00

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0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

Antibody Dilution (ug/ml)

Mean

Cha

nnel

Fluo

resc

ence

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

Sign

al to

Noi

se R

atio 4694

Control

S/N Ratio

1:8

00

1:4

00

1:2

00

1:1

00

1:5

0

1:2

5

MEK1/2 (red)actin (green)DNA (blue)HeLa cells

EGF Receptor Inhibition

Untreated Iressa

H1975 cellsL858R + T790M

HCC827 cellsDel 746-750, amp.

EGFR Mutant NSCLC Cell Lines

phospho-Tyrosine (red, Alexa647 conjugate)phospho-S6 (green, Alexa488 conjugate)

GPCR Activation

C6 cells

merge

phospho-Erk

phospho-Akt

DNA

MEK PI3K inhibitor inhibitor

LPA treatment 0 min 2 min 5 min 15 min 15 min 15 min

Neuroscience - Development

Postnatal Day 1 Postnatal Day 14

P4 P14P1

Postnatal Rat Brain

p-Histone H3Red=GFAPBlue=Doublecortin

G1 Phase S Phase

Cell Cycle / Checkpoint

Kip1

CDK2

CyclinE

E2F1H3

Aurora

Rad17

p53

H2Acdc2

#2558 p-Kip2 (T310)

Untreated Dexamethasone

CDK4/6

CyclinD

Cdc25A

DP1

AblHDAC

Rb

R

Rb

E2F1DP1OFF ON

Chk1/2

G2 Phase M PhaseCyclinB

cdc2

cdc25BATM

DNA Damage

X

P

P

P

P

P

P

PPP P

P

#9309 Rb (4H1) #9308 p-Rb (S807/811)

Normal Colon Colon CarcinomaUntreated UV UV+PPT

#3421 p-Rad17 (S645)#9289 p-p53 (Ser37)#9719 p-H2A.X (S139)

UV

#9309 Rb (4H1)#3068 p-Aurora A/B/C #4718 Aurora A/AIK

Metaphase Anaphase

CST Conjugated Antibodies

Conjugated antibodies helpful for multiplex analyses

CST conjugates are optimized for cytometric applications• high-quality pre-validated antibodies

• bright photostable Alexa dyes

• F/P trials to ensure bright signal

• antibody titration

• stability tested (accelerated and real-time)

• screened with flow cytometry, HCA, and IF

• lot-to-lot stability

Survivin (Alexa488-conjugate)

• inhibits apoptosis and regulates mitosis

• over-expressed in most human cancers

• expression correlates with both accelerated relapse and chemotherapy resistance

CST’s conjugated Survivin antibody is currently being used to screen patient samples

P1 Rat Brain

Caspase-3

Survivin

Smac/Diablo

Caspase-12

Caspase-9

Caspase-7

Cyto C

PARPLamin A DFFa-Fodrin

Caspase-6

[Ca++]

FADDCaspase-8/10

ER Stress Mitochondria

FAS, TNFa

The Bigger Picture

Single well immunofluorescence

+ ability to examine subcellular (co)localization in 4-dimension (XYZt)

- difficult to quantify without specialized software

- imaging is time consuming and data files become massive

High Content Analysis

+ some systems able to analyze localization

+ rapid scanning (comparatively), sensitive, and quantifiable

+ ability to multiplex and dissect various pathways in tandem

High Content Analysis

• automated plate-based image analysis

• quantifiable signal intensity and subcellular localization

• more predictive of drug activity in a cellular environment compared to ELISAs

• can be used to determine:

efficacy and therapeutic dose

cell-permeability of drugs

potential toxicity (DNA damage, apoptosis, micronuclei)

downstream effects of drug/target interaction

off-target effects

Untreated

PDGF

U0126+PDGF

LY/Wort+PDGF

Untreated

PDGF

U0126+PDGF

LY/Wort+PDGF

1:2

5

1:5

0

1:1

00

1:2

00

1:4

00

1:8

00

p-E

rkp-

Akt

Current platforms have increased colors and resolution in an attempt to quantify complex events

Example: nuclear translocation of Erk or NFkB

Requirements

• nuclear marker

• total antibody

• high resolution optics

• complex software

• lots of data storage

Using a phospho antibody will eliminate these costly requirements and speed up the screen (on/off as opposed to determination of localization)

KEY: reliable simple affordable assay with clear robust results

Value of CST Antibodies in HCS

Multiple, Parallel Analyses by Cellular Imaging (ICW)

Unt

reat

ed

PM

A

PD

GF

Gle

evec

Gle

evec

/PD

GF

LY29

4002

PD

GF

U01

26/P

DG

F

blank

p-PDGFR

p-Erk

p-Akt

RTK signaling analysis using LI-COR Odyssey

Untreated Anisomycin UV

p-p38

p-Jnk

p-ATF2

p-H2A.X

DNA damage profiling analysis using LI-COR Odyssey

Future Plans: Broad Signal Profiling by HCA

• large antibody panel for profiling screens

• can be used on any cytometric platform

• detection = fluorescent, chromogenic, chemiluminescence

• up to 96 antibodies per platetotal and phosphorylation-specific antibodies

MAPK, Akt, NFkB, Jak/Stat, etc.

receptor tyrosine kinases (EGFR, VEGFR, FGFR, IGF-IR, cKit)

adaptor proteins and downstream targets

transcription factors

motif antibodies (general serine or tyrosine phosphorylation)

• can be customized for analysis of different biological processestoxicity

cell cycle/arrest

apoptosis

cell adhesion

96 prediluted CST antibodies

cells treated with compound X

Starved

EGF

Anisomycin

UV

Antibody Development: FoxO1 Screening

CytoplasmicNuclear

IGF-1 + serum(AKT on)

PI3K inhibitor(Akt off)

nuclear trigger(half-width intensity)

How can we generate antibodies that work well for cytometric applications?

Screen using cytometric applications

HTS & HCS Platforms

Outline


Imaging Cytometry




Flow Cytometry

• protocols

• clinical assays

Optimization: Fixation/Permeabilization

CST 2-4% formaldehyde/90% methanol

Fix&Perm Kits 0.25-4% formaldehyde/detergent (triton or saponin)

Krutzik and Nolan (2004) Cytometry 55A:61-70.

2% Formaldehyde90% Methanol

4% Formaldehyde0.3% Triton X-100

Commercial Fix&Perm Kit(aldehyde/saponin)

p-Erk

p-p38

Fixation/Permeabilization

Surface and Signaling Markers

• Methanol diminishes or abolishes signal from some key surface markers

• Many signaling event are very transient so immediate fixation is critical, no time to prelabel with surface markers, lyse RBCs, or perform FiColl separations

• Staggered protocol: fix cells with aldehyde to stop all enzymes and preserve phosphoepitopes, label with surface markers, permeabilize with methanol, and then label with intracellular signaling antibodies (destroys some fluorochromes)

• Need a better solution…

New Fix & Perm Protocol

4% Formaldehyde + 0.1% Triton X-100 + 50% Methanol

Untreated

PMA

CD19 (FITC)

Protocol Comparison

CD19 (FITC)

3% PFA at 37ºC for 10m

90% ice cold MeOH at -20ºC for 10m

4%PFA at RT for 10m

0.1% Triton X-100 at RT for 30m

50% ice cold MeOH at 4ºC for 10m

Flow Cytometry: Clinical Applications

• Staining of intracellular signaling molecules can be easily incorporated with traditional cell surface marker labeling

4% Formaldehyde for 10m0.1 Trinton X-100 ft RT for 30m

50% ice cold MeOH at 4oC for 10m

• Important implications in the Clinic

• Flow Cytometry and Disease-specific Signaling• Chronic myelogenous leukemia (CML)• Chronic lymphocytic leukemia (CLL)• Acute myelogenous leukemia (AML)

Bcr/Abl Pathway Profiling by Flow Cytometry

0 hr 1 hr 24 hr 48 hr

phospho-Bcr

phospho-Stat5

cleaved-Casp3

CML cells (K562) + Gleevec

Large Scale Bcr/Abl Pathway Profiling

K562 cells +/- Gleevecbar height represents amount of Gleevec inhibition

Biomarkers for CLL

*

*

Crespo et al. (2003) N Engl J Med 348:1746-75

Staudt’s lab first identified Zap-70 as a potential biomarker of the aggressive form of CLL

CLL patients with Zap-70 (+) B-cells have poorer prognosis

CLL Assay using Zap-70 (136F12) RmAb

Difficulties persist in Zap-70 analysis - Zap-70 levels decline significantly after blood is drawn - Inconclusive results with weak expression - Additional biomarkers to make assay more reliable?

B cells (Ramos)T cells (Jurkat)

In House: In the Clinic:

Primary CLL Cells:

Zap-70 (low)(slow growing)

Zap-70 (high)(fast growing)

Source: Esoterix Center for Innovation

Alternative CLL Biomarkers

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6.00

p-PLCg(Y783)

p-FGFR(y653/654)

PAK Zap70 p-Stat1(S727)

p-Stat3(S727)

p-NFkB p65(S536)

p-IGF-IR(Y1131)

Me

an

Flu

ore

sce

nce

(n

orm

aliz

ed)

CLL23LB4 (Zap70-)

MO1043 (Zap70+)

Rosenwald et al. (2001) J Exp Med. 194(11):1639-1647.0.00

1.00

2.00

3.00

4.00

5.00

6.00

p-PLCg(Y783)

p-FGFR(y653/654)

PAK Zap70 p-Stat1(S727)

p-Stat3(S727)

p-NFkB p65(S536)

p-IGF-IR(Y1131)

Mea

n F

luor

esce

nce

(no

rmal

ized

)

CLL23LB4 (Zap70-)

MO1043 (Zap70+)

Scheijen and Griffin (2002) Oncogene 21:3314-33

Flt3

Flt3 Signaling

• Mutation or overexpression of Flt3 kinase is observed in AML, (~70%), B-ALL, T-ALL, and some blast phase CML

• Most common mutations are internal tandem duplications (ITD) and active loop mutations (AL), both of which result in constitutive activation

• Phospho-specific antibodies can be used to profile downstream signaling and to monitor the efficacy of specific Flt3 inhibitors

Dose Response (MOLM14)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.01 0.1 0.5 2

Concentration of Flt3 inhibitor (µM)

No

rmal

ized

mea

n f

luo

resc

en

ce

p-S6

p-Flt3

p-Stat5

p-Stat3

PY

p-AKT

Dose Response (SEM)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.01 0.1 0.5 2

Concentration of Flt3 inhibitor (µM)

No

rmal

ized

mea

n f

luo

resc

enc

e

p-S6

p-Flt3

p-Stat5

p-Stat3

p-Erk

PY

p-AKT

Phospho-Flt3Alexa488 Conjugate

(SEM cells)

uninhibited Flt3 inhibitor

Flt3 ITD(MOLM-14 Cells)

Overexpressed Flt3(SEM Cells)

Percentage of Cleaved Caspase-3 positive cells

0

10

20

30

40

50

60

70

80

0 2 3 5 22

Time (hours)

Pe

rce

nt

(%)

Flt3 inhibitor decreases downstream signaling, causes cell cycle arrest, and induces apoptotic cell death

Conclusion:Phospho-S6 is a key indicator of Flt3 inhibition, regardless of mutation

This antibody is a reliable robust biomarker for receptor tyrosine kinase (RTK) activity

Flt3 Inhibitor Pharmacodynamic Assay Design

Conclusion

• Activation state-specific antibodies are very useful in multiplex _phosphoproteomics analyses on various fluorescence based platforms

• stringent validation assures highest quality antibody and lot-to-lot reproducibility

• determine prognostic significance of signaling anomalies

• monitor patients for resistance or recurrence

• study effectiveness and optimal dosage of specific kinase inhibitors

• allow tumor “typing” to determine best treatment option

• improve clinical trial and basic research design (outcome and economics)

Gregory InnocentiClinical Applications/Image [email protected]

Thank You

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