Analysis of Cellular Signaling using Activation-State Specific Antibodies

Greg Innocenti

Cell Signaling Technology

December 19, 2006

Wnt 5’

β-Catenin (C-term)

Insulin

Akt (pan) (11E7)

Total Antibodies

Untreated

LY294002

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Phospho-specific Antibodies

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

EGF Receptor (FITC)

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Untreated Staurosporine

Caspase-3 Cleavage in Apoptosis

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

Cleaved-Caspase 3

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Antibody Validation for Immunofluorescence/HCA

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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

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

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

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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)

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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

What are activation state-specific antibodies?

Imaging Cytometry

• immunofluorescence & antibody validation

• antibody conjugation

• high content analysis (HCA)

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