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Cytometry of Cell Signaling: Simultaneous Analysis of Multiple Signaling Pathways in AML. T. Vincent Shankey, Ph.D. Systems Research/Life Sciences Division Beckman Coulter, Inc Miami, FL [email protected] Control. Control. FA/Triton X-100. FA/TX/MeOH. 40 uM PMA. 40 uM PMA.

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Cytometry of Cell Signaling: Simultaneous Analysis of Multiple Signaling Pathways in AML

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Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Cytometry of Cell Signaling:Simultaneous Analysis of Multiple Signaling Pathways in AML

T. Vincent Shankey, Ph.D.

Systems Research/Life Sciences Division

Beckman Coulter, Inc

Miami, FL

[email protected]


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Control

Control

FA/Triton X-100

FA/TX/MeOH

40 uM PMA

40 uM PMA

P-ERK-Alexa 488

P-ERK-Alexa 488


Advantages of whole blood sampling for signal transduction pathway analysis

Advantages of Whole Blood Sampling for Signal Transduction Pathway Analysis

  • Sample Processing Speed

    • No cell separation step(s)

    • Rapid fixation minimizes potential for spontaneous de-phosphorylation of target epitopes (cytoplasmic phosphatases)

    • Ideal for use in clinical setting

  • Minimal Cell Loss

    • Cell separation techniques can deplete specific cell types

  • Keeps Target Cell Populations in Contact with Pathway Inhibitors (Targeted Therapeutics)

    • Rapid loss/reversal of in vivo pathway inhibition after removal of cells from serum


Measurement of cell signaling bone marrow

Measurement of Cell SignalingBone Marrow


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Acute Myeloid Leukemia (AML)(used with CD45, CD34, CD13/33, CD117)

(GDC-0941)

David Hedley, Princess Margaret Hospital


Hematopoietic differentiation

Hematopoietic Differentiation

CD34+

CD117+/-

Peripheral Circulation


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Gating/Analysis Protocol for Bone Marrow Signaling Analysis

+SCF

+GM-SCF


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Rapid Activation/Inactivation of P-ERK in Normal Bone Marrow CD34+/CD117+ Cells

James Jacobberger, Case Western Reserve University


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Signaling Responses in Normal CD34+/CD117+ cells

James Jacobberger, Case Western Reserve University


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Signaling Responses in Normal CD34+/CD117+ cells


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Signaling Response in

AML Bone Marrows


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

8

7

6

5

4

Interpolated

3

2

1

0

2.0

0

1.5

1.0

20

0.5

40

0.0

60

AML1 = M4

AML2 = M2

AML3 = M4eo

AML4 = M5b

AML5 = M1

SCF stimulated P-S6 and P-Erk

Flt3L-stimulated P-S6 and P-Erk

AML1

AML3

AML5

NBM

pS6 (MFI)

pERK (MFI)

Time (min)

James Jacobberger, Case Western Reserve University


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

C

B

A

CD34+CD117+

Non Lymphs

Lymphs

Fig 1

CD34 ECD

CD45 APC Alexa 750

CD64 APC

CD34-CD117+

Stem Enrich

Blast

Side Scatter

CD117 PeCy5.5

Side Scatter

D

E

Monocytes

Mature Myeloid

CD13 PeCy7

CD64 APC

Intermediate Myeloid

Myeloid Enrich

Immature Myeloid

CD16 Alexa 700

Side Scatter

Gating/Analysis Protocol for Bone Marrow Signaling Analysis

Chuck Goolsby, Northwestern University


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Growth Factor Receptor Expression Profiles for the Six

Non-Lymphoid Cell Populations from Normal Bone Marrow

Chuck Goolsby, Northwestern University


Normal bone marrow p erk s n

Normal Bone Marrow: P-ERK (S/N)

Chuck Goolsby, Northwestern Univ


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Normal Bone Marrow: P-STAT5 (S/N)

Chuck Goolsby, Northwestern Univ


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Normal Bone Marrow: P-STAT3 (S/N)

Chuck Goolsby, Northwestern Univ


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Normal bone marrow cells show highly reproducible signaling pathways that correlate with the differentiation state and the presence of specific cell surface cytokine receptors


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Constitutive Activation

P-STAT5

AML – Categories of Abnormal Bone Marrow Signaling

Receptor Dysregulation

GM-CSF

P-Akt

Abnormal Kinetics

SCF

P-Akt

Chuck Goolsby, Northwestern University


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Aberrant Signaling Patterns in AML Bone Marrow Samples


Measurement of cell signaling whole blood

Measurement of Cell SignalingWhole Blood


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Acute Myeloid Leukemia (AML)(used with CD45, CD34, CD13/33, CD117)

(GDC-0941)

David Hedley, Princess Margaret Hospital


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

David Hedley, Princess Margaret Hospital


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

David Hedley, Princess Margaret Hospital


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

David Hedley, Princess Margaret Hospital


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

David Hedley, Princess Margaret Hospital


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

David Hedley, Princess Margaret Hospital


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

David Hedley, Princess Margaret Hospital


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Pre-dose

Day 8

Day 29

Day 211

15%

0.17%

0.03%

0.8%

CD117+

Blasts

CD117

Control

ENMD2076

1.6uM

P-STAT5

Patient #106 FLT3/ITD

Daily Oral Dose 225mg

David Hedley, Princess Margaret Hospital

pSTAT5


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Signaling Classification of AML(Work in Progress)Real-time Monitoring of Molecular Targeted Therapeutics


Monitoring bcr abl kinase inhibitor imatinib in cml patients

+

+

CD34

CD34

cells

cells

Pre

Pre

-

-

therapy

therapy

Count

Count

Count

p

p

-

-

Stat5

Stat5

Three weeks

Three weeks

Post

Post

-

-

therapy

therapy

Count

Count

Count

p

p

p

-

-

-

Stat5

Stat5

Stat5

Three weeks

Three weeks

Post

Post

-

-

therapy

therapy

Count

Count

Count

In vitro

In vitro

imatinib

imatinib

treated

treated

p

p

p

-

-

-

Stat5

Stat5

Stat5

Monitoring Bcr/Abl kinase inhibitor Imatinib in CML patients

Sequential flow data shows target inhibition in this patient, but incomplete as additional treatment with Imatinib ex vivo causes further decrease in p-STAT5.

Implication is that if we had this information, we would adjust the drug dose

D.W.Hedley, C. Goolsby, and T.V. Shankey. Tox Pathol 36;133-139, 2008


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

AML Blast Response to Gleevec

Patient #2 –SCF activation

David Hedley, Princess Margaret Hospital


Summary aml blast response to in vivo gleevec treatment

Summary - AML Blast Response to in vivo Gleevec Treatment

P-Akt levels at D4/t2 predicts clinical response to subsequent Chemotherapy

(p=0.008)


Aml conclusions

AML - Conclusions

  • Normal bone marrow stem cells, monocytic and myeloid cells have distinct and restricted signaling “fingerprints”

    • AML blasts (bone marrow or peripheral blood) have signaling patterns distinct from normal

  • Signaling characteristics of peripheral blood and bone marrow stem-like cells appear similar (needs validation)

  • Real-time monitoring of signaling pathways is useful in following response to therapies


Need for automation

Need for Automation!


Manual assay kinetics

Manual Assay Kinetics


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Cell Signaling Sample Preparation

  • Throughput requirements:

    • No info.

Blood sample in vacutainer

Aliquot up to100 uL per tube (up to 32 tubes/patient)

Add 5 uL of activator (LPS @ 37C or RT) and/or inhibitor to activation tubes or 5 uL of PBS to the control tube

Incubate at 37 C for 10-60 min

Add 65uL of 10% formaldehyde at RT

Vortex

Incubate for 10 min (exact) at RT

Add 1 mL of 0.1165% Triton X-100 in PBS at RT

Pippet up and down

Incubate for 15 min at 37C

Add 2 mL of cold (4 C, possibly RT) PBS+4%FCS

Spin at 1000xg, 3 min

Up to 2 washes

Remove supernatant/resuspendpellet with residual buffer

Add 1 mL of “RT” 50-80% MeOH in PBS

Pipette up and down right after addition of MeOH, incubate at ? C for ? min

Spin at 1000xg, 3 min

Remove supernatant

Add 2 mL of PBS+4%FCS (cold)

Spin at 1000xg, 3 min

Remove supernatant as much as possible

Add Abs and cold (4C) PBS+4%FCS to a final volume of 100 uL

Incubate at RT for 30 min in dark

Add 2 mL of cold (4 C) wash buffer

Spin at 1000xg, 3 min

Remove supernatant

Resuspend cells in 1 mL wash buffer

Place the tubes (barcoded) on a 32 tube carousel

Analyze on a FC500 or CRS

June 30, 2009


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Assay Automation Tools

Gallios

Biomek NXp

Centrifuge

Accessories

Deck Layout


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Shaking/Temperature Cycling Peltier

48 deep-well

plate

Adapter

Peltier


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Temperature Cycling in Wells

15 min

10 min


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Temperature Cycling in Wells

Modified Shaking Peltier w fluid interface

5 min

2 min


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

3 min fixation

5 min fixation

4 min fixation

2 min fixation

1 min fixation

Impact of fixation time at 37 deg C on light scatter profiles


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

Signal to Noise for Fixation Kinetic study at 37°C Wet Coupling Biomek

16.00

14.00

12.00

10.00

S/N Biomek (p38)

S/N

8.00

Controls (p38)

6.00

4.00

2.00

0.00

1

2

3

4

5

Fixation Time (mins)

Impact of fixation time at 37 deg C on P-p38 S/N


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

S/N = 7.86

S/N = 6.50

Comparison of Manual vs Automated Signaling Assays

Manual Assay

SS

Automated Assay

P-ERK

CD14 PC7


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

CV’s of Current Automated Assay for P-ERK, P-p38 and P-S6


Cytometry of cell signaling simultaneous analysis of multiple signaling pathways in aml

S/N = 17.80

S/N = 14.50

S/N = 16.01

S/N = 14.70

S/N = 25.10

10 LPS Activation Comparison of 2 Biomeks

Biomek NXp1

S/N = 27.35

Biomek NXp 2

P-ERK Alexa 647

P-p38 Alexa 488

P-S6 Pac Blue


Collaborators

Collaborators

ACCG/Cytometry Consortium

David Hedley/Sue Chow /Qing Chang– Ontario Cancer Institute, UHN, Toronto, Ont.

Chuck Goolsby/James Marvin – Northwestern University, Chicago, IL

Jim Jacobberger/Phil Woost - Case Western Reserve Univ, Cleveland, OH

Beckman Coulter

Patty Grom, Lilly Lopez – Advanced Technology/Systems Research

Meryl Forman & Co (Ltd) – Advanced Technology

Bob Zigon/Ernie Anderson – Kaluza Software Development


Systems research automation group

Systems Research Automation Group

Kelechi Eluwa

Valentin Quesada

Bob Auer

Lilly Lopez

Sergei Gulnik

T. Vincent Shankey


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