Cytometry of Cell Signaling:Simultaneous Analysis of Multiple

Signaling Pathways in AML

T. Vincent Shankey, Ph.D.Systems Research/Life Sciences Division

Beckman Coulter, IncMiami, FL

Vincent.Shankey@Coulter.com

Control

P-ERK-Alexa 488

FA/Triton X-100

40 uM PMA

FA/TX/MeOH

Control

40 uM PMA

P-ERK-Alexa 488

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

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

David Hedley, Princess Margaret Hospital

(GDC-0941)

Hematopoietic Differentiation

Peripheral Circulation

CD34+CD117+/-

Gating/Analysis Protocol for Bone Marrow Signaling Analysis

+SCF

+GM-SCF

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

James Jacobberger, Case Western Reserve University

Signaling Responses in Normal CD34+/CD117+ cells

James Jacobberger, Case Western Reserve University

Signaling Responses in Normal CD34+/CD117+ cells

Signaling Response in AML Bone Marrows

01

23

4

5

0

1

2

0

20

40

60

Interpolated

James Jacobberger, Case Western Reserve University

SCF stimulated P-S6 and P-Erk

AML1 = M4AML2 = M2AML3 = M4eoAML4 = M5bAML5 = M1

0

20

40

60

0

1

2

3

4

5

6

7

8

0.0

0.51.0

1.52.0

AML1 AML3 AML5 NBM

pS6

(MF

I)

pERK (M

FI)

Time (min)

Flt3L-stimulated P-S6 and P-Erk

A B C

Side Scatter

Fig 1

100

101

102

103

104

100

101

102

103

104

0 16384 32768 49152 6553610

0

101

102

103

104

CD64

APC

CD117 PeCy5.5Side Scatter

CD34

ECD

CD13

Pe

Cy7

CD64

APC

CD16 Alexa 700Side Scatter

D E

Lymphs

Non Lymphs

Stem EnrichBlast

CD34+CD117+

CD34-CD117+

Monocytes

Myeloid Enrich Immature Myeloid

Intermediate Myeloid

Mature Myeloid

CD45

APC

Al

exa

750

Gating/Analysis Protocol for Bone Marrow Signaling Analysis

Chuck Goolsby, Northwestern University

Chuck Goolsby, Northwestern University

Growth Factor Receptor Expression Profiles for the Six Non-Lymphoid Cell Populations from Normal Bone Marrow

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

34+/117+ 34-/117+ Mono G1 G2 G302468

10121416

SCF

FLT3

IL3

GMCSF

GCSF

13.3

8.8

1.5 2.2 2.2 2.5

11.99.8

12.4

2.2 2.2 2.6

12.2

1

15.6

5.4 6.3 6.7

10.6

3.5

12.1

6.2

9.411.3

10.2

7.4

1

56.5 6.8

SCF FLT3 IL3 GMCSF

Chuck Goolsby, Northwestern Univ

34+/117+ 34-/117+ Mono G1 G2 G30

5

10

15

20

25

30

SCF

FLT3

IL3

GMCSF

GCSF

1 1 1 1 1 1

1 1 1 1 1 1

19.8

12.3 12.7 13.8 14.217.7

20.5

14.3 13.616.8 18.3

24.1

25.2

13.5

6.58.7 9.7

11.7

SCF FLT3 IL3 GMCSF

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

Chuck Goolsby, Northwestern Univ

34+/117+ 34-/117+ Mono G1 G2 G30

5

10

15

SCF

FLT3

IL3

GMCSF

GCSF

1 1 1 1 1 1

1 1 1 1 1 1

2.91 1 1 1 1

1 1 1

7.8 7.69.2

10.2 10.4

6.9

14 14.2 14.8

SCF FLT3 IL3 GMCSF

Chuck Goolsby, Northwestern Univ

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

Normal bone marrow cells show highly reproducible signaling pathways that

correlate with the differentiation state and the presence of specific cell surface

cytokine receptors

Chuck Goolsby, Northwestern University

AML – Categories of Abnormal Bone Marrow Signaling

Constitutive Activation

P-STAT5 P-Akt

Receptor Dysregulation

Abnormal Kinetics

GM-CSF

P-Akt

SCF

Aberrant Signaling Patterns in AML Bone Marrow Samples

Measurement of Cell Signaling

Whole Blood

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

David Hedley, Princess Margaret Hospital

(GDC-0941)

David Hedley, Princess Margaret Hospital

David Hedley, Princess Margaret Hospital

David Hedley, Princess Margaret Hospital

David Hedley, Princess Margaret Hospital

David Hedley, Princess Margaret Hospital

David Hedley, Princess Margaret Hospital

Patient #106 FLT3/ITD

pSTAT5

Daily Oral Dose 225mg

FL2 INT LOG

SS

INT

LIN

100 101 102 103 104

0

256

512

768

1024

Control

FL2 INT LOG

SS

INT

LIN

100 101 102 103 104

0

256

512

768

1024

FL2 INT LOG

SS

INT

LIN

100 101 102 103 104

0

256

512

768

1024

FL2 INT LOG

SS

INT

LIN

100 101 102 103 104

0

256

512

768

1024

P-STAT5FL2 INT LOG

SS

INT

LIN

100 101 102 103 104

0

256

512

768

1024

ENMD20761.6uM

FL5 INT LOG

SS

INT

LIN

100 101 102 103 104

0

256

512

768

1024

Pre-dose

CD117+Blasts

Day 8

FL5 INT LOG

SS

INT

LIN

100 101 102 103 104

0

256

512

768

1024

FL5 INT LOG

SS

INT

LIN

100 101 102 103 104

0

256

512

768

1024

FL5 INT LOG

SS

INT

LIN

100 101 102 103 104

0

256

512

768

1024

Day 29 Day 211 15% 0.17% 0.03% 0.8%

CD117

David Hedley, Princess Margaret Hospital

Signaling Classification of AML(Work in Progress)

Real-time Monitoring of Molecular Targeted Therapeutics

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

p-Stat5

Count

p-Stat5

Count

p-Stat5

Count

CD34+ cellsPre-therapy

Three weeksPost-therapy

Three weeks Post-therapy In vitro imatinibtreated

p-Stat5

Count

Count

p-Stat5

Count

p-Stat5

Count

p-Stat5

Count

p-Stat5

Count

CD34+ cellsPre-therapy

Three weeksPost-therapy

Three weeks Post-therapy In vitro imatinibtreated

Patient #2 –SCF activation David Hedley, Princess Margaret Hospital

AML Blast Response to Gleevec

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

• 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!

Manual Assay Kinetics

Tube Contents LPS / 37º Add LPSAdd

Formaldehyde Add Triton

1 14 +p38 - no lps 0.00 31.20 46.20

2 14+p38 LPS Test 2 2.00 29.10 31.10 46.10

3 14+p38 LPS Test 3 4.00 27.00 31.00 46.00

4 14+p38 LPS Test 4 6.00 24.50 30.50 45.50

5 14+p38 LPS Test 5 8.00 22.40 30.40 45.40

6 14+p38 LPS Test 6 10.00 20.30 30.30 45.30

7 14+p38 LPS Test 7 15.00 15.20 30.20 45.20

8 14+p38 LPS Test 8 20.00 10.10 30.10 45.10

9 14+p38 LPS Test 9 30.00 0 min 30.00 45.00

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 downIncubate for 15 min at 37C

Add 2 mL of cold (4 C, possibly RT) PBS+4%FCSSpin at 1000xg, 3 min

Remove supernatant/resuspend pellet with residual buffer

Add 1 mL of “RT” 50-80% MeOH in PBS

Spin at 1000xg, 3 min

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

Remove supernatant

Spin at 1000xg, 3 minRemove 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 minRemove supernatant

Place the tubes (barcoded) on a 32 tube carouselAnalyze on a FC500 or CRS

Resuspend cells in 1 mL wash buffer

Cell Signaling Sample Preparation

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

Up to 2 washes

June 30, 2009

Biomek NXp

AccessoriesDeck Layout

Centrifuge

Gallios

Assay Automation Tools

Shaking/Temperature Cycling Peltier

48 deep-wellplate

Adapter

Peltier

0

5

10

15

20

25

30

35

40

45

90.00 100.00 110.00 120.00 130.00 140.00 150.00 160.00 170.00 180.00

Elapse Time

Tem

p C

deg

Column 1

Column 2

Column 3

Column 4

Column 5

Column 6

Column 7

Column 8

Temperature Cycling in Wells

10 min

15 min

2 min 2 min 5 min 5 min 10 min 10 min 15 min 15 min 30 min 30 min0

2

4

6

8

10

12

14

16

Automation LPS Kinetics P-p38+P-ERK

P-p38 37 deg Contr

P-p38 LPS

Time Interval

MF

I

0

5

10

15

20

25

30

35

40

45

0.50 2.50 4.50 6.50 8.50 10.50 12.50 14.50 16.50 18.50 20.50 22.50 24.50 26.50 28.50 30.50 32.50 34.50 36.50 38.50

"°C"1

"°C"2

"°C"3

"°C"4

"°C"5

"°C"6

"°C"7

"°C"8

Temperature Cycling in WellsModified Shaking Peltier w fluid interface

2 min5 min

5 min fixation 4 min fixation 3 min fixation

2 min fixation 1 min fixation

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

0

5

10

15

20

25

30

35

40

45

0.50 2.50 4.50 6.50 8.50 10.50 12.50 14.50 16.50 18.50 20.50 22.50 24.50 26.50 28.50 30.50 32.50 34.50 36.50 38.50

"°C"1

"°C"2

"°C"3

"°C"4

"°C"5

"°C"6

"°C"7

"°C"8

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

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

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

1 2 3 4 5

Fixation Time (mins)

S/N S/N Biomek (p38)

Controls (p38)

S/N = 7.86

S/N = 6.50

SS

CD14 PC7 P-ERK

Automated Assay

Manual Assay

Comparison of Manual vs Automated Signaling Assays

pERK Signal after Biomek NXp 2 minutes LPS activation. Cells reconstitution in

Wash buffer or Formaldehyde

0

10

20

30

40

50

Well 1 Well 2 Well 3 Well 4

Well

MF

I

Wash Buffer 0.1% Form 0.5% Form

pS6 Signal after Biomek NXp 2 minutes LPS activation. Cells reconstitution in

Wash buffer or Formaldehyde

0

8

16

24

32

Well 1 Well 2 Well 3 Well 4

Well

MF

I

Wash Buffer 0.1% Form 0.5% Form

p38 Signal after Biomek NXp 2 minutes LPS activation. Cells reconstitution in

Wash buffer or Formaldehyde

0

8

16

24

32

Well 1 Well 2 Well 3 Well 4

Well

MF

I

Wash Buffer 0.1% Form 0.5% Form

%CV of Biomek NXp 2min LPS Activation Assay. Four Replicates of pERK, pS6 and

p38 Signals for cells reconstituted in Wash Buffer of Formaldehyde

0.00

4.00

8.00

12.00

16.00

WashBuffer

0.1% Form 0.5% Form

Reconstitution Medium

%C

VpERK

pS6

p38

pERK Signal after Biomek NXp 2 minutes LPS activation. Cells reconstitution in

Wash buffer or Formaldehyde

0

10

20

30

40

50

Well 1 Well 2 Well 3 Well 4

Well

MF

I

Wash Buffer 0.1% Form 0.5% Form

pS6 Signal after Biomek NXp 2 minutes LPS activation. Cells reconstitution in

Wash buffer or Formaldehyde

0

8

16

24

32

Well 1 Well 2 Well 3 Well 4

Well

MF

I

Wash Buffer 0.1% Form 0.5% Form

p38 Signal after Biomek NXp 2 minutes LPS activation. Cells reconstitution in

Wash buffer or Formaldehyde

0

8

16

24

32

Well 1 Well 2 Well 3 Well 4

Well

MF

I

Wash Buffer 0.1% Form 0.5% Form

%CV of Biomek NXp 2min LPS Activation Assay. Four Replicates of pERK, pS6 and

p38 Signals for cells reconstituted in Wash Buffer of Formaldehyde

0.00

4.00

8.00

12.00

16.00

WashBuffer

0.1% Form 0.5% Form

Reconstitution Medium

%C

VpERK

pS6

p38

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

Biomek NXp 2

Biomek NXp1

S/N = 25.10 S/N = 14.70 S/N = 16.01

S/N = 17.80 S/N = 27.35 S/N = 14.50

P-ERK Alexa 647 P-S6 Pac BlueP-p38 Alexa 488

10 LPS Activation Comparison of 2 Biomeks

Collaborators

ACCG/Cytometry Consortium

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

Chuck Goolsby/James Marvin – Northwestern University, Chicago, ILJim Jacobberger/Phil Woost - Case Western Reserve Univ, Cleveland, OH

Beckman Coulter

Patty Grom, Lilly Lopez – Advanced Technology/Systems ResearchMeryl Forman & Co (Ltd) – Advanced TechnologyBob Zigon/Ernie Anderson – Kaluza Software Development

Systems Research Automation Group

Kelechi Eluwa

Valentin Quesada

Bob Auer

Lilly Lopez

Sergei Gulnik

T. Vincent Shankey