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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.

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Presentation on theme: "Cytometry of Cell Signaling: Simultaneous Analysis of Multiple Signaling Pathways in AML T. Vincent Shankey, Ph.D. Systems Research/Life Sciences Division."— Presentation transcript:

1 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

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3 Control P-ERK-Alexa 488 FA/Triton X uM PMA FA/TX/MeOH Control 40 uM PMA P-ERK-Alexa 488

4 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) 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

5 Measurement of Cell Signaling Bone Marrow

6 Acute Myeloid Leukemia (AML) (used with CD45, CD34, CD13/33, CD117) David Hedley, Princess Margaret Hospital (GDC-0941)

7 Hematopoietic Differentiation Peripheral Circulation CD34+ CD117+/-

8 Gating/Analysis Protocol for Bone Marrow Signaling Analysis +SCF +GM-SCF

9 Rapid Activation/Inactivation of P-ERK in Normal Bone Marrow CD34+/CD117+ Cells James Jacobberger, Case Western Reserve University

10 Signaling Responses in Normal CD34+/CD117+ cells James Jacobberger, Case Western Reserve University

11 Signaling Responses in Normal CD34+/CD117+ cells

12 Signaling Response in AML Bone Marrows

13 Interpolated James Jacobberger, Case Western Reserve University SCF stimulated P-S6 and P-Erk AML1 = M4 AML2 = M2 AML3 = M4eo AML4 = M5b AML5 = M1 AML1 AML3 AML5 NBM pS6 (MFI) pERK (MFI) Time (min) Flt3L-stimulated P-S6 and P-Erk

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15 A B C Side Scatter Fig 1 CD64 APC CD117 PeCy5.5 Side Scatter CD34 ECD CD13 PeCy7 CD64 APC CD16 Alexa 700 Side Scatter D E Lymphs Non Lymphs Stem Enrich Blast CD34+CD117+ CD34-CD117+ Monocytes Myeloid Enrich Immature Myeloid Intermediate Myeloid Mature Myeloid CD45 APC Alexa 750 Gating/Analysis Protocol for Bone Marrow Signaling Analysis Chuck Goolsby, Northwestern University

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

17 Normal Bone Marrow: P-ERK (S/N) Chuck Goolsby, Northwestern Univ

18 Normal Bone Marrow: P-STAT5 (S/N) Chuck Goolsby, Northwestern Univ

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

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

21 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

22 Aberrant Signaling Patterns in AML Bone Marrow Samples

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24 Measurement of Cell Signaling Whole Blood

25 Acute Myeloid Leukemia (AML) (used with CD45, CD34, CD13/33, CD117) David Hedley, Princess Margaret Hospital (GDC-0941)

26 David Hedley, Princess Margaret Hospital

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32 Patient #106 FLT3/ITD pSTAT5 Daily Oral Dose 225mg Control P-STAT5 ENMD uM Pre-dose CD117+ Blasts Day 8 Day 29 Day %0.17%0.03%0.8% CD117 David Hedley, Princess Margaret Hospital

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

34 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; , 2008 p-Stat5 Count p-Stat5 Count p-Stat5 Count CD34 + cells Pre-therapy Three weeks Post-therapy Three weeks Post-therapy In vitroimatinib treated p-Stat5 Count p-Stat5 Count p-Stat5 Count p-Stat5 Count p-Stat5 Count CD34 + cells Pre-therapy Three weeks Post-therapy Three weeks Post-therapy In vitroimatinib treated

35 Patient #2 –SCF activation David Hedley, Princess Margaret Hospital AML Blast Response to Gleevec

36 Summary - AML Blast Response to in vivo Gleevec Treatment P-Akt levels at D4/t2 predicts clinical response to subsequent Chemotherapy (p=0.008)

37 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

38 Need for Automation!

39 Manual Assay Kinetics TubeContentsLPS / 37ºAdd LPS Add Formaldehyde Add Triton 114 +p38 - no lps p38 LPS Test p38 LPS Test p38 LPS Test p38 LPS Test p38 LPS Test p38 LPS Test p38 LPS Test p38 LPS Test min

40 Throughput requirements: No info. Blood sample in vacutainer Aliquot up to100 uL per tube (up to 32 tubes/patient) Add 5 uL of activator 37C or RT) and/or inhibitor to activation tubes or 5 uL of PBS to the control tube Incubate at 37 C for min Add 65uL of 10% formaldehyde at RT Vortex Incubate for 10 min (exact) at RT Add 1 mL of % 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 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 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 Place the tubes (barcoded) on a 32 tube carousel Analyze 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

41 Biomek NXp Accessories Deck Layout Centrifuge Gallios Assay Automation Tools

42 Shaking/Temperature Cycling Peltier 48 deep-well plate Adapter Peltier

43 Temperature Cycling in Wells 10 min 15 min

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45 Temperature Cycling in Wells Modified Shaking Peltier w fluid interface 2 min 5 min

46 5 min fixation 4 min fixation 3 min fixation 2 min fixation1 min fixation Impact of fixation time at 37 deg C on light scatter profiles

47 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 Fixation Time (mins) S/N S/N Biomek (p38) Controls (p38)

48 S/N = 7.86 S/N = 6.50 SS CD14 PC7 P-ERK Automated Assay Manual Assay Comparison of Manual vs Automated Signaling Assays

49 CVs of Current Automated Assay for P-ERK, P-p38 and P-S6

50 Biomek NXp 2 Biomek NXp1 S/N = S/N = 14.70S/N = 16.01S/N = S/N = S/N = P-ERK Alexa 647P-S6 Pac BlueP-p38 Alexa LPS Activation Comparison of 2 Biomeks

51 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

52 Systems Research Automation Group Kelechi Eluwa Valentin Quesada Bob Auer Lilly Lopez Sergei Gulnik T. Vincent Shankey


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