530 likes | 741 Views
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 Vincent.Shankey@Coulter.com. Control. Control. FA/Triton X-100. FA/TX/MeOH. 40 uM PMA. 40 uM PMA.
E N D
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 Vincent.Shankey@Coulter.com
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 • 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
Acute Myeloid Leukemia (AML)(used with CD45, CD34, CD13/33, CD117) (GDC-0941) David Hedley, Princess Margaret Hospital
Hematopoietic Differentiation CD34+ CD117+/- Peripheral Circulation
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 Response in AML Bone Marrows
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
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
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) Chuck Goolsby, Northwestern Univ
Normal Bone Marrow: P-STAT5 (S/N) Chuck Goolsby, Northwestern Univ
Normal Bone Marrow: P-STAT3 (S/N) Chuck Goolsby, Northwestern Univ
Normal bone marrow cells show highly reproducible signaling pathways that correlate with the differentiation state and the presence of specific cell surface cytokine receptors
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
Acute Myeloid Leukemia (AML)(used with CD45, CD34, CD13/33, CD117) (GDC-0941) David Hedley, Princess Margaret Hospital
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
Signaling Classification of AML(Work in Progress)Real-time Monitoring of Molecular Targeted Therapeutics
+ + 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
AML Blast Response to Gleevec Patient #2 –SCF activation David Hedley, Princess Margaret Hospital
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
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
Assay Automation Tools Gallios Biomek NXp Centrifuge Accessories Deck Layout
Shaking/Temperature Cycling Peltier 48 deep-well plate Adapter Peltier
Temperature Cycling in Wells 15 min 10 min
Temperature Cycling in Wells Modified Shaking Peltier w fluid interface 5 min 2 min
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
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
S/N = 7.86 S/N = 6.50 Comparison of Manual vs Automated Signaling Assays Manual Assay SS Automated Assay P-ERK CD14 PC7
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