Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Analysis of Cellular Signaling usingActivation-State Specific Antibodies               Greg Innocenti         Cell Signali...
OutlineWhat are activation state-specific antibodies?Imaging Cytometry   • immunofluorescence & antibody validation   • an...
OutlineWhat are activation state-specific antibodies?Imaging Cytometry   • immunofluorescence & antibody validation   • an...
Activation State-Specific Antibodies                                                               phospho-p38Total (bind ...
Activation State-Specific Antibodies                                                               phospho-p38Total (bind ...
Total Antibodies        Untreated   Wnt 5’                              Whole BloodHeLa Cells                             ...
Phospho-specific Antibodies                            Untreated                    EGF-Treated        Phospho-EGFR (PE)  ...
Caspase-3 Cleavage in Apoptosis                     Untreated                   Staurosporine          TUNEL              ...
Antibody Validation & ApplicationsCST’s activation-specific antibodies are purified to distinguish  between only one or tw...
OutlineWhat are activation state-specific antibodies?Imaging Cytometry   • immunofluorescence & antibody validation   • an...
Antibody Validation for Immunofluorescence/HCA             Part I - Titration to determine                                ...
Antibody Validation for Immunofluorescence/HCA               [Ab]                                                         ...
EGF Receptor Inhibition                        EGFR Mutant NSCLC Cell Lines                        Untreated          Ires...
GPCR Activation                                                               MEK         PI3K                            ...
Neuroscience - Development     Postnatal Day 1            Postnatal Day 14                                                ...
Cell Cycle / Checkpoint                Untreated         Untreated                                   UV                   ...
CST Conjugated AntibodiesConjugated antibodies helpful for multiplex analysesCST conjugates are optimized for cytometric a...
Survivin (Alexa488-conjugate)                                                                    FAS, TNFa• inhibits apopt...
The Bigger PictureSingle well immunofluorescence   + ability to examine subcellular (co)localization in 4-dimension   (XYZ...
High Content Analysis• automated plate-based image analysis• quantifiable signal intensity and subcellular                ...
Value of CST Antibodies in HCSCurrent platforms have increased colors and resolution in an attempt toquantify complex even...
Multiple, Parallel Analyses by Cellular Imaging (ICW)                                                         Gleevec/PDGF...
Future Plans: Broad Signal Profiling by HCA• large antibody panel for profiling screens• can be used on any cytometric pla...
Antibody Development: FoxO1 Screening                                         PI3K inhibitor            IGF-1 + serum     ...
OutlineWhat are activation state-specific antibodies?Imaging Cytometry   • immunofluorescence & antibody validation   • an...
Optimization: Fixation/Permeabilization          CST 2-4% formaldehyde/90% methanolFix&Perm Kits 0.25-4% formaldehyde/dete...
Fixation/Permeabilizationp-p38p-Erk        2% Formaldehyde   4% Formaldehyde     Commercial Fix&Perm Kit         90% Metha...
Surface and Signaling Markers• Methanol diminishes or abolishes signal from some key surface markers• Many signaling event...
New Fix & Perm Protocol                                                     Untreated                                     ...
Protocol Comparison                                           4%PFA at RT for 10m     3% PFA at 37ºC for 10m           0.1...
Flow Cytometry: Clinical Applications•    Staining of intracellular signaling molecules can be easily     incorporated wit...
Bcr/Abl Pathway Profiling by Flow Cytometry                             CML cells (K562) + Gleevec                phospho-...
Large Scale Bcr/Abl Pathway Profiling                         K562 cells +/- Gleevec                bar height represents ...
Biomarkers for CLL                                                                                        *               ...
CLL Assay using Zap-70 (136F12) RmAbIn House:                                 In the Clinic:            B cells (Ramos)   ...
Alternative CLL Biomarkers                                 6.00                                 5.00Mean Fluorescence (nor...
Flt3 Signaling                      Flt3                        • Mutation or overexpression of Flt3 kinase               ...
Flt3 Inhibitor Pharmacodynamic Assay Design                                                                               ...
Conclusion• Activation state-specific antibodies are very useful in multiplex_phosphoproteomics analyses on various fluore...
Thank You
Upcoming SlideShare
Loading in …5
×

Cyto gi bioke4

507 views

Published on

  • Be the first to comment

  • Be the first to like this

Cyto gi bioke4

  1. 1. Analysis of Cellular Signaling usingActivation-State Specific Antibodies Greg Innocenti Cell Signaling Technology December 19, 2006
  2. 2. OutlineWhat are activation state-specific antibodies?Imaging Cytometry • immunofluorescence & antibody validation • antibody conjugation • high content analysis (HCA)Flow Cytometry • protocols • clinical assays
  3. 3. OutlineWhat are activation state-specific antibodies?Imaging Cytometry • immunofluorescence & antibody validation • antibody conjugation • high content analysis (HCA)Flow Cytometry • protocols • clinical assays
  4. 4. Activation State-Specific Antibodies phospho-p38Total (bind regardless of activation state)Phospho-specific (eg. phospho-EGFR) p38Cleavage-specific (eg. cleaved Caspase-3)Acetylation-specific (eg. acetyl-Stat3) + - + -Methylation-specific (eg. methyl-Histone H3)Substrate-specific (eg. phospho-Akt Substrate)Motif-specific (eg. phospho-Tyrosine)
  5. 5. Activation State-Specific Antibodies phospho-p38Total (bind regardless of activation state)Phospho-specific (eg. phospho-EGFR) p38Cleavage-specific (eg. cleaved Caspase-3)Acetylation-specific (eg. acetyl-Stat3) + - + -Methylation-specific (eg. methyl-Histone H3)Substrate-specific (eg. phospho-Akt Substrate)Motif-specific (eg. phospho-Tyrosine)
  6. 6. Total Antibodies Untreated Wnt 5’ Whole BloodHeLa Cells CD5 β-Catenin (C-term) CD13 LY294002 Insulin Side Scatter C2C12 Cells Akt (pan) (11E7) CD4
  7. 7. Phospho-specific Antibodies Untreated EGF-Treated Phospho-EGFR (PE) EGF Receptor (FITC)Green = Phospho-EGF Receptor (Tyr1068)Blue = DRAQ5
  8. 8. Caspase-3 Cleavage in Apoptosis Untreated Staurosporine TUNEL Cleaved-Caspase 3Green = Cleaved Caspase-3 (Asp175) (5A1) RmAbRed = F-Actin (Phalloidin)Blue = DRAQ5
  9. 9. Antibody Validation & ApplicationsCST’s activation-specific antibodies are purified to distinguish between only one or two posttranslationally modified amino acid residuesMost antibodies raised against such modified sites have a lower affinity when compared with antibodies recognizing whole proteinsThus, all product development follows rigorous in-house testing on a wide range of assay applications by our clinical applications specialists  Stringent validation requirements  Protocol optimization  Cross platform functionality
  10. 10. OutlineWhat are activation state-specific antibodies?Imaging Cytometry • immunofluorescence & antibody validation • antibody conjugation • high content analysis (HCA)Flow Cytometry • protocols • clinical assays
  11. 11. Antibody Validation for Immunofluorescence/HCA Part I - Titration to determine Part II - Specificity Testing optimal dilution/concentration • 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) Confocal Imaging on Chamber Slides Titration Curve 45000.00 6.00 40000.00 5.00 35000.00 30000.00 4.00 2966Mean Channel Fluorescence Signal to Noise Ratio 25000.00 3.00 Control 20000.00 S/N Ratio 15000.00 2.00 10000.00 1.00 5000.00 0.00 0.00 0 5 10 15 20 25 30 35 40 45 Antibody Dilution (ug/ml)
  12. 12. Antibody Validation for Immunofluorescence/HCA [Ab] Antibody Isotype Antibody1:25 1:50 1:100 1:200 1:400 1:800 16000.00 4.50 14000.00 4.00 12000.00 3.50 3.00 10000.00 Isotype 4694Mean Channel Fluorescence 2.50 Signal to Noise Ratio 8000.00 Control 2.00 6000.00 S/N Ratio 1.50 4000.00 1.00 2000.00 0.50 0.00 0.00 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 Antibody Dilution (ug/ml) MEK1/2 (red) actin (green) DNA (blue) HeLa cells
  13. 13. EGF Receptor Inhibition EGFR Mutant NSCLC Cell Lines Untreated Iressa HCC827 cellsDel 746-750, amp. H1975 cells L858R + T790M phospho-Tyrosine (red, Alexa647 conjugate) phospho-S6 (green, Alexa488 conjugate)
  14. 14. GPCR Activation MEK PI3K inhibitor inhibitor LPA treatment 0 min 2 min 5 min 15 min 15 min 15 min mergephospho-Erkphospho-Akt DNA C6 cells
  15. 15. Neuroscience - Development Postnatal Day 1 Postnatal Day 14 Postnatal Rat Brain p-Histone H3 Red=GFAP Blue=DoublecortinP1 P4 P14
  16. 16. Cell Cycle / Checkpoint Untreated Untreated UV Normal Colon Dexamethasone Colon Carcinoma Metaphase UV UV+PPT Anaphase #9309 Rb (4H1) #3068 p-Aurora #2558 p-Kip2 (T310) p-Rb (S807/811) p-p53 (Ser37) p-Rad17 (S645) #9309 Rb (4H1) #9719 p-H2A.X (S139) Aurora A/AIK A/B/C #9308 #4718 #9289 #3421 P Chk1/2 p53 Aurora Cdc25A Kip1 P cdc25B P ATM P CDK4/6 CDK2 cdc2 CyclinD CyclinE CyclinBG1 Phase R S Phase G2 Phase M Phase P P P Rad17 Abl HDAC P Rb P X P P Rb cdc2 H2A H3 E2F1 E2F1 OFF DP1 ON DP1 DNA Damage
  17. 17. CST Conjugated AntibodiesConjugated antibodies helpful for multiplex analysesCST 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
  18. 18. Survivin (Alexa488-conjugate) FAS, TNFa• inhibits apoptosis and regulates mitosis• over-expressed in most human cancers FADD• expression correlates with both accelerated Caspase-8/10 ER Stress Mitochondria relapse and chemotherapy resistance [Ca++] Smac/P1 Rat Brain Diablo Cyto C Caspase-12 Survivin Caspase-9 Caspase-3 Caspase-6 Caspase-7 Lamin A a-Fodrin DFF PARP CST’s conjugated Survivin antibody is currently being used to screen patient samples
  19. 19. The Bigger PictureSingle 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 massiveHigh Content Analysis + some systems able to analyze localization + rapid scanning (comparatively), sensitive, and quantifiable + ability to multiplex and dissect various pathways in tandem
  20. 20. High Content Analysis• automated plate-based image analysis• quantifiable signal intensity and subcellular 1:800 1:100 1:400 1:200 1:50 1:25 localization Untreated• more predictive of drug activity in a cellular PDGF p-Erk environment compared to ELISAs U0126+PDGF LY/Wort+PDGF• can be used to determine: Untreated efficacy and therapeutic dose PDGF p-Akt U0126+PDGF cell-permeability of drugs LY/Wort+PDGF potential toxicity (DNA damage, apoptosis, micronuclei) downstream effects of drug/target interaction off-target effects
  21. 21. Value of CST Antibodies in HCSCurrent platforms have increased colors and resolution in an attempt toquantify complex eventsExample: nuclear translocation of Erk or NFkB Requirements • nuclear marker • total antibody • high resolution optics • complex software • lots of data storageUsing a phospho antibody will eliminate these costly requirements andspeed up the screen (on/off as opposed to determination of localization) KEY: reliable simple affordable assay with clear robust results
  22. 22. Multiple, Parallel Analyses by Cellular Imaging (ICW) Gleevec/PDGF U0126/PDGF LY294002 Untreated Gleevec PDGF PDGF PMA blank 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
  23. 23. Future Plans: Broad Signal Profiling by HCA• large antibody panel for profiling screens• can be used on any cytometric platform• detection = fluorescent, chromogenic, chemiluminescence Starved• up to 96 antibodies per plate total and phosphorylation-specific antibodies MAPK, Akt, NFkB, Jak/Stat, etc. receptor tyrosine kinases (EGFR, VEGFR, FGFR, IGF-IR, cKit) adaptor proteins and downstream targets EGF transcription factors motif antibodies (general serine or tyrosine phosphorylation)• can be customized for analysis of different biological processes toxicity cell cycle/arrest Anisomycin apoptosis cell adhesion 96 prediluted cells treated with CST antibodies compound X UV
  24. 24. Antibody Development: FoxO1 Screening PI3K inhibitor IGF-1 + serum (Akt off) (AKT on)How can we generate antibodies thatwork well for cytometric applications?Screen using cytometric applications HTS & HCS Platforms Nuclear Cytoplasmic nuclear trigger (half-width intensity)
  25. 25. OutlineWhat are activation state-specific antibodies?Imaging Cytometry • immunofluorescence & antibody validation • antibody conjugation • high content analysis (HCA)Flow Cytometry • protocols • clinical assays
  26. 26. Optimization: Fixation/Permeabilization CST 2-4% formaldehyde/90% methanolFix&Perm Kits 0.25-4% formaldehyde/detergent (triton or saponin) Krutzik and Nolan (2004) Cytometry 55A:61-70.
  27. 27. Fixation/Permeabilizationp-p38p-Erk 2% Formaldehyde 4% Formaldehyde Commercial Fix&Perm Kit 90% Methanol 0.3% Triton X-100 (aldehyde/saponin)
  28. 28. 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…
  29. 29. New Fix & Perm Protocol Untreated PMA4% Formaldehyde + 0.1% Triton X-100 + 50% Methanol
  30. 30. Protocol Comparison 4%PFA at RT for 10m 3% PFA at 37ºC for 10m 0.1% Triton X-100 at RT for 30m90% ice cold MeOH at -20ºC for 10m 50% ice cold MeOH at 4ºC for 10m CD19 (FITC) CD19 (FITC)
  31. 31. Flow Cytometry: Clinical Applications• Staining of intracellular signaling molecules can be easily incorporated with traditional cell surface marker labeling 4% Formaldehyde for 10m 0.1 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)
  32. 32. Bcr/Abl Pathway Profiling by Flow Cytometry CML cells (K562) + Gleevec phospho- Bcr phospho- Stat5 cleaved- Casp3 0 hr 1 hr 24 hr 48 hr
  33. 33. Large Scale Bcr/Abl Pathway Profiling K562 cells +/- Gleevec bar height represents amount of Gleevec inhibition
  34. 34. Biomarkers for CLL * * Staudt’s lab first identified Zap-70 as a potential biomarker of the aggressive form of CLL CLL patients with Zap-70 (+) B-cells have poorer prognosisCrespo et al. (2003) N Engl J Med 348:1746-75
  35. 35. CLL Assay using Zap-70 (136F12) RmAbIn House: In the Clinic: B cells (Ramos) T cells (Jurkat)Primary CLL Cells: Source: Esoterix Center for Innovation Difficulties persist in Zap-70 analysis - Zap-70 levels decline significantly after blood is drawn - Inconclusive results with weak expression Zap-70 (low) Zap-70 (high) - Additional biomarkers to make assay more reliable?(slow growing) (fast growing)
  36. 36. Alternative CLL Biomarkers 6.00 5.00Mean Fluorescence (normalized) 4.00 CLL23LB4 (Zap70-) 3.00 MO1043 (Zap70+) 2.00 1.00 0.00 p-PLCg p-FGFR PAK Zap70 p-Stat1 p-Stat3 p-NFkB p65 p-IGF-IR (Y783) (y653/654) (S727) (S727) (S536) (Y1131) CLL23LB4 (Zap70-) MO1043 (Zap70+) Rosenwald et al. (2001) J Exp Med. 194(11):1639-1647.
  37. 37. Flt3 Signaling Flt3 • Mutation or overexpression of Flt3 kinase is observed in AML, (~70%), B-ALL, T- ALL, and some blast phase CML • Most common mutations are internal tandem duplications (ITD) and active loop mutations (AL), both of which result in constitutive activation • Phospho-specific antibodies can be used to profile downstream signaling and to monitor the efficacy of specific Flt3 inhibitorsScheijen and Griffin (2002) Oncogene 21:3314-33
  38. 38. Flt3 Inhibitor Pharmacodynamic Assay Design Phospho-Flt3 Flt3 ITD Overexpressed Flt3 Alexa488 Conjugate (MOLM-14(MOLM14) Dose Response Cells) (SEM Cells) Dose Response (SEM) (SEM cells) 1.4 1.4Normalized mean fluorescence Normalized mean fluorescence 1.2 1.2 1 1 p-S6 p-S6 p-Flt3 p-Flt3 0.8 0.8 p-Stat5 p-Stat5 0.6 p-Stat3 p-Stat3 0.6 PY p-Erk 0.4 p-AKT PY 0.4 p-AKT uninhibited Flt3 inhibitor 0.2 0.2 0 0 0 0.01 0.1 0.5 2 0 0.01 0.1 0.5 2 Concentration of Flt3 inhibitor (µM) Concentration of Flt3 inhibitor (µM) Conclusion: Phospho-S6 is a key indicator of Flt3 inhibition, regardless of mutation This antibody is a reliable Flt3 inhibitor decreases downstream signaling, Percentage of Cleaved Caspase-3 positive cells robust biomarker for receptor causes cell cycle 80 70 tyrosine kinase (RTK) activity 60 arrest, and induces Percent (%) 50 apoptotic cell death 40 30 20 10 0 0 2 3 5 22 Time (hours)
  39. 39. Conclusion• Activation state-specific antibodies are very useful in multiplex_phosphoproteomics analyses on various fluorescence based platforms • stringent validation assures highest quality antibody and lot-to-lot reproducibility • determine prognostic significance of signaling anomalies • monitor patients for resistance or recurrence • study effectiveness and optimal dosage of specific kinase inhibitors • allow tumor “typing” to determine best treatment option • improve clinical trial and basic research design (outcome and economics) Gregory Innocenti Clinical Applications/Image Cytometry ginnocenti@cellsignal.com 978-867-2475
  40. 40. Thank You

×