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Single-cell mass cytometry adapted to measurements of the cell cycle

Single-cell mass cytometry adapted to measurements of the cell cycle. S. G2. G0/1. Cell cycle analysis by mass cytometry. Method for cell cycle analysis Markers of cell cycle phases S-phase G0 G1, G2, M Validation with cycling T Cells

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Single-cell mass cytometry adapted to measurements of the cell cycle

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  1. Single-cell mass cytometry adapted to measurements of the cell cycle S G2 G0/1

  2. Cell cycle analysis by mass cytometry • Method for cell cycle analysis • Markers of cell cycle phases • S-phase • G0 • G1, G2, M • Validation with cycling T Cells • System-level analysis of cell cycle in normal and malignant hematopoiesis • SPADE clustering • 35 parameter analysis of normal bone marrow cell cycle • Application to hematologic malignancies

  3. Mass cytometry DNA staining G0 G1 (pentamethylcyclopentadienyl)-Ir(III)-dipyridophenazine S G2 Hoechst IrIntercalator

  4. IdU incorporates rapidly into S-phase cells Fluorescent Cytometry Mass Cytometry Uridine Uridine 56.5% 47.6% IrIntercalator Hoechst

  5. IdU incorporates rapidly into S-phase cells Fluorescent Cytometry Mass Cytometry Uridine Uridine 56.5% 47.6% IrIntercalator Hoechst

  6. IdU incorporates rapidly into S-phase cells 0.3% 21.9% 20.6% 24.2% No IdU 5 min 10 min 15 min 24.3% 28.9% 26.6% IdU I127 Intercalator Ir193 30 min 60 min 120 min

  7. Additional markers allow for complete cell cycle state assignment p-histone H3 -Andrew Hughes, Gene TherMol Biol., 2006; 10:41

  8. Additional markers allow for complete cell cycle state assignment p-histone H3 p-Rb (S807/S811) G0 Uridine

  9. Additional markers allow for complete cell cycle state assignment G2 p-histone H3 p-histone H3 S Cyclin B1 G0/G1 Uridine

  10. Additional markers allow for complete cell cycle state assignment p-histone H3 p-histone H3 M p-Histone H3 (S28) Uridine

  11. Cell cycle assessment is robust and consistent across multiple cell types human T cells U937 cells HL60 cells NALM6 cells A G0 IdU I127 p-Rb Ho165 B S IdU I127 Cyclin B1 Dy164 G0/G1 G2/M C M IdU I127 p-Histone H3 Er168

  12. Cell cycle assessment is robust and consistent across multiple cell types human T cells U937 cells HL60 cells NALM6 cells A G0 IdU I127 p-Rb Ho165 B S IdU I127 Cyclin B1 Dy164 G0/G1 G2/M C M IdU I127 p-Histone H3 Er168

  13. Phosphorylated Rb (S807/S811) discriminates G0 and G1 phase cells C A 23.8% Pyronin Y Pyronin Y 76.3% Hoechst IdU FITC B D 27.6% 24.4% p-RbAlexa 647 p-RbAlexa 647 p-Rb Ho165 p-Rb Ho165 75.6% 72.3% IdU I127 Hoechst IdU FITC Intercalator Ir193

  14. The same cell cycle markers can be used for fluorescence cytometry Fluorescence Cytometry S G0 Mass Cytometry IdU G0 S IdU G2/M G0/G1 pRb Cyclin B1 G2/M G0/G1 S pRb Cyclin B1 Pyronin Y IdU G0 G0/G1 G2/M Hoechst Hoechst

  15. Mass cytometry cell cycle analysis is equivalent to fluorescent methodologies Fluorescent cytometry Mass cytometry 0h 28h 48h

  16. Mass cytometry cell cycle analysis is equivalent to fluorescent methodologies Fluorescent cytometry Mass cytometry 0h 28h 48h

  17. Mass cytometry cell cycle analysis is equivalent to fluorescent methodologies Fluorescent cytometry Mass cytometry 0h 28h 48h

  18. Mass cytometry cell cycle analysis is equivalent to fluorescent methodologies 0h 28h 48h

  19. Cell cycle analysis by mass cytometry • Method for cell cycle analysis • Markers of cell cycle phases • S-phase • G0 • G1, G2, M • Validation with cycling T Cells • System-level analysis of cell cycle in normal and malignant hematopoiesis • SPADE clustering • 35 parameter analysis of normal bone marrow cell cycle • Application to hematologic malignancies

  20. Panel for analysis of cell cycle in human marrow

  21. Biaxial plots are not a scalable solution Parameters: 4 8 14 32 Plots: 6 28 91 496 Sean Bendall, Erin Simonds. Science,May 2011

  22. SPADE: Spanning-tree Progression Analysis of Density-normalized Events – Peng Qiu • Determine Tree Structure • Overlay regions with surface marker expression levels

  23. SPADE clustering of normal bone marrow mirrors immunophenotypic differentiation. CD45

  24. SPADE clustering of normal bone marrow mirrors immunophenotypic differentiation. NK Naïve CD8 T Naïve CD4 T CD4 T NKT CD8 T CD45 CD3

  25. SPADE clustering of normal bone marrow mirrors immunophenotypic differentiation. Myeloblasts CD45 CMP Myelocytes Late Early Promyelocytes CD15 Meta-myelocytes Mature Myeloid

  26. SPADE clustering of normal bone marrow mirrors immunophenotypic differentiation. Mature Monocytes Early Monocytes Macrophages Pro-monocytes PC-DCs Monoblasts CD45 CD14

  27. SPADE clustering of normal bone marrow mirrors immunophenotypic differentiation. CD45 Pre-BI Pre-BII Pro-B Immature B cells Plasma Mature B cells CD20

  28. SPADE clustering of normal bone marrow mirrors immunophenotypic differentiation. MEP CD45 Pro-erythroblasts Erythroblasts Nucleated RBCs CD235

  29. SPADE clustering of normal bone marrow mirrors immunophenotypic differentiation. Vasc. Progen? MPP HSC MEP GMP Monoblasts Myeloblasts CD45 Pre-BI CMP Pro-erythroblasts Pro-B CD34

  30. SPADE clustering of normal bone marrow mirrors immunophenotypic differentiation. Vasc. Progen? NK Mature Monocytes MPP Naïve CD8 T HSC Naïve CD4 T Early Monocytes CD4 T Macrophages MEP NKT Pro-monocytes CD8 T GMP PC-DCs Monoblasts CD45 Myeloblasts Pre-BI Pre-BII CMP Pro-erythroblasts Pro-B Erythroblasts Immature B cells Plasma Myelocytes Nucleated RBCs Mature B cells Late Early Promyelocytes Meta-myelocytes Mature Myeloid

  31. B cell proliferation is concentrated in pre-BII population G1 CD34 Pro-B CD10 Pre-BI S Pre-BII CD19 G2 Normal Human Bone Marrow Colored for CD45 CD20 = 200 Cells = 67% Mature B

  32. Erythroid cell proliferation is concentrated in erythroblast population MEP Pro- erythro- blast Nucleated RBC Erythro- blast CD34 CD71 CD235 Normal Human Bone Marrow Colored for CD45 = 200 Cells G1 S G2 = 67%

  33. Myelocyte proliferation peaks at early promyelocyte stage Late Promyelo- cytes Early Promyelo- cytes G1 CD11b Myelocyte Metamyelocyte S CD15 Normal Human Bone Marrow Colored for CD45 Mature myelocyte = 3500 Cells = 67% G2 CD16

  34. SPADE analysis allows for identification of distinct AML immunophenotypes HSC / Early Progenitor Mature Monocytic Promyelocyte / Myelocyte Promonocyte Mature Myeloid ?Pro-B / Pre-B Myleo/mono- blast NK Cell T Cell B Cell Normal human bone marrow Clustered alongside AML samples Colored for CD45

  35. Cell cycle distribution varies across the immunophenotypic subsets within each AML sample AML5 AML9 CD34 S = 20%

  36. Conclusions • Validated methodology for using mass cytometry to asses cell cycle state in combination with high-parameter immunophenotypic analysis • System-wide analysis of proliferation across normal human hematopoiesis • The ability to combine cell cycle state with multiple other variables in the monitoring of cellular responses at the single-cell level • We intend to use this methodology to characterize the cell cycle within complex human cancer samples

  37. Acknowledgements • Nolan Lab • Garry Nolan • Wendy Fantl • Sean Bendall • Erin Simmonds • Rachel Fink • Matt Clutter • Angelica Trejo • Matt Hale • Stanford • Michael Linderman • Sylvia Plevritis • MD Anderson • Peng Qiu • Hematology • Bruno Medeiros • Peter Greenberg • Beverly Mitchell • AparnaRaval • Cytobank • NikeshKotecha

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