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© 2011 J. Simonds Tools and approaches for mass cytometry data analysis Erin Simonds, PhD W. A. Weiss Lab, UCSF May 23, 2013 Biaxial plots are not a scalable solution Parameters: 32 14 8 4 Plots: 91 28 6 496 How can we explore in 30+ dimensions? The cytometrist’s toolbox Transform raw data Histogram Biaxial plot 3D plot Radar PCA Gemstone SPADE viSNE Wanderlust Box plot Heatmap FLAME SamSPECTRAL Reduce dimensionality Summarize statistics Identify clusters FlowClust FlowMerge FLOCK Vetting the CyTOF: In a fair fight, nearly identical data Experimental design 7 Surface Markers (CD 3, 4, 8, 20, 33, 45RA, 56) 2 Functional Markers (pSTAT3 & 5) Bendall, Simonds, et al. Science, May 2011 The cytometrist’s toolbox Transform raw data Histogram Biaxial plot 3D plot Radar PCA Gemstone SPADE viSNE Wanderlust Box plot Heatmap FLAME SamSPECTRAL Reduce dimensionality Summarize statistics Identify clusters FlowClust FlowMerge FLOCK Marker 1 SPADE SPADE: Spanning-tree Progression Analysis of Density-normalized Events Marker 1 Marker 2 Marker 2 Qiu et al., Nature Biotechnology, Oct. 2011 SPADE projects bone marrow as a continuum of phenotypes CD123 CD11b CD45 CD34 CD33 CD20 CD19 CD8 CD4 CD3 0 Intensity (%max) 100 Bendall, Simonds, et al. Science, May 2011 Rediscovery of canonical signaling pathways Basal IL-7 pSTAT5 0 Intensity (%max) 100 Bendall, Simonds, et al. Science, May 2011 Dasatinib differentially affects immune cell subsets IL7 (pSTAT5) PVO4 (pSTAT5) + Dasatinib Abl / Src-family kinase inhibitor Bendall, Simonds, et al. Science, May 2011 The cytometrist’s toolbox Transform raw data Histogram Biaxial plot 3D plot Radar PCA Gemstone SPADE viSNE Wanderlust Box plot Heatmap FLAME SamSPECTRAL Reduce dimensionality Summarize statistics Identify clusters FlowClust FlowMerge FLOCK viSNE preserves high-dimensional, non-linear relationships viSNE map (in 2D) viSNE-2 Raw data (in 3D) z y x viSNE-1 Amir et al., Nature Biotechnol. ePub 19 May 2013 viSNE maps healthy marrow as discrete populations CD11b-hi monocyte CD4+ T CD11b-lo monocyte CD20+ B CD20- B NK CD8+ T Amir et al., Nature Biotechnol. ePub 19 May 2013 viSNE can help detect rare subpopulations Barcode A: Healthy bone marrow cells Barcode B: ALL cells spiked into sample at 0.25% frequency (1/ 400 cells) ALL cells Amir et al., Nature Biotechnol. ePub 19 May 2013 Same data, different dimensionality reduction algorithms Amir et al., Nature Biotechnol. ePub 19 May 2013 viSNE is still stochastic, but much more reproducible SPADE’s stochasticity causes difference between runs So why should I ever use SPADE? Clustering allows comparison between samples Amir et al., Nature Biotechnol. ePub 19 May 2013 The cytometrist’s toolbox Transform raw data Histogram Biaxial plot 3D plot Radar PCA Gemstone SPADE viSNE Wanderlust Box plot Heatmap FLAME SamSPECTRAL Reduce dimensionality Summarize statistics Identify clusters FlowClust FlowMerge FLOCK Goal: Characterize the GCSF-responsive subpopulation G-CSF pSTAT3 pSTAT5 pSTAT5 Who are these cells? Are they the same in every patient? pSTAT3 Surface marker profiling of G-CSF responders in AML Differential gene expression pSTAT5 Nonresponders CD14 CD32 CD36 CD86 CD93 CD11a CD1d HLA-DR pSTAT3 G-CSF pSTAT5 pSTAT5 Basal + markers from literature Responders CD46 CD69 CD117 CD133 CD155 CD321 LSCs CD13 CD15 CD33 CD34 CD38 CD44 CD45 CD47 CD64 CD114 CD123 CD11b CXCR4 TIM3 Screen pediatric AML samples (18 diagnosis, 3 relapse, 7 healthy) Gate G-CSF responders Identify enriched markers CD46 pSTAT3 pSTAT3 with Faye Hsu, Yury Goltsev pSTAT5 Leukemic GCSF responders have distinct surface profiles Universal markers pSTAT3 Healthy Growth factor receptors CD34 LSC markers with Rob Bruggner & Sean Bendall The cytometrist’s toolbox Transform raw data Histogram Biaxial plot 3D plot Radar PCA Gemstone SPADE viSNE Wanderlust Box plot Heatmap FLAME SamSPECTRAL Reduce dimensionality Summarize statistics Identify clusters FlowClust FlowMerge FLOCK Human B cell development is less understood than in the mouse Surface Markers Intracellular Markers IgH Genes CD34 CD38 CD10 IL7R CD24 CD19 CD179a Rag TdT DJH VDJH Adapted – Cobaleda, BioEssays 2009 The predicted trajectory ‘rediscovers’ human B cell development TDT CD24 Maturity Trajectory “Wanderlust” algorithm: El-ad Amir, Dana Pe’er (Columbia) 3 4 2 5 TdT CD38 Following the predicted B cell trajectory 1 CD34 CD24 • Fluorescent panel design Sorting qPCR • VDJ rearrangement confirms the trajectory: (unrearranged) 1 2 3 4 5 (rearranged) Sean Bendall, Kara Davis New insights and resolution in human B cell development I Surface Markers Cytoplasmic Markers IgH Genes CD34 CD38 CD10 IL7R IIa IIb III IV Va Vb ✓ CD24 ✓ CD19 Ki67 CD179b CD179a Rag TdT pSTAT5 DJH VDJH Sean Bendall, Kara Davis Summary Transform raw data Histogram Biaxial plot 3D plot Radar PCA Gemstone SPADE viSNE Wanderlust Box plot Heatmap FLAME SamSPECTRAL Reduce dimensionality Summarize statistics Identify clusters FlowClust FlowMerge FLOCK Prof. Garry P. Nolan Sean Bendall Kara Davis Harris Fienberg Astraea Jager Rob Bruggner Rachel Finck Bernd Bodenmiller ( U. Zurich) Eli Zunder Greg Behbehani Wendy Fantl and the entire Nolan lab stanford.edu/group/nolan Columbia El-Ad Amir Jacob Levine Dana Pe’er St. Jude Children’s Amanda Gedman Ina Radtke James Downing Mount Sinai Michael Linderman Stanford Peng Qiu ( MD Anderson) Sylvia Plevritis