tools and approaches for mass cytometry · the cytometrist’s toolbox radar flame pca heatmap...

Erin Simonds, PhD W. A. Weiss Lab, UCSF

May 23, 2013

Tools and approaches for mass cytometry

data analysis

© 2011 J. Simonds

Biaxial plots are not a scalable solution

Parameters: 4 8 14 32

Plots: 6 28 91 496 2010 2000 1989 1983

How can we explore in 30+ dimensions?

The cytometrist’s toolbox

Radar

FLAME

PCA

Heatmap

Biaxial plot Histogram

FLOCK SamSPECTRAL FlowMerge FlowClust

Gemstone SPADE viSNE

Transform raw data

3D plot

Reduce dimensionality

Summarize statistics

Identify clusters

Wanderlust

Box plot

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


Radar

FLAME

PCA

Heatmap




Transform raw data

3D plot



Identify clusters

Wanderlust

Box plot

SPADE: Spanning-tree Progression Analysis of Density-normalized Events

Qiu et al., Nature Biotechnology, Oct. 2011

Marker 1

Mar

ker 1

Marker 2

Marker 2

SPADE

CD45 CD3 CD4 CD8 CD34 CD19 CD20 CD33 CD11b CD123

SPADE projects bone marrow as a continuum of phenotypes

0 100 Intensity (%max)


pSTAT5

0 100 Intensity (%max)

Rediscovery of canonical signaling pathways

Basal IL-7


+ Dasatinib Abl / Src-family kinase inhibitor

Dasatinib differentially affects immune cell subsets


IL7 (pSTAT5)

PVO4 (pSTAT5)


Transform raw data



Identify clusters

Radar

FLAME

PCA

Heatmap




3D plot

Wanderlust

Box plot

viSNE preserves high-dimensional, non-linear relationships

Raw data (in 3D) viSNE map (in 2D)

z

y x

viSNE-1

viS

NE

-2

Amir et al., Nature Biotechnol. ePub 19 May 2013

viSNE maps healthy marrow as discrete populations


CD4+ T

CD11b-hi monocyte

CD11b-lo monocyte

NK

CD20+ B

CD8+ T

CD20- B


viSNE can help detect rare subpopulations

ALL cells

Barcode A: Healthy bone marrow cells Barcode B: ALL cells spiked into sample at 0.25% frequency (1/ 400 cells)


Same data, different dimensionality reduction algorithms

viSNE is still stochastic, but much more reproducible


SPADE’s stochasticity causes difference between runs

So why should I ever use SPADE? à Clustering allows comparison between samples


Transform raw data



Identify clusters

Radar

FLAME

PCA

Heatmap




3D plot

Wanderlust

Box plot

pSTA

T5

pSTAT3

Goal: Characterize the GCSF-responsive subpopulation

G-CSF

pSTAT3 pS

TAT5

Who are these cells?

Are they the same in every

patient?

Surface marker profiling of G-CSF responders in AML

with Faye Hsu, Yury Goltsev

pSTAT3

pSTA

T5

CD14 CD32 CD36 CD86 CD93 CD11a CD1d HLA-‐DR

CD46 CD69 CD117 CD133 CD155 CD321

CD13 CD15 CD33 CD34 CD38 CD44 CD45

Non- responders

+ markers from literature

CD47 CD64 CD114 CD123 CD11b CXCR4 TIM3

Responders

Differential gene expression

Screen pediatric AML samples (18 diagnosis, 3 relapse, 7 healthy)

LSCs

Gate G-CSF responders

Identify enriched markers

pSTAT3

pSTA

T5

Basal G-CSF

pSTAT3

pSTA

T5

CD46

Leukemic GCSF responders have distinct surface profiles

with Rob Bruggner & Sean Bendall

Growth factor

receptors

Universal markers

CD34

LSC markers

pSTAT3

pSTA

T5

Healthy


Transform raw data



Identify clusters

Radar

FLAME

PCA

Heatmap




3D plot

Wanderlust

Box plot

Human B cell development is less understood than in the mouse

CD34

CD10

CD24 IL7R

CD38

CD179a

Surface Markers

Intracellular Markers

Rag TdT

IgH Genes

CD19

Adapted – Cobaleda, BioEssays 2009

DJH

VDJH

Maturity Trajectory

The predicted trajectory ‘rediscovers’ human B cell development

CD34

TDT

“Wanderlust” algorithm: El-ad Amir, Dana Pe’er (Columbia)

CD24

CD34

CD

38

Following the predicted B cell trajectory

1

•  Fluorescent panel design à Sorting à qPCR

•  VDJ rearrangement confirms the trajectory: (unrearranged) 1 à 2 à 3 à 4 à 5 (rearranged)

Sean Bendall, Kara Davis

CD24

TdT

4

2 5

3

New insights and resolution in human B cell development

CD34

CD10

CD24 IL7R

CD38

CD179a

Surface Markers

Cytoplasmic Markers

Rag TdT

IgH Genes

CD19

DJH VDJH

I IIa IIb III IV Va Vb

✓

✓

CD179b Ki67

pSTAT5

Sean Bendall, Kara Davis

Summary

Radar

FLAME

PCA

Heatmap




Transform raw data

3D plot



Identify clusters

Wanderlust

Box plot

Sean Bendall Kara Davis Harris Fienberg Astraea Jager Rob Bruggner Rachel Finck

stanford.edu/group/nolan

Mount Sinai Michael Linderman

Columbia El-Ad Amir Jacob Levine Dana Pe’er

St. Jude Children’s Amanda Gedman Ina Radtke James Downing

Stanford Peng Qiu (à MD Anderson) Sylvia Plevritis

Prof. Garry P. Nolan Bernd Bodenmiller (à U. Zurich) Eli Zunder Greg Behbehani Wendy Fantl and the entire Nolan lab

tools and approaches for mass cytometry · the cytometrist’s toolbox radar flame pca heatmap...

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