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

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

G0/1

S

G2

• 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

Cell cycle analysis by mass cytometry

Mass cytometry DNA staining

G0

G1

G2

S

Hoechst Ir Intercalator

(pentamethylcyclopentadienyl)-Ir(III)-dipyridophenazine

IdU incorporates rapidly into S-phase cells

56.5% 47.6%

Ir Intercalator

Uri

din

e

FluorescentCytometry

Mass Cytometry

Hoechst

Uri

din

e

Intercalator Ir193IdU

I127

No IdU 5 min 10 min 15 min

30 min 60 min 120 min

0.3% 21.9% 20.6% 24.2%

24.3% 26.6% 28.9%

IdU incorporates rapidly into S-phase cells

-Andrew Hughes, Gene Ther Mol Biol., 2006; 10:41

p-histone H3

Additional markers allow for complete cell cycle state assignment

p-histone H3


p-R

b (

S807/S

811)

Uridine

G0

p-histone H3


p-histone H3

Uridine

Cyclin

B1

G0/G1

S

G2

p-histone H3


p-histone H3

Uridinep-H

isto

ne H

3 (

S28)

M

B

A

C

Cyclin B1 Dy164

IdU

I127

p-Rb Ho165

IdU

I127

p-Histone H3 Er168

IdU

I127

human T cells

G0

M

S

G2/MG0/G1

U937 cells HL60 cells NALM6 cells

Cell cycle assessment is robust and consistent across multiple cell types

U937 cells HL60 cellshuman T cells

B

A

CCyclin B1 Dy164

IdU

I127

p-Rb Ho165

IdU

I127

p-Histone H3 Er168

IdU

I127

G0

M

S

G2/MG0/G1

NALM6 cells

G0 G1 S G2 M0

10

20

30

40

50

60

70

Fluorescence Cytometry

Mass Cytome-try

Cell cycle assessment is robust and consistent across multiple cell types

Pyro

nin

Y

Hoechst

Hoechst

p-R

b A

lexa 6

47

Pyro

nin

Y

IdU FITC

IdU FITC

p-R

b A

lexa 6

47

p-R

b H

o1

65

IdU I127

p-R

b H

o1

65

Intercalator Ir193

A

B

C

D24.4%

75.6%

23.8%

76.3%

72.3%

27.6%

Phosphorylated Rb (S807/S811) discriminates G0 and G1 phase cells

The same cell cycle markers can be used for fluorescence cytometry

IdU

Cyclin B1pRb

Mass Cytometry

S

G0/G1 G2/M

G0

Hoechst

Cyclin B1pRb

Fluorescence Cytometry

IdU

Pyro

nin

Y

Hoechst

S

S

G0/G1

G0/G1

G2/M

G2/M

G0

G0

IdU

0h

28h

48h

Mass cytometry cell cycle analysis is equivalent to fluorescent methodologies

Fluorescent cytometry Mass cytometry

0h

28h

48h

G0 G1 S G2 M0

20

40

60

80

100

Fluorescent Cytometry

Mass Cytome-try

G0 G1 S G2 M0

20

40

60

80

100


Mass Cy-tometry

G0 G1 S G2 M0

20

40

60

80

100


Mass cytome-try

Mass cytometry cell cycle analysis is equivalent to fluorescent methodologies

• 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

Cell cycle analysis by mass cytometry

Panel for analysis of cell cycle in human marrow

Biaxial plots are not a scalable solution

Parameters:

481432

Plots: 62891496

Sean Bendall, Erin Simonds. Science, May 2011

SPADE: Spanning-tree Progression Analysis of Density-normalized Events – Peng Qiu

1. Determine Tree Structure

2. Overlay regions with surface marker expression levels

SPADE clustering of normal bone marrow mirrors immunophenotypic differentiation.

CD45

Naïve CD4 T

CD4 T

NK

CD8 T

Naïve CD8 T

NKT


CD3

CD45

Early

Mature Myeloid

Meta-myelocytes

Myelocytes

LatePromyelocytes

Myeloblasts

CMP


CD15

CD45

Macrophages

Mature Monocytes

EarlyMonocytes

PC-DCs

Pro-monocytes

Monoblasts


CD14

CD45

Mature B cells

ImmatureB cells

Pre-BII

Plasma

Pre-BI

Pro-B


CD20

CD45

Erythroblasts

NucleatedRBCs

Pro-erythroblasts

MEP


CD235

CD45

HSC

Vasc. Progen?MPP

Pre-BI

Pro-B

Pro-erythroblasts

MonoblastsMyeloblasts

CMP

MEP

GMP


CD34

CD45

HSC

Macrophages

Mature Monocytes

EarlyMonocytes

PC-DCs

Pro-monocytes

Naïve CD4 T

CD4 T

NK

CD8 T

Naïve CD8 T

Vasc. Progen?MPP

Mature B cells

ImmatureB cells

Pre-BII

Plasma

Pre-BI

Pro-B

Erythroblasts

NucleatedRBCs

Early

Mature Myeloid

Meta-myelocytes

Myelocytes

LatePromyelocytes

Pro-erythroblasts

MonoblastsMyeloblasts

CMP

MEP

GMPNKT


CD45

B cell proliferation is concentrated in pre-BII population

CD34

CD10

CD19

CD20

Pro-B

Pre-BI

Pre-BII

Mature B

Normal Human Bone MarrowColored for CD45

G1

S

G2

= 200 Cells

= 67%

Erythroid cell proliferation is concentrated in erythroblast population


G1 S G2

CD34 CD71 CD235

MEP

Pro-erythro-blast

Erythro-blast

NucleatedRBC

= 200 Cells

= 67%

Myelocyte proliferation peaks at early promyelocyte stage


CD11b

CD15

CD16

EarlyPromyelo-cytes

Metamyelocyte

Mature myelocyte

LatePromyelo-cytes

Myelocyte

= 3500 Cells

= 67%

S

G2

G1

SPADE analysis allows for identification of distinct AML immunophenotypes

NK Cell

T Cell

Mature Myeloid

Promyelocyte /Myelocyte

HSC /Early Progenitor

B Cell

?Pro-B /Pre-B

Mature Monocytic

Promonocyte

Normal human bone marrowClustered alongside AML samples

Colored for CD45

Myleo/mono-blast

Cell cycle distribution varies across the immunophenotypic subsets within each AML sample

AML9AML5

CD34

S

= 20%

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

• Hematology– Bruno Medeiros– Peter Greenberg– Beverly Mitchell

• Aparna Raval

• Cytobank– Nikesh Kotecha

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

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

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