Metastatic breast cancer stem cells

Christophe GINESTIER, PhD

Centre de Recherche en Cancérologie de Marseille Laboratoire d’Oncologie Moléculaire Breast Stem Cell Group U1068/Inserm/Institut Paoli-Calmettes Marseille

Normal breast

epithelium Preneoplasia Invasive

Carcinoma Metastasis Carcinoma

in situ

Different step of the breast carcinogenesis

Initiation of carcinogenesis

Screening and Prevention

Diagnostic and

Treatment

“BRCA1 regulates human mammary stem/progenitor cell fate” (Liu, Ginestier et al. PNAS, 2008)

ALDH

BAAA

DEAB

ALDEFLUOR + cells

With DEAB Without DEAB

6%

With DEAB Without DEAB

R1 R2 R1 R2

ALDEFLUOR- 50,000 cells 5000 cells 500 cells

ALDEFLUOR+ 50,000 cells 5000 cells 500 cells

ALDEFLUOR + 500 cells

ALDEFLUOR – 50,000 cells

ALDEFLUOR- ALDEFLUOR+

ALDEFLUOR+ population and tumorigenicity

5% to 10% of ALDEFLUOR + population in Breast tumors

Ginestier et al. Cell stem cell, 2007

ALDEFLUOR+ population and tumorigenicity

ALDEFLUOR + ALDEFLUOR -

ALDEFLUOR+ population regenerates heterogeneity of the initial tumor

Ginestier et al. Cell stem cell, 2007

Cancer stem cells and metastasis

matrigel

DMEM with 10% human serum

Cell invasion assay

ALDEFLUOR-positive or

ALDEFLUOR-negative

0

1000

2000

3000

4000

5000

6000

7000

8000

SUM-149 HCC1954 SUM-159 MDAMB 453 MDAMB 436 BRCAMZ 01Num

ber o

f tot

al c

ells

inva

ded/

10,

000

cells

pla

ted ALDEFLUOR-positive ALDEFLUOR-negative

Cell invasion assay

Intracardiac injections

R2 R1

Lentivirus expressing Luciferase

0

25

50

75

100

1 3 5 7 9 11 13 15

Weeks after injection

100,000 ALDELFUOR-positive

100,00 ALDEFLUOR-negative

100,000 Unseparated

Weeks after injections

Nor

mal

ized

pho

ton

flux

ALDEFLUOR+ (100k)

Unsorted (100K)

ALDEFLUOR- (100K)

0

25

50

75

100

1 3 5 7 9 11 13 15

Weeks after injection

50,000 ALDELFUOR-positive

50,00 ALDEFLUOR-negative

50,000 Unseparated

Nor

mal

ized

pho

ton

flux

Intracardiac injections

Invasive Carcinoma Metastasis

Anti-CSC therapy

Conventional therapy

Breast Cancer Stem Cell Gene Expression Signature

184a1 HCC1954 SUM149 SUM159 MDAMB453 MDAMB231 BrCaMZ01 SKBR7

184a1 HCC1954 SUM149 SUM159 MDAMB453 MDAMB231 BrCaMZ01 SKBR7

8 Breast cell lines

184a

1 H

CC

1954

SU

M14

9 SU

M15

9 M

DAM

B45

3

MD

AMB

231

BrC

a-M

Z-01

B

rCa-

MZ-

01

SUM

149

SKB

R7

SKB

R7

184a

1 SU

M15

9 H

CC

1954

M

DAM

B45

3 M

DAM

B23

1

413 genes breast cancer stem cell signature

33 genes overexpressed in the ALDEFLUOR-positive populations 380 genes overexpressed in the ALDEFLUOR-negative populations

NOTCH2 NFYA TPRXL RBM15 PCNX

FBXO21 CAMK2B ADRBK1 UGCCL1

CXCR1 CD300LB GIPC3 TAS2R14

RAD51 ARID1B

ZFP41 FAM49B PSORS1C2

Role in stem cell biology

Cell Signaling

Membrane protein

DNA repair/ Chromatin remodeling

Unknown

Genes up-regulated in ALDEFLUOR-positive populations

184a

1 H

CC

1954

SU

M14

9 SU

M15

9 M

DAM

B45

3

MD

AMB

231

BrC

a-M

Z-01

B

rCa-

MZ-

01

SUM

149

SKB

R7

SKB

R7

184a

1 SU

M15

9 H

CC

1954

M

DAM

B45

3 M

DAM

B23

1

MRPL41 PDCD10 PDCD5

ATP5S NDUFA2 ATP5J2 IMMP1L CCNL1 PRPF39 LSM3 SFRS7 PRPF4B

NACA

ALG5 HBLD2 GART DC2 COMMD6 UBL5

Cell Signaling

Apoptosis

Differentiation

Oxidative phosphorylation

RNA splicing

Genes up-regulated in ALDEFLUOR-negative

populations

Breast Cancer Stem Cell Gene Expression Signature

CXCR1

Charafe-Jauffret et al., Cancer Research, 2009

CXCR1/IL8RA

CXCR1 is a receptor for Interleukin-8 involves in the recruitment of leukocytes to sites of inflammation

CXCR1 is increased in variety of cancers including breast cancer

CXCR1/IL-8 promotes: Tumor cells motility/metastasis Tumor cells proliferation

Repertaxin a small molecule inhibitor of IL8 signaling

Repertaxin CXCR1/IL8RA

CXCR2/IL8RB

Ginestier et al. JCI, 2010 Ginestier et al. JCI, 2010

0

1

2

3

4

5

6

7

8

9

-40 -30 -20 -10 0 10 20 30

98 7 6 5 4 3 2 1 0 -40 -30 -20 -10 0 10 20 30

Days after injection

Tum

or s

ize

(mm

)

Control Repertaxin Docetaxel Repertaxin/Docetaxel

0

4

8

12

16

20

Control

Repertaxin

Docetaxel

epertaxin/Docetaxel

20

16

12

8

4

0

ALD

EFLU

OR

-pos

itive

pop

ulat

ion

(%)

0

0.5

1

1.5

10,000 1,000 100Tu

mor

siz

e (m

m)

Control

Repertaxin

Docetaxel

Repertaxin/Docetaxel

10,000 1,000 100

15

10

5

0

Number of cells injected

Xenotransplantation

4mm

Repertaxin

28 days

Tumor size

% Cancer stem cells

Effect of Repertaxin treatment on cancer cells survival

HCC1954 MDA-MB-453 SUM159

IL8 - - - + + + - - - + + +

ALDEFLUOR + + + + + + - - - - - -

IL-8 induces invasion of breast CSC

Charafe-Jauffret et al., Cancer Research, 2009

Effect of CXCR1 blockade on metastasis formation

Repertaxin

Control

0

100

200

300

400

500

1 2 3 4 5

Repertaxin

Control

SUM159 Control Repertaxin

Nor

mal

ized

Pho

ton

Flux

Weeks after injection

0

100

200

300

400

500

1 2 3 4 5 6

HCC1954

Nor

mal

ized

Pho

ton

Flux

Weeks after injection

Control Repertaxin

Ginestier et al. JCI, 2010

Time from birth (weeks)

3 5 7 12

b-oestradiol

Humanization

Human fibroblasts

Passages

«Clearing»

Patient-derived xenograft (PDX)

+ Matrigel Tumoral growth monitoring

Primary Tumor

Dissociation

> 200 Primary tumor injections

> 50 established PDXs

Xenobank

CRCM 224 PT

CRCM 224X

HES

HES

ER

ER PR

PR ERBB2

ERBB2

PDX retains histological/molecular features of corresponding primary tumors

0 10 20 30 40

0.0

0.2

0.4

0.6

0.8

1.0

Months

met

asta

sis-

free

su

rviv

al

LogRank p = 0.00352Grp 1 : 0 N=35, 3-Year survival = 96%, 95%CI = [0.88-1]Grp 2 : 1 N=18, 3-Year survival = 66%, 95%CI = [0.47-0.92]

p=0.00352

PT engrafter

Met

asta

sis-

free

sur

viva

l

PT non-engrafter

Months after surgery

Prop

ortio

n of

tum

or o

utgr

owth

ALDH1+

ALDH1-

p=0.007e-5

0 100 200 300 400

0.0

0.2

0.4

0.6

0.8

1.0

Days

Pris

e (%

)

LogRank p = 1.00e-05 Grp 1 : 0 N=29, 5-Year survival = 100%, 95%CI = [1-1]

0 1

Days after PT implantation

1

0.8

0.6

0.4

0.2

0

ALDH1

ALDH1+

ALDH1-

0 100 200 300 400 0 100 200 300 400

0.0

0.2

0.4

0.6

0.8

1.0

Days

Pris

e (%

)

GRD

123

Prop

ortio

n of

tum

or o

utgr

owth

Days after PT implantation

SBR Grade

I II

III p=0.0005

1

0.8

0.6

0.4

0.2

0

0 100 200 300 400

Variable Hazard ratio (95% CI) p-value

ER 0.78 [0.27-2.31] 0,66

PR 0.67 [0.23-1.92] 0,45

SBR Grade 2.94 [1.02-8.44] 4,60E-02 KI67 1.32 [0.46-3.82] 0,61

ALDH1 3.61 [1.12-11.65] 3,20E-02

Charafe-Jauffret et al., Submitted

25% engrafment rate

GFP +

Lentiviral infection

Mammary fat pad implantation

Xenograft removal

Xenograft removal Mammary fat pad implantation

Xenograft removal

Bioluminescent imaging

PDX model for metastasis study

GFP+ Cell sorting

CV

Fo Ce

Po

Ra Re

Ut

TD

Co Th poumons 11%

rein 0,6%

rate 0,2% uterus

8%

tube digestif

14% ganglion

44%

pancréas 22%

CRCM389X

CRCM318X

foie 11%

uterus 4%

cerveau 2%

tube digestif

11% os 72%

Ce

Fo

Ut Po

Pa

Re

Ra

TD

Co

TD Fo Co

Ra

Re

Ut

Ce

Po

Pa Gg

CRCM341X

Metastatic patterns

Bone

Liver Brain Uterus

Digestive tract

Lymph node

Pancreas Lung

Uterus

Digestive tract

poumons 100% Lung

0

25

50

75

100

Bone

Genita

l trac

t

GI trac

tLiv

erLu

ngBrai

nKidn

ey

Spleen

Heart

Lymph

Nod

e

Pancre

as

Adrena

l Glan

d

0

25

50

75

100

Bone

Genita

l trac

t

GI trac

tLiv

erLu

ngBr

ain

Kidn

ey

Splee

nHea

rt

Lymph

Nod

e

Panc

reas

Adrena

l Glan

d

0

25

50

75

100

Bone

Genita

l trac

t

GI tract

Liver

Lung

Brain

Kidney

Spleen

Heart

Lymph N

ode

Pancre

as

Adrena

l Glan

d

0

25

50

75

100

Bone

Genital tr

act

GI tract

Liver

Lung

BrainKidn

ey

SpleenHea

rt

Lymph

Node

Pancreas

Adrenal G

land

0

25

50

75

100

Bone

Genita

l trac

t

GI tract

Liver

Lung

Brain

Kidney

Spleen

Heart

Lymph

Nod

e

Pancre

as

Adrena

l Glan

d

0

25

50

75

100

Bone

Genita

l trac

t

GI trac

tLiv

erLu

ngBr

ain

Kidn

ey

Splee

nHea

rt

Lymph

Nod

e

Panc

reas

Adre

nal G

land

* * * * *

* * *

* *

*

*

*

**

T412 (5 mice) Lung Mets-enriched

T434 (5 mice, 2 to come) Lung Mets-reduced

T389 (6 mice) Lung Mets-enriched Brain Mets-reduced

T318 (3 mice, 1 to come) Lymph node Mets-enriched

T341 (7 mice) Liver Mets-enriched Brain Mets-enriched

T226 (3 mice, 2 to come) Liver Mets-enriched Lung Mets-enriched

Max WICHA Gabriela DONTU Suling LIU Hasan KORKAYA Marc DIEBEL

Daniel BIRNBAUM Emmanuelle CHARAFE-JAUFFRET Marion SALVADOR Simon LAUNAY Rita EL HELOU Julien WICINSKI Olivier CABAUD Emmanuelle JOSSELIN

University of Michigan

J-F Saunière

François BERTUCCI Nathalie CERVERA Pascal FINETTI

Mofitt Cancer center

Said Sebti

IRIS

Stephane Depil Helene Lelièvre Laurence Kraus-Berthier