Liu S, Ginestier C, Ou SJ, Clouthier SG,

Patel SH, Monville F, Korkaya H, Heath A,

Dutcher J, Kleer CG, Jung Y, Dontu G, Taichman R, Wicha MS. 

Cytokine networks mediate interaction of mesenchymal stem

cells and breast cancer stem cells

Department of Internal Medicine-Hematology/OncologyComprehensive Cancer Center

University of MichiganAnn Arbor, MI

DIF

FER

EN

TIA

TIO

N

Early Progenitor

Late Progenitor

Luminal cells

Myoepithelial cells

Alveolar cells

Stem cell

Quiescent pool of stem cell

“stem cell” Capacity to self-renew Capacity to differentiate into multiple cell lineages

DIF

FER

EN

TIA

TIO

NEarly Progenitor

Late Progenitor

Luminal cells

Myoepithelial cells

Alveolar cells

Stem cell

Quiescent pool of stem cell

Cancer stem cell

Cancer stem cell

ALDH

• Aldehyde dehydrogenase metabolizes aldehydes, main substrate retinoic acid

• Role in differentiation: Inhibition of aldehyde dehydrogenase induces

the expansion of human hematopoietic stem cells

• Marker of stem/progenitor cells in neural and hematopoietic systems

• Marker of cancer stem cells in brain tumors, leukemias and multiple myeloma

ALDH

BAAA

DEAB

ALDEFLUOR + cells in Normal breast epithelium

With DEAB Without DEAB

~ 5% of ALDEFLUOR positive cells in breast cancer cell line SUM159

4.25%

ALDEFLUOR+ population and tumorigenicity

ALDEFLUOR +ALDEFLUOR -

ALDEFLUOR+ population regenerates the phenotypicheterogeneity of the initial tumor

Ginestier et al. Cell stem cell, 2007

Bone Marrow Mesenchymal Stem Cell• The Microenvironment (“Stem cell niche”) contains fibroblasts, endothelial cells, inflammatory cells and mesenchymal stem cells recruited from bone marrow.• The Microenvironment plays an important role in normal mammary development as well as tumor growth and metastasis. > The growth of human mammary structures in NOD/SCID mice is greatly facilitated by the addition of irradiated human mammary fibroblasts (Liu 2006). > Tumor associated fibroblasts (TAFs) enhance carcinogenesis and tumor growth (Bissell 2001). > Bone marrow mesenchymal stem cells were demonstrated in gastric carcinoma and gliomas (Menon 2007). > Bone marrow derived mesenchymal stem cells are recruited to the tumors where they constitute an important component of the tumor microenvironment in mouse model of breast cancer (Karnoub 2007). • Cytokines from microenvironment regulate cancer stem cells. > IL4 plays a role in regulating colon cancer stem cells (Francipane 2008). > IL6 has been recently reported to increase mammary stem cell number as determined by mammosphere formation (Sanson 2008).

Mesenchymal Stem Cells (MSC) increase

breast cancer stem/progenitor cells in vitro

02468

10121416

Alone Co-culture Transwell-culture C.M. from MSC C.M. from Co-culture C.M. from Transwell-culture

co-culture C.M. from MSC

C.M. from Co-culture

C.M. from transwell-

culture

ALD

EFLO

UR

-pos

itive

SU

M15

9 ce

lls (%

)

Alone Transwell-culture

A

B

0

1

2

3

4

5

6

Alone Co-culture Transwell-culture

ALD

EFLO

UR

-pos

itive

MSC

(%)

ALD

EFLO

UR

-pos

itive

MSC

(%

)

Osteogenic induction

Adipogenic induction

Control

ALDH+

ALDH-

B

A

Co-culture

02468

1012141618

SUM159-DsRedalone

SUM159-DsRedmix-cultured w .HMSC-bm ALDH+

SUM159-DsRedmix-cultured w .HMSC-bm ALDH-

ALD

EFLO

UR

-pos

itive

SU

M15

9 ce

lls (%

)

SUM159-DsRedalone

SUM159-DsRedCo-cultured w.

MSC ALDH+

SUM159-DsRedCo-cultured w.

MSC ALDH-

ALD

EFLO

UR

-po

sitiv

eSU

M15

9 ce

lls (%

)

Hierarchy of Mesenchymal Stem Cells

Fold increase(SUM159 from co-culture

vs SUM159 alone)Cytokines

CXCL5 (ENA-78)

CXCL6 (GCP-2)

CXCL1 (GRO-)

IL6

IL8

2015

2.9

17.2

6.2

2140

CXCL7 (NAP-2) NEG

CytokinesFold increase

(MSC from co-culture vs MSC alone)

CXCL5 (ENA-78)

CXCL6 (GCP-2)

CXCL1 (GRO-)

IL6

IL8

53.9

12.8

10.2

2.8

21.5

CXCL7 (NAP-2) 6

CytokinesFold increase

(MSC from co-culture vs MSC alone)

CXCL5 (ENA-78)

CXCL6 (GCP-2)

CXCL1 (GRO-)

IL6

IL8

14.5

8.6

5.6

2.6

3.4

CXCL7 (NAP-2) NA

Fold increase(SUM159 from co-culture

vs SUM159 alone)Cytokines

CXCL5 (ENA-78)

CXCL6 (GCP-2)

CXCL1 (GRO-)

IL6

IL8

42

9

3

79

CXCL7 (NAP-2) NA

2

0

25

50

75

100

125

150

175

200

MSC alone SUM159 alone SUM159 alone +MSC alone (1:1)

Co-culture

Norm

aliz

ed p

rote

in le

vel (

%)

CXCL1 CXCL5 CXCL6CXCL7 IL8 IL6

MSC alone(A)

SUM159 alone(B)

A+B (1+1)

Affy microarray RT-PCR Antibody array

Cytokines upregulated in co-/transwell-culture

of MSC and SUM159

0

4

8

12

16

SUM159alone

Control 100 ug/mlanti-CXCL5

50 ug/mlanti-CXCL6

20 ug/mlanti-CXCL7

100 ug/mlanti-IL6

10 ug/mlanti-IL8

ALD

EFLO

UR

-pos

itive

cel

ls (%

)A

LDEF

LOU

R-p

ositi

veSU

M15

9 ce

lls (%

)

SUM159 alone

N.T. 100 ug/mlanti-CXCL5

50 ug/mlanti-CXCL6

20 ug/mlanti-CXCL7

100 ug/mlanti-IL6

10 ug/mlanti-IL8

Co-culture

0

2

4

6

8

10

12

14

Co-culture

Control 2 ug/mlCXCL1

1 ug/mlCXCL5

10 ng/mlCXCL6

10 ng/mlCXCL7

100 ng/mlIL6

100 ng/mlIL8

ALD

EFLO

UR

-pos

itive

cel

ls (%

)

ALD

EFLO

UR

-pos

itive

SU

M15

9 ce

lls (%

)

Co-culture

N.T. 2 ug/mlCXCL1

1 ug/mlCXCL5

10 ng/mlCXCL6

10 ng/mlCXCL7

100 ng/mlIL8

100 ng/mlIL6

SUM159

A

B

Effects of Cytokines on breast cancer stem/progenitor cell

population

0

25

50

75

100150

175

200

225

SUM159 alone Co-culture SUM159 treated w.10ng/ml CXCL7

Co-culture treated w.20ug/ml anti-CXCL7

Nor

mal

ized

pro

tein

leve

l (%

)

CXCL1 CXCL5CXCL6 CXCL7IL8 IL60 0 XBreak at

0

2

4

6

8

10

12

Control CXCL1 CXCL5 CXCL6 IL6 IL8

MSC

Nor

mal

ized

CXC

L7 p

rote

in le

vel

(%)

Nor

mal

ized

CXC

L7 p

rote

in le

vel

(%)

N.T. 2 ug/mlCXCL1

1 ug/mlCXCL5

10 ng/mlCXCL6

100 ng/mlIL8

100 ng/mlIL6

MSC

MS

C w

/ CXC

L1 (+

SU

M15

9 al

one)

MS

C+S

UM

159

co-c

ultu

red

MS

C w

/ IL6

(+ S

UM

159

alon

e)

MS

C w

/ CXC

L7 (+

SU

M15

9 al

one)

MS

C w

/ IL8

(+ S

UM

159

alon

e)

MS

C w

/ CXC

L6 (+

SU

M15

9 al

one)

MS

C a

lone

+ S

UM

159

alon

e

MS

C w

/ CXC

L5 (+

SU

M15

9 al

one)

A C

B

CXCL7 and IL6, two crucial regulators

MS

C+S

UM

159

co-c

ultu

red

MS

C w

/ IL6

(+ S

UM

159

alon

e)

MS

C w

/ CXC

L7 (+

SU

M15

9 al

one)

SUM159

Cancer Stem Cell

Bulk Tumor CellsMesenchymal

Cells

CXCL7

CXCR1

Stem cell self renewal

IL-6

IL-6Rgp130

CXCL5CXCL1

CXCL6 IL-8 IL-6

ALDH-

ALDH+

IL-6Rgp130

ALDH-

ALDH-

CXCR2

Hypothetic model of cytokine networks

between MSC and breast cancer cells

A

SU

M15

9 w

/ CXC

L7 (+

MS

C a

lone

)

MS

C+S

UM

159

mix

ed w

/ ant

i-IL6

+CXC

L7

MS

C+S

UM

159

co-c

ultu

re

MS

C+S

UM

159

co-c

ultu

re w

/ ant

i-CXC

L7

MS

C a

lone

+ S

UM

159

alon

e

MS

C+S

UM

159

co-c

ultu

re w

/ ant

i-IL6

B

Cancer Stem Cell

Bulk Tumor CellsMesenchymal

Cells

CXCL7

CXCR1

Stem cell self renewal

IL-6

IL-6Rgp130

CXCL5CXCL1

CXCL6 IL-8 IL-6

ALDH-

ALDH+

IL-6Rgp130

ALDH-

ALDH-

CXCR2X

Anti-CXCL7

MS

C+S

UM

159

co-c

ultu

re w

/ ant

i-CXC

L7

MS

C a

lone

+ S

UM

159

alon

e

A

SU

M15

9 w

/ CXC

L7 (+

MS

C a

lone

)

MS

C+S

UM

159

mix

ed w

/ ant

i-IL6

+CXC

L7

MS

C+S

UM

159

co-c

ultu

re

MS

C+S

UM

159

co-c

ultu

re w

/ ant

i-CXC

L7

MS

C a

lone

+ S

UM

159

alon

e

MS

C+S

UM

159

co-c

ultu

re w

/ ant

i-IL6

B

Cancer Stem Cell

Bulk Tumor CellsMesenchymal

Cells

CXCL7

CXCR1

Stem cell self renewal

IL-6

IL-6Rgp130

CXCL5CXCL1

CXCL6 IL-8 IL-6

ALDH-

ALDH+

IL-6Rgp130

ALDH-

ALDH-

CXCR2

X

Anti-IL6

MS

C+S

UM

159

co-c

ultu

re W

/ ant

i-CX

CL7

MS

C a

lone

+ S

UM

159

alon

e

MS

C+S

UM

159

co-c

ultu

re w

/ ant

i-IL6

A

SU

M15

9 w

/ CXC

L7 (+

MS

C a

lone

)

MS

C+S

UM

159

mix

ed w

/ ant

i-IL6

+CXC

L7

MS

C+S

UM

159

co-c

ultu

re

MS

C+S

UM

159

co-c

ultu

re w

/ ant

i-CXC

L7

MS

C a

lone

+ S

UM

159

alon

e

MS

C+S

UM

159

co-c

ultu

re w

/ ant

i-IL6

B

Cancer Stem Cell

Bulk Tumor CellsMesenchymal

Cells

CXCL7

CXCR1

Stem cell self renewal

IL-6

IL-6Rgp130

CXCL5CXCL1

CXCL6 IL-8 IL-6

ALDH-

ALDH+

IL-6Rgp130

ALDH-

ALDH-

CXCR2

X

Anti-IL6 SU

M15

9 W

/ CX

CL7

(+M

SC

alo

ne)

MS

C+S

UM

159

co-c

ultu

re w

/ ant

i-IL6

+CXC

L7

MS

C+S

UM

159

co-c

ultu

re

CXCL7

CXCL5CXCL1

CXCL6 IL-8 IL-6

Tum

or s

ize

(cm

)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 7 14 21 28 35 42 49 56 63 70 77 84

Days after injection

Tum

or s

ize

(cm

)

SUM159-DsRed ALDH+ alone (1k)

SUM159-DsRed ALDH+ + HMSC-bm Total (1k)

SUM159-DsRed ALDH+ + HMSC-bm ALDH+ (1k)

SUM159-DsRed ALDH+ + HMSC-bm ALDH- (1k)

HMSC-bm Total alone (1k)

HMSC-bm ALDH+ alone (1k)

HMSC-bm ALDH- alone (1k)

Days after injection

SUM159 alone (1k)

SUM159 + HMSC-bm Total (1k)

SUM159 + HMSC-bm ALDH+ (1k)

SUM159 + HMSC-bm ALDH- (1k)

HMSC-bm Total alone(1k)

HMSC-bm ALDH+ alone (1k)

HMSC-bm ALDH- alone (1k)

C

A

SUM159-DsRed alone

SUM159-DsRed + MSC-GFP

DsRed (SUM159) ALDH1GFP (MSC)DAPI Merge

0

1

2

3

4

5

SUM159alone

SUM159 +MSC Total

SUM159 +MSC ALDH-

SUM159 +MSC ALDH+

ALD

EFLO

UR

-pos

itive

SU

M15

9 ce

lls (%

)A

LDEF

LOU

R-

posi

tive

SUM

159

cells

(%)

BMSC stimulate breast tumor growth in NOD/SCID mice

DsRed (MSC) ALDH1DAPI Merge

With

out M

SCIn

tra-

tibia

inje

ctio

nW

ith M

SCIn

tra-

tibia

inje

ctio

n

DsRed (MSC)

A

B

700

600

500

400

300

200

100

x103

3.957x1061.812x106 3.525x106 C

MSCs traffic from Bone marrow to breast primary tumor sites

Conclusions• MSCs increase breast cancer stem/progenitor cells (CSCs) in vitro.• MSCs stimulate the growth of breast xenografts by increasing their stem cell components. • MSC stimulate CSCs through a positive feedback loop: > IL6 produced by the tumor cells stimuates MSC to produce CXCL7. > MSC-derived CXCL7 induces the synthesis of a family of cytokines by both MSC and tumor cells which in turn regulates the cancer stem/progenitor cell population.

These studies suggest an important mechanistic link between inflammation and carcinogenesis occurs at the level of cytokine mediated stem cell regulation, and suggest that blocking the interaction of MSC and CSC in the stem cell “niche” may provide a novel approach to target the cancer stem cells. Since these cells drive tumorigenesis and metastasis, this may lead to improved outcomes for women with advanced breast cancer.

Acknowledgements• Max S. Wicha’s Lab Christophe Ginestier Hasan Korkaya Shawn Clouthier Gabriela Dontu

Julie Dutcher Stacey Swancutt Jenny Ou Wasim Nasir Dang Vu Phan Khaled Hassan Qin Zen Amanda Paulson Maduri Kakarala • Dan Hayes’s Lab Marty Brown

• Kenneth Pienta’s Lab Chris Neeley Natalie McGregor

• Celina Kleer

• UM Vector CoreThomas LaniganTonya Kopas

• UM Flow Cytometry CoreDavid AdamsAnn Marie Des LauriersMartin J. White

Karen A. Peterson Michael Pihalja

• NIH grants and DOD grant (M Wicha):CA101860 5 P 30 CA46592 BC030214

Taubman Institute

• Susan G. Komen Fellowship (S Liu)