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Liu S, Ginestier C, Ou SJ, Clouthier SG, Patel SH, Monville F, Korkaya H, Heath A,

Cytokine networks mediate interaction of mesenchymal stem cells and breast cancer stem cells. 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. .

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Liu S, Ginestier C, Ou SJ, Clouthier SG, Patel SH, Monville F, Korkaya H, Heath A,

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  1. Cytokine networks mediate interaction of mesenchymal stem cells and breast cancer stem cells 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.  Department of Internal Medicine-Hematology/OncologyComprehensive Cancer Center University of MichiganAnn Arbor, MI

  2. “stem cell” Self-renewal Stem cell Quiescent pool of stem cell Early Progenitor DIFFERENTIATION Late Progenitor Luminal cells Myoepithelial cells Alveolar cells • Capacity to self-renew • Capacity to differentiate into multiple cell lineages

  3. Self-renewal Self-renewal Self-renewal Self-renewal Stem cell Cancer stem cell Quiescent pool of stem cell Early Progenitor DIFFERENTIATION Late Progenitor Cancer stem cell Luminal cells Myoepithelial cells Alveolar cells

  4. 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

  5. ALDEFLUOR + cells in Normal breast epithelium With DEAB Without DEAB 4.25% ~ 5% of ALDEFLUOR positive cells in breast cancer cell line SUM159 BAAA ALDH DEAB

  6. ALDEFLUOR+ population and tumorigenicity ALDEFLUOR + ALDEFLUOR - ALDEFLUOR+ population regenerates the phenotypic heterogeneity of the initial tumor Ginestier et al. Cell stem cell, 2007

  7. 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).

  8. Mesenchymal Stem Cells (MSC) increase breast cancer stem/progenitor cells in vitro ALDEFLOUR-positive SUM159 cells (%) Transwell-culture C.M. from MSC co-culture Alone C.M. from Co-culture C.M. from transwell-culture ALDEFLOUR-positive MSC (%) A B

  9. ALDEFLOUR-positive SUM159 cells (%) SUM159-DsRed Co-cultured w. MSC ALDH- SUM159-DsRed alone SUM159-DsRed Co-cultured w. MSC ALDH+ Hierarchy of Mesenchymal Stem Cells Adipogenic induction Osteogenic induction Control A ALDH+ ALDH- B

  10. Fold increase (SUM159 from co-culture vs SUM159 alone) Fold increase (MSC from co-culture vs MSC alone) Cytokines Cytokines CXCL5 (ENA-78) CXCL5 (ENA-78) 3.4 79 CXCL6 (GCP-2) 14.5 CXCL6 (GCP-2) 42 CXCL1 (GRO-a) 5.6 CXCL1 (GRO-a) 9 CXCL7 (NAP-2) CXCL7 (NAP-2) NA NA IL6 IL6 2.6 3 2 IL8 8.6 IL8 Fold increase (SUM159 from co-culture vs SUM159 alone) Fold increase (MSC from co-culture vs MSC alone) Cytokines Cytokines CXCL5 (ENA-78) CXCL5 (ENA-78) 2140 21.5 CXCL6 (GCP-2) CXCL6 (GCP-2) 53.9 2015 CXCL1 (GRO-a) 17.2 10.2 CXCL1 (GRO-a) 6 CXCL7 (NAP-2) CXCL7 (NAP-2) NEG IL6 6.2 2.8 IL6 12.8 IL8 2.9 IL8 MSC alone (A) SUM159 alone (B) A+B (1+1) Cytokines upregulated in co-/transwell-culture of MSC and SUM159 Affy microarray RT-PCR Antibody array

  11. ALDEFLOUR-positive SUM159 cells (%) Co- culture N.T. 10 ng/ml CXCL6 10 ng/ml CXCL7 100 ng/ml IL8 2 ug/ml CXCL1 1 ug/ml CXCL5 100 ng/ml IL6 SUM159 ALDEFLOUR-positive SUM159 cells (%) SUM159 alone N.T. 20 ug/ml anti-CXCL7 100 ug/ml anti-IL6 10 ug/ml anti-IL8 100 ug/ml anti-CXCL5 50 ug/ml anti-CXCL6 Co-culture Effects of Cytokines on breast cancer stem/progenitor cell population A B

  12. CXCL7 and IL6, two crucial regulators Normalized CXCL7 protein level (%) C A MSC w/ CXCL1 (+ SUM159 alone) MSC+SUM159co-cultured MSC w/IL6 (+ SUM159 alone) MSC w/ CXCL7 (+ SUM159 alone) MSC w/ IL8 (+ SUM159 alone) MSC w/ CXCL6 (+ SUM159 alone) MSC alone + SUM159 alone MSC w/ CXCL5 (+ SUM159 alone) N.T. 10 ng/ml CXCL6 100 ng/ml IL8 2 ug/ml CXCL1 1 ug/ml CXCL5 100 ng/ml IL6 MSC B MSC+SUM159 co-cultured MSC w/ IL6 (+ SUM159 alone) MSC w/ CXCL7 (+ SUM159 alone) SUM159

  13. Hypothetic model of cytokine networks between MSC and breast cancer cells CXCL7 Bulk Tumor Cells Mesenchymal Cells CXCR2 ALDH+ IL-6R gp130 IL-6 CXCL1 CXCL5 ALDH- ALDH- ALDH- CXCL6 IL-8 IL-6 CXCR1 IL-6R gp130 Stem cell self renewal Cancer Stem Cell

  14. Anti-CXCL7 ALDH- ALDH- ALDH- CXCL1 CXCL5 CXCL6 IL-8 IL-6 A B X SUM159 w/ CXCL7 (+ MSC alone) MSC+SUM159 mixed w/ anti-IL6+CXCL7 MSC+SUM159 co-culture MSC+SUM159 co-culture w/ anti-CXCL7 MSC alone + SUM159 alone MSC+SUM159 co-culture w/ anti-IL6 CXCL7 Bulk Tumor Cells Mesenchymal Cells MSC+SUM159 co-culture w/ anti-CXCL7 MSC alone + SUM159 alone CXCR2 ALDH+ IL-6R gp130 IL-6 CXCR1 IL-6R gp130 Stem cell self renewal Cancer Stem Cell

  15. Anti-IL6 CXCL7 ALDH- ALDH- ALDH- CXCL1 CXCL5 CXCL6 IL-8 IL-6 A B SUM159 w/ CXCL7 (+ MSC alone) MSC+SUM159 mixed w/ anti-IL6+CXCL7 MSC+SUM159 co-culture MSC+SUM159 co-culture w/ anti-CXCL7 MSC alone + SUM159 alone MSC+SUM159 co-culture w/ anti-IL6 Bulk Tumor Cells MSC+SUM159 co-culture W/ anti-CXCL7 MSC alone + SUM159 alone MSC+SUM159 co-culture w/ anti-IL6 Mesenchymal Cells CXCR2 ALDH+ X IL-6R gp130 IL-6 CXCR1 IL-6R gp130 Stem cell self renewal Cancer Stem Cell

  16. Anti-IL6 CXCL7 CXCL7 ALDH- ALDH- ALDH- CXCL1 CXCL1 CXCL5 CXCL5 CXCL6 CXCL6 IL-8 IL-6 IL-8 IL-6 A B SUM159 w/ CXCL7 (+ MSC alone) MSC+SUM159 mixed w/ anti-IL6+CXCL7 MSC+SUM159 co-culture MSC+SUM159 co-culture w/ anti-CXCL7 MSC alone + SUM159 alone MSC+SUM159 co-culture w/ anti-IL6 Bulk Tumor Cells Mesenchymal Cells SUM159 W/ CXCL7 (+MSC alone) MSC+SUM159 co-culture w/ anti-IL6+CXCL7 MSC+SUM159 co-culture CXCR2 ALDH+ X IL-6R gp130 IL-6 CXCR1 IL-6R gp130 Stem cell self renewal Cancer Stem Cell

  17. MSC stimulate breast tumor growth in NOD/SCID mice Tumor size (cm) SUM159 alone (1k) ALDEFLOUR-positive SUM159 cells (%) 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) Days after injection B A DAPI Merge DsRed (SUM159) GFP (MSC) ALDH1 C SUM159-DsRed alone SUM159-DsRed + MSC-GFP

  18. MSCs traffic from Bone marrow to breast primary tumor sites 3.525x106 1.812x106 3.957x106 700 600 500 400 300 200 100 x103 A C B Merge DsRed (MSC) DsRed (MSC) ALDH1 DAPI Without MSC Intra-tibia injection With MSC Intra-tibia injection

  19. 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.

  20. Acknowledgements • Celina Kleer • UM Vector Core • Thomas Lanigan • Tonya Kopas • UM Flow Cytometry Core • David Adams • Ann Marie Des Lauriers • Martin 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) • 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

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