Lung-Derived Factors Mediate Breast Cancer Cell Migration through CD44 Receptor-Ligand Interactions in a Novel Ex Vivo System for Analysis of Organ-Specific Soluble Proteins 1,2 Jenny E. Chu * ,†,3 , Ying Xia * ,3 , Benjamin Chin-Yee * , David Goodale * , Alysha K. Croker * ,† and Alison L. Allan * ,†,‡,§ *London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada; † Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; ‡ Department of Oncology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada; § Lawson Health Research Institute, London, Ontario, Canada Abstract Breast cancer preferentially metastasizes to lung, lymph node, liver, bone, and brain. However, it is unclear whether properties of cancer cells, properties of organ microenvironments, or a combination of both is responsible for this observed organ tropism. We hypothesized that breast cancer cells exhibit distinctive migration/growth patterns in organ microenvironments that mirror common clinical sites of breast cancer metastasis and that receptor-ligand interactions between breast cancer cells and soluble organ-derived factors mediate this behavior. Using an ex vivo model system composed of organ-conditioned media (CM), human breast cancer cells (MDA-MB-231, MDA-MB-468, SUM149, and SUM159) displayed cell line–specific and organ-specific patterns of migration/proliferation that corresponded to their in vivo metastatic behavior. Notably, exposure to lung-CM increased migra- tion of all cell lines and increased proliferation in two of four lines (P < .05). Several cluster of differentiation (CD) 44 ligands including osteopontin (OPN) and L-selectin (SELL) were identified in lung-CM by protein arrays. Immunodepletion of SELL decreased migration of MDA-MB-231 cells, whereas depletion of OPN decreased both migration and proliferation. Pretreatment of cells with a CD44-blocking antibody abrogated migration effects (P < .05). “Stemlike” breast cancer cells with high aldehyde dehydrogenase and CD44 (ALDH hi CD44 + ) responded in a distinct chemotactic manner toward organ-CM, preferentially migrating toward lung-CM through CD44 receptor-ligand interactions (P < .05). In contrast, organ-specific changes in migration were not observed for ALDH low CD44 - cells. Our data suggest that interactions between CD44 + breast cancer cells and soluble factors present in the lung microenvironment may play an important role in determining organotropic metastatic behavior. Neoplasia (2014) 16, 180–191 Abbreviations: ALDH, aldehyde dehydrogenase; ANOVA, analysis of variance; AP, alkaline phosphatase; bFGF, basic fibroblast growth factor; BM, bone marrow; BMSC, bone marrow stromal cell; BrdU, bromodeoxyuridine; CD, cluster of differentiation; CM, conditioned media; COX-2, cyclooxygenase 2; DAPI, 4′,6-diamidino-2-phenylindole; DEAB, diethylamino- benzaldehyde; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; ER, estrogen receptor; EREG, epiregulin; FITC, fluorescein isothiocyanate; HPF, high- powered field; LN, lymph node; LNSC, lymph node stromal cell; NIH, National Institutes of Health; OPN, osteopontin; PR, progesterone receptor; RT, room temperature; SELE, E-selectin; SELL, L-selectin; SELP, P-selectin; uPA, urokinase-type plasminogen activator; VCAM-1, vascular cell adhesion molecule 1; VEGFA, vascular endothelial growth factor A Address all correspondence to: Alison L. Allan, PhD, London Regional Cancer Program, Room A4-132, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6. E-mail: alison.allan@lhsc.on.ca 1 This work was supported by grants from the Canadian Breast Cancer Foundation-Ontario Region, the Canada Foundation for Innovation (No. 13199), and donor support from John and Donna Bristol through the London Health Sciences Foundation (to A.L.A.). Studentship and fellowship support were provided by an Ontario Graduate Scholarship (Province of Ontario, to J.E.C.), the Canadian Institutes of Health Research (CIHR)–Strategic Training Program (to J.E.C. and B.C-Y.), and the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit at the London Regional Cancer Program (to J.E.C., Y.X., and A.K.C.). A.L.A. is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation. 2 This article refers to supplementary materials, which are designated by Tables W1 and W2 and Figures W1 to W5 and are available online at www.neoplasia.com. 3 These two authors contributed equally to the manuscript. Received 22 December 2013; Revised 5 February 2014; Accepted 6 February 2014 Copyright © 2014 Neoplasia Press, Inc. All rights reserved 1522-8002/14/$25.00 DOI 10.1593/neo.132076 www.neoplasia.com Volume 16 Number 2 February 2014 pp. 180–191 180