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and B.C-Y.), as well as 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.). migration and proliferation. Pretreatment of cells with RAB5A a CD44-blocking antibody abrogated migration effects (< .05). Stemlike breast malignancy cells with high aldehyde dehydrogenase and CD44 (ALDHhiCD44+) responded in a distinct chemotactic manner toward organ-CM, preferentially migrating toward lung-CM through CD44 receptor-ligand interactions (< .05). In contrast, organ-specific changes in migration were not observed for ALDHlowCD44- cells. Our data suggest that interactions between CD44+ breast malignancy cells and soluble factors present in the AT7867 lung microenvironment may play an important role in determining organotropic metastatic behavior. Introduction Breast malignancy remains a leading cause of morbidity and mortality in women [1], mainly due to the propensity of primary breast tumors to metastasize to distant sites and the failure of most therapies in the metastatic setting. Further insight into the biology of metastasis is usually therefore essential to gain a greater understanding of this process and to develop better cancer therapies. Metastasis is usually a complex process, and tumor cells must successfully negotiate a series of sequential steps to establish clinically relevant macrometastases. These actions include dissemination from the primary tumor through blood or lymphatic systems, survival within the circulation, extravasation into secondary sites, initiation of growth into micrometastases, and maintenance of growth as vascularized macro-metastases [2]. Clinical observations indicate that many cancers show an organ-specific pattern of metastasis, termed selection and genetic analysis of the MDA-MB-231 human breast malignancy cell line, this group exhibited that particular genes can mediate experimental breast cancer metastasis in an organ-specific manner to lung [10], bone [9], and brain [8] and validated that these genes reflect organ-specific metastatic disease in patients with breast malignancy. Although these studies contribute valuable knowledge regarding the contribution of the cancer cell (seed) to organ tropism of breast cancer, the factors contributed by the metastatic microenvironment (ground) still remain poorly understood. In addition, these studies do not take into account the concepts of tumor cell heterogeneity and the cancer stem AT7867 cell hypothesis. Despite the deadly nature of metastasis, it is an inherently inefficient process [2,11]. This suggests that only a small subset of cells can successfully navigate the metastatic cascade. We believe that these metastasis-initiating cells may in fact be cells with stemlike properties [12]. In breast malignancy, tumor-initiating cells have been isolated from primary tumors and pleural effusions on the basis of a cluster of differentiation (CD) 44-positive-CD24-unfavorable (CD44+CD24-) phenotype [13] and/or high aldehyde dehydrogenase (ALDH) activity [14]. Our group as well as others have demonstrated that breast malignancy cells with an ALDHhiCD44+ phenotype show enhanced metastatic behavior and compared to their ALDHlowCD44- counterparts [15C17]. However, the role of such cells in mediating organ-specific metastasis has not been investigated. In the current study, we hypothesized that breast cancer cells exhibit distinctive growth and migration patterns in organ microenvironments that mirror common clinical sites of breast cancer metastasis and that receptor-ligand interactions between breast malignancy cells and specific soluble organ-derived factors can mediate this behavior. We first developed and validated a comprehensive model system for investigating the influence of organ-specific soluble factors on metastatic behavior of human breast malignancy cells. Our results indicate that human breast malignancy cells with varying genetic backgrounds exhibit differential migration and growth patterns toward specific organ conditions. Notably, these patterns reflect the known metastatic dissemination patterns of these cell lines and spotlight the lung as an important source of soluble factors that mediate metastatic behavior. Furthermore, our results suggest for the first time that interactions between subpopulations of CD44-expressing breast malignancy cells (including ALDHhiCD44+ cells) and soluble ligands present in the lung microenvironment may play an important role in determining organotropic metastatic AT7867 behavior. Materials and Methods Cell Culture and Reagents MDA-MB-231 cells [18] were obtained from American.