Conversely, an elevated fibronectin level in ECM-Sdc1 was responsible for the enhanced attachment of the breast carcinoma cells. culture surfaces, we forced the Sdc1-negative fibroblasts to produce ECM with parallel fiber organization, mimicking the architecture observed in ECM-Sdc1. We found that the fiber topography governs carcinoma cell migration directionality. Conversely, an elevated fibronectin level in ECM-Sdc1 was responsible for the enhanced attachment of the breast carcinoma cells. These observations suggest that Sdc1 expression in breast carcinoma stromal fibroblasts promotes the assembly of an architecturally abnormal ECM that is permissive to breast carcinoma directional migration and invasion. Epithelial-stromal interactions play crucial roles in directing mammary gland development and in maintaining normal tissue homeostasis. Conversely, during tumorigenesis, the stroma accelerates carcinoma growth and progression. The predominant cell type within the stromal compartment is the fibroblast, which synthesizes, organizes, and maintains a three-dimensional (3D) network of glycoproteins and proteoglycans known as the extracellular matrix (ECM). Normal stromal fibroblasts and their ECM are believed to exert an inhibitory constraint on tumor growth 5-Iodotubercidin and progression.1,2Major alterations occur in the stromal fibroblasts and ECM during neoplastic transformation, giving rise to a permissive and supportive microenvironment for carcinomas. Compared with their quiescent normal counterpart, carcinoma-associated fibroblasts display an activated phenotype, which is characterized by the expression of smooth muscle markers, an enhanced proliferative and migratory potential, and altered gene expression profiles. Carcinoma-associated fibroblasts produce and deposit elevated amounts and abnormal varieties of ECM components.35Recent evidence6,7indicates that not only ECM composition but also ECM architecture are altered in carcinomas and that these changes may promote tumor progression. However, the contribution of these stromal modifications to tumor development and the molecular mechanisms and signaling events underlying these alterations are incompletely understood. Syndecans (Sdcs) constitute a family of transmembrane heparan sulfate proteoglycans with four known members (Sdc1-4). Via their heparan sulfate glycosaminoglycan (HS-GAG) chains, Sdcs interact with a wide variety of proteins, including growth factors and ECM constituents.810Consequently, they play roles in cell growth, adhesion, migration, and morphogenesis. Sdc2 appears to be required to assemble laminin and fibronectin (FN) into a fibrillar matrix.11Syndecan-4 has also been implied to participate in FN matrix assembly. Concomitant engagement of Sdc4 and integrins promotes Rho GTPase and focal adhesion kinase (FAK) activities, which are crucial for efficient initiation of FN matrix assembly.1215Sdc1 is expressed primarily by epithelial and plasma cells of healthy adult tissue.16Recently, we and others17,18observed the induction of Sdc1 in stromal fibroblasts of invasive breast carcinomas. Syndecan-1, Rabbit Polyclonal to ELOA3 aberrantly expressed by stromal fibroblasts in breast carcinomas, participates in a reciprocal carcinoma growthpromoting feedback loop that requires proteolytic shedding of its ectodomain.17,19,20Although the role of Sdc1 in matrix assembly has not been investigated, 5-Iodotubercidin this molecule has interacted with various ECM components, including FN, fibrillar collagens, laminin, vitronectin, thrombospondin, and tenascin.810 In the present study, we explore the possibility that Sdc1 expression by stromal fibroblasts may be causally involved in altered matrix production of tumor stroma. We find that in mammary stromal fibroblasts, Sdc1 regulates ECM assembly and determines ECM fiber architecture. We further show that cell-free 3D ECMs produced by Sdc1-expressing fibroblasts facilitate the directional migration of mammary carcinoma cells and link this activity to the parallel fiber architecture. == Materials and Methods == == Human Breast Carcinoma Samples == Paraffin sections from a tissue microarray containing duplicate tumor samples from 207 patients with breast carcinoma were 5-Iodotubercidin immunoperoxidase labeled with an antibody to Sdc1, as previously described.17The immunolabeled slides were examined by bright field microscopy and scored manually, using a method developed by Harvey and coworkers.21Sections from the same tumor blocks and those from MMTV-Wnt1induced mammary tumors (provided by C. Alexander, Ph.D.), were stained with picro-sirius red (sirius red F3B [C.I. 35782; 0.1% w/v] in saturated picric acid aqueous solution) for 60 minutes, followed by two washes in glacial acetic acid (0.5% v/v). Images acquired during polarization microscopy were overlaid with a predesigned template that defined nine evenly distributed measurement points. The fiber closest to each of these points was identified, and the intersection angle of the nearest crossing fiber was measured using an angle measurement tool (ImageJ;http://rsbweb.nih.gov/ij/). Two observers measured the angles in the tissue microarray slides independently in a blinded fashion (intraclass correlation coefficient, 0.82). == Cell Culture == The human breast carcinoma cell lines MDA-MB-231 were from A.C. Rapraeger, T47D cells were from M. Gould, Ph.D., and NIH-3T3 fibroblasts were from J.S. Malter, MD (University of WisconsinMadison). Immortalized human.