Breast cancer affects approximately 1 in 8 US women, making it the most common type of female cancer. Despite numerous advances in screening programs and treatment, breast cancer remains the second leading cause of cancer deaths in women. Until recently, most research focused on targeting transformed breast epithelial cells, yet mounting evidence now attributes a large role for the surrounding breast tumor stroma in cancer progression. The breast stroma comprises myoepithelial cells that surround the epithelial cell-lined ducts of the breast, as well as fibroblasts and the extracellular matrix (ECM). Our studies have focused on the ECM glycoprotein tenascin-C (TN-C), which is generally not expressed in the normal adult breast stroma Initially, we investigated the expression pattern of TN-C via immunohistochemistry (IHC) in breast cancer tissue microarrays encompassing ˜200 patient samples, which revealed stromal TN-C expression in the majority of infiltrating ductal carcinoma cases, as well as other breast cancer types. Having established that TN-C is induced in human breast cancer stroma, we then used 3-D organotypic cultures of human MCF-10A mammary epithelial cells to model how TN-C might affect epithelial cell behavior. Accordingly, MCF-10A cells were cultured for 3-6 days in a reconstituted basement membrane cocktail (Matrigel), with or without purified human TN-C protein. Changes in 3-D epithelial cell cultures were monitored using phase contrast, immunofluorescence and confocal microscopy to examine the effects of TN-C on proliferation (via Ki-67), polarity (via laminin-5 and integrin α6), cell shape (via phospho-ezrin/radixin/moesin (P-ERM) and phalloidin), apoptosis (via caspase-3), and cell migration (via pan-keratin). Also, we developed an algorithm to provide a 3-D reconstruction of epithelial structures that were used to quantify the effects of TN-C on epithelial cell architecture. Whereas control cultures underwent normal 3-D mammary morphogenesis forming polarized spheroid structures with a centrally located lumen, TN-C treated cultures formed significantly larger, dysmorphic structures that displayed increased cell proliferation and migration, decreased apoptosis and defects in basement membrane deposition. Thus, TN-C is expressed in the stroma in a variety of breast cancers, and exerts a profound effect on normal human mammary tissue architecture.