basement membrane matrix (Matrigel) to assess growth and invasion. Epithelial cells extracted from mammary gland(s) of wild type mice formed spherical ‘cysts’ without branches during the course of 24 hour culture (Figure 6D). In contrast, cells isolated from Syk +/2 mice formed invasive, branching processes after 24 h (Figures 6D, arrow and 6E, F). Thus, both proliferation and invasion were enhanced in primary mouse epithelial cells in heterozygote glands compared with wild type. Mammary hyperplasia and tumor formation in heterozygote animals. In order to determine whether aged heterozygote animals develop tumors, five virgin Syk +/2 mice and virgin wild type mice were examined at one year. Whole mounts of the fourth inguinal wild type mammary glands revealed limited ductal structure in wild type glands, whereas those of the Syk +/2 animals were densely filled with epithelial structure (Figure 7A). Loci of hyperplasias were found in the mammary gland whole mounts of the Syk +/2 animals (Figures 7A and B). The epithelial structures were clusters of lobules each containing a small central lumen, whereas the hyperplasias contained densely packed solid cores of irregularly shaped lobules (Figure 7B, X-Y views). At one year, each of the five Syk +/2 females had formed mammary tumors associated with the 3rd or 4th inguinal glands whereas none of the Syk +/+ wild type females had formed them. The tumors, one in each animal, were attached to the mammary fat pad (not shown). The tumors were invasive, intraductal carcinoma (IDC). A representative view of one of the tumors is shown in Figure 7C. At one year, at least one animal has shown the development of lung metastases. Discussion The progression cell line series of MCF10A, MCF10AneoT, and DCIS.com exhibited a progressive loss of Syk. This result mirrors the loss of Syk observed in breast tissues from patients [4]. Strikingly, knockdown of Syk using either siRNA or shRNA consistently enhanced proliferation, motility, invasion, and anchorage-indepen- dent growth in each of these cell lines along the progression series to DCIS.com, a cell line modeling ductal carcinoma in situ (DCIS). Thus, changes in levels of Syk potently mediate their effects on these activities and suggest that Syk must be present in epithelial cells at all stages of progression including benign tissues in order to suppress invasive, tumor-like cell behavior. The results in human breast epithelial cells are supported by the results in the mouse mammary gland where loss of one allele had an impressive effect on mammary ductal structure and ultimately on carcinoma development. In both human and mouse epithelial cells, we demonstrated increased cellular proliferation and invasion. The mouse heterozygote model further demonstrates that partial systemic loss of Syk can result in hyperplastic growth, tumor formation, and in some cases metastasis. The results on Syk knockdown in MCF10A cells support the interpretation that Syk is required in the epithelial cells of normal mouse mammary glands to control proper growth and invasion during puberty although it is possible that additional factors contribute since Syk is present in other tissues including immune cells and endothelium [1]. Multiple lines of evidence suggest that loss of Syk triggers EMT. EMT-related phenotypic changes are evidenced by change in cellular morphology and size in Matrigel and collagen I matrix. In addition, vimentin expression was strongly up-regulated following loss of Syk in the MCF10A cell lines. Vimentin loss is associated with the invasive and mesenchymal phenotype observed following EMT in multiple breast cancer cell types [19,28]. Our observations of spontaneous EMT and vimentin upregulation in subconfluent cultures of MCF10a are consistent with previous Syk Loss in Breast Cells Figure 7. Heterozygous Syk +/2 mice develop mammary gland hyperplasia and ductal carcinoma. (A) At one year in virgin females, 4th inguinal mammary glands reveal a denser epithelial network in heterozygote compared to wild type glands. Insets at right are higher magnification views of the distal ductal tree. An area of hyperplasia in the heterozygote gland (Syk +/2) indicated by a red box is enlarged. Scale bars correspond to 5 mm and 250 mm insets at right and 1 mm inset below. (B) Multiphoton imaging of carmine red autofluorescence in whole mounts of the Syk +/2 gland revealed that the branching epithelium is over developed but lumens are visible (X-Y, inset at left). The hyperplastic portion contains no lumens (X-Y, inset at right). Scale bars correspond to 50 mm. (C) Hemotoxylin/eosin stained paraffin section from an invasive ductal carcinoma taken from a one year, virgin female Syk +/2. Scale bar is 50 mm. doi:10.1371/journal.pone.0007445.g007 PLoS ONE | www.plosone.org 9 October 2009 | Volume 4 | Issue 10 | e7445