S1). Thus, loss of Syk enhances migration in normal breast epithelial cells as well as in tumor-forming cell lines. Effects of Syk knockdown on invasion and branching in 3D matrices. To determine whether loss of Syk in normal breast epithelial cells would lead to acquisition of an invasive phenotype in 3D Matrigel, we cultured cells in Matrigel following siRNA knockdown. After 48 hours of culture, MCF10A cells treated with control siRNA formed small round cell aggregates whereas Syk siRNA dramatically increased the size of the colonies which took on an irregular shape similar to initial stages of in vitro branching (Figure 2A). The effect was not as dramatic in the MCF10AneoT and DCIS.com cells, but in all three cell lines, the differences were statistically significant (Figure 2A). Similarly, we determined the morphology of the epithelial cells in 3.0 mg/ml bovine collagen I gels, a setting that is more closely akin to the stromal cell environment. Following Syk knockdown, MCF10A cells took on a more fibroblast-like morphology in collagen, extending more cellular protrusions (Figure 2B). Cells became significantly less round in shape as shown in the representative phalloidin-stained cells and as quantified by cell shape (Figure 2B). Cell migration on the collagen I gels was significantly enhanced by siRNA targeting Syk compared with control siRNA (Figure 2C). siRNA knockdown experiments, in summary, have demonstrated that loss of Syk significantly enhances cell proliferation, migration, and invasive morphologies in normal and transformed breast epithelial cells alike, and the changes in migration and invasion can be observed in assays on plastic, or in 3D matrix settings such as Matrigel or collagen I mats. Loss of Syk increases tumorigenic and metastatic potential. Soft agar assay and chemoinvasion assays through Matrigel in Boydon chambers are more accurate predictors of increased metastatic potential. Therefore, we created shRNA knockdown cell lines for MCF10A, MCF10AneoT, and DCIS.com to obtain stably transfected cells for use in longer term experiments (Figure 3A). Similar to siRNA knockdown of Syk, shRNA knockdown enhanced cell proliferation by 2–3 fold in each of the cell lines measured by the MTS assay (Figure 3B). In Boydon chamber chemoinvasion assays, both siRNA and shRNA Syk were effective in substantially increasing chemoinvasion (Figure 3C). Similarly, loss of Syk in shRNA stably-expressing cell lines was associated with significantly enhanced colony growth within soft agar (Figures 3D and S2). We conclude from this series of experiments, that Syk loss results in increased chemoinvasion and enhanced colony formation in normal and transformed human mammary epithelial cells. Syk knockdown gene expression changes in MCF10A cells related to epithelial-to-mesenchymal transition (EMT), growth factor and stress pathways, and invadopodia formation. To explore the mechanisms whereby loss of Syk promotes invasion, microarray analysis was performed on MCF10A cells transiently transfected with control or Syk siRNA and cultured on a mat of collagen I. After Syk knockdown and culture on collagen, the phenotype of cells does become less epithelial and more mesenchymal (Figure 2) as indicated by loss of keratin genes (Keratin 6B, 10, 23), genes associated with stratified epithelium (small proline-rich protein 1A, involucrin, sciellin) and luminal mammary epithelium (CD24), the extracellular matrix (CD36, the thrombospondin receptor) and gain of genes related to integrin/ECM signaling (integrin beta 5, integrin-linked kinase, laminin gamma 1), collagen biosynthesis and crosslinking (lysyl oxidase (LOX), lysyl oxidase-like 2 (ENTPD4), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), procollagen-proline, 2- oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide I (P4HA1)), and growth factor signaling (EGFR, Syk Loss in Breast Cells VEGF, TGFb1) (Table S1). This suggests that Syk loss releases inhibition of signaling pathways that are normally repressed in epithelial cells and that promote increased proliferation, motility, and invasiveness. Consistent with the changes in phenotype that we observed, i.e., following epithelial-to-mesenchymal transition (EMT), we found that vimentin protein expression was dramatically increased following Syk siRNA knockdown in MCF10A, MCF10AneoT, and DCIS.COM (Figure 4A). To assess overall gene expression changes due to Syk loss in more detail, we performed an Ingenuity Pathway analysis using as input all of the genes that were differentially expressed on collagen following Syk knockdown. The analysis resulted in the identifica- tion of a number of networks and their interactions (Table S2). Several of these networks were linked and identified differentially expressed genes related to EMT and Syk signaling including EGFR, TGFb1, and NFkB networks (Figure S3 and Table S2). These interactive networks are summarized in Figure S4 with ‘‘hubs’’ that include EGFR, TGFb1, NFkB, integrin, collagen, and Rac. Therefore, microarray analysis supports the role of these pathways and their contribution to the changes in phenotype and behavior induced following Syk loss. Invadopodia-related gene expression. Using the same microarray data, we asked whether expression of invadopodia- related genes was enhanced in MCF10A Syk knockdown cells cultured on collagen. We prepared a list of proteins (total 63, 117 probe sets) required for localized matrix degradation, a functional identifier of the presence of invadopodia [15] and determined that a subset were differentially expressed following Syk knockdown and culture on collagen. CORO1A, WASL, GRB2, and PLAUR were down regulated and ITGB1, PAK1, MAPK7, LAMP1, WASF1, VEGFA, ADAM12, and TGFb1 were up regulated (Table S3). The multilayered relationships connecting the gene networks differentially regulated by Syk identified in Figures S1 and S2 and the invadopodia gene network (data not shown) are shown in Figure S1 (blue lines). Since the epidermal growth factor receptor (EGFR) mRNA was up-regulated (Table S1), but did not appear on the invadopodia network (not shown), we validated up regulation of EGFR at the protein level. We found that EGFR levels increased markedly in the MCF10AneoT (ras-transformed) cells (Figures 4B and S5A). Phosphorylation of Y1045 EGFR was up regulated slightly (less than 2 fold) in both Syk siRNA and Syk shRNA knockdown cells on collagen in each of the cell lines (Figures 4B and S5A). Functional invadopodia. To determine whether invadopodia formation is linked to EMT and increased cellular invasion, we asked whether invadopodia were induced or increased following Syk loss. Since MMP14 is required for invadopodia-mediated matrix degradation and invasion on both collagen and gelatin [16,17], we first measured MMP 14 (MT1-MMP) using flow cytometry (FACS). Cell surface expression was dramatically increased following Syk knockdown in cells cultured on plastic (Figure 4C). Next, we performed a localized degradation assay to measure invadopodia activity by culturing cells on fluorescent, crosslinked films [18]. Evidence that cells underwent EMT was further obtained by immuno-staining cell monolayers with phalloidin to detect F-actin and cortactin (Figure 4D) in cells seeded on fluorescent crosslinked gelatin to detect invadopodia, or F-actin, cortactin, and vimentin (Figure 4E). We found in cells seeded at confluent density that control siRNA-treated cells were cobble- stone in appearance and polarized and the cell-cell borders were delineated by adjacent rings of actin filaments (Figures 4D, E, and S5B, con siRNA). In contrast, the Syk siRNA-treated cells were disorganized in appearance with regions of fibroblast-like cell spreading (Figures 4D, E and S5B, Syk siRNA). PLoS ONE | www.plosone.org 3 October 2009 | Volume 4 | Issue 10 | e7445