Figure 3. siRNA and shRNA knockdown of Syk enhances chemoinvasion and anchorage independent proliferation. (A) MCF10A, MCF10AneoT, and DCIS.com cells were transduced with Syk or control shRNA in a lentiviral vector. Western blot analysis of puromycin- selected cell populations is shown. (B) The MTS proliferation assay revealed that shRNA cell lines behave similarly to the siRNA transiently transfected cells with respect to Syk knockdown; cell proliferation was enhanced. (C) Chemoinvasion assays were performed in parallel using cells transiently transfected with control or Syk siRNA or stably transduced with lentiviral control or Syk shRNA. Loss of Syk increases chemoinvasion in every case. (D). In soft agar assays, each of the Syk shRNA knockdown cell lines experienced increased colony formation. Representative images are shown in Figure S3. (B–D) Data are represented as means +/2 SEM. P values are significant by the student’s t-test for each cell line. doi:10.1371/journal.pone.0007445.g003 Syk Loss in Breast Cells The formation of invadopodia-induced holes in the fluorescent matrix was quantified. Syk siRNA knockdown followed by plating at non-confluent (40–45%) conditions elevated hole formation 5.6 fold over the control siRNA-treated cells, whereas at confluent (90–95%) conditions, Syk siRNA knockdown elevated hole formation 5.9 fold over the control cells (Figures 4D and F). Thus, loss of Syk increased invadopodia-mediated matrix degradation. Invadopodia formation and vimentin expression are correlated with Syk negativity in breast cancer cells [19–21]. To determine the relationship between vimentin expression and the ability to form invadopodia, we stained cells with anti-vimentin antibody, phalloi- din, and DAPI (Figure 4E). 34% of cells cultured at sub-confluent density on gelatin films contained vimentin-positive filaments and the percentage of vimentin-positive cells increased to 48% after Syk knockdown (Figure 4G, Non-confluent). The percentage of vimentin-positive cells increased from 11% to 27% after Syk knockdown in confluent cells (Figure 4G, Confluent). Examining individual cells and the subtending matrix, we determined that there was no correlation between cells that expressed vimentin filaments and those that formed invadopodia since 43% of Syk knockdown cells forming holes were vimentin filament-positive and the remaining cells were vimentin filament-negative. Overlap of invadopodia and breast cancer stem cell markers. Participation of CD44 (hyaluronan receptor) and CD49f (a6 integrin) in invadopodia (tumor cells) or podosomes (immune cells) has been previously reported [22–24]. We asked whether the cell surface expression of CD44 or CD49f was altered in MCF10A following Syk knockdown in concert with CD24 expression changes. CD24 is a luminal epithelial marker, and is used as a breast cancer stem cell marker in combination with other stem/progenitor cell markers such as CD49f (integrin alpha6) and CD44 [25–27]. FACS co-staining analysis of CD44/CD24 and CD49f/CD24 revealed that CD44 and CD49f were dramatically enhanced at the cell surface, whereas CD24 was modestly suppressed or unaffected (Figures 4H and S5C). In vivo studies Syk loss increases ductal branching and proliferation in the Syk +/2 mouse mammary gland. To determine the effect of Syk loss during normal mammary gland development in vivo, we examined Syk hetero-knockout (Syk +/2) mice (129 background) [12]. Homozygous Syk knockout mice are not viable, with most mice dying at approximately 16.5 days of gestation [12]. At 10 and 12 weeks of age, mammary glands from virgin Syk +/2 female mice showed increased ductal branches and alveolar development compared with Syk +/+ mice in the 4rth inguinal gland as shown in the whole mount analysis (Figures S6A–C). As expected, Syk protein expression was strongly decreased in mammary gland and spleen from virgin Syk +/2 female mice compared with those tissues of wild type (WT) female mice (129 back ground) (Figure S6D). These data suggest that Syk loss might enhance ductal development in mammary gland. Syk +/2 (FVB) mammary glands show strongly enhanced branching morphogenesis. The Syk +/2 mice (129 background) were crossed with eGFP +/+ mice (FVB background) and loss of Syk expression in heterozygote female mammary glands was confirmed (Figure 5A). Using immunohistochemistry, Syk expression was strongly detected in mammary glands of WT female mice where it localized to luminal epithelium and mammary gland lymph nodes in subsets of lymphocytes (Figure S5A, Syk +/+) and in the spleen (not shown). At those same sites, Syk was decreased in Syk +/2 mice (Figure S7A, Syk +/2). To examine the morphology of ductal development during puberty, whole mount analysis was performed on Syk +/2 and WT PLoS ONE | www.plosone.org 5 October 2009 | Volume 4 | Issue 10 | e7445