E-Cadherin Expression in GBM Figure 2. E-cadherin protein expression occurs in a subset of xenograft glioma lines. Nineteen glioma cell lines propagated as xenografts in mouse flank were examined by Western Blot for expression of various cadherin and catenin proteins. Actin serves as a loading control. Positive control lysates are from MCF7 cells (E-cadherin, b-catenin), UMRC3 cells (N-cadherin), and MDA231 cells (cadherin-11). Low levels of E-cadherin expression were detected in a small subset of these glioma xenografts. doi:10.1371/journal.pone.0013665.g002 E-cadherin expression in conventional GBM cell lines To further probe the role of E-cadherin in GBMs, we wanted to apply standard cell biology techniques, including using interfering RNA (RNAi) technology to knockdown E-cadherin expression. Because conventionally cultured cell lines are more easily manipulated, a panel of nineteen conventionally cultured glioma cell lines was screened by Western blot for the presence of E- cadherin, N-cadherin, cadherin-11, b-catenin, and p120 catenin (Figure 4). As was seen with the xenograft cultured GBM lines, b- catenin and p120 catenin were expressed in all lines and most lines expressed N-cadherin and/or cadherin-11. Only the SF767 cell line, derived from a recurrent anaplastic astrocytoma [41], was found to express E-cadherin. Importantly, identical results were Figure 3. E-cadherin protein expression correlates with glioma cell invasiveness in an orthotopic xenograft mouse model. Xenograft cell lines 5, 6, 8, 10, 12, 14, 15, 16, 22, 26, 28, 34, 36, 38, 43, 44, 46, and 59 were examined for relative invasiveness following orthotopic injection into mouse brain. Lines were categorized as highly, moderately, minimally, or non invasive. The level of E-cadherin expression (relative to actin expression) determined by Western blot for each cell line was then plotted vs. relative glioma invasiveness. The lines through the data indicate the median for each invasiveness category; *indicates a statistical difference (one-tailed, unpaired t test) between the two categories of invasiveness at p,0.05. doi:10.1371/journal.pone.0013665.g003 obtained using the HECD1 anti-E-cadherin antibody (data not shown). SF767 cells did not express either of the other two cadherins evaluated. An original clone of the SF767 cell line was independently obtained from the line’s original source (the UCSF/ Neurosurgery Tissue Bank) and showed an identical pattern of cadherin expression (Lewis-Tuffin, data not shown). SF767 cells co-segregate with several other GBM cell lines and primary GBM tumors in gene expression studies [42]. Therefore, subsequent experiments probing E-cadherin’s role were carried out in the SF767 cell line under conventional culture conditions. Role of E-cadherin in SF767 cell growth and motility Initially, the localization of E-cadherin was assessed in SF767 cells using immunofluorescence (Figure 5). When grown under conventional culture conditions, these cells exhibit a ‘‘spiky’’ appearance in which the cells are surrounded by filopodia. This morphology is relatively common in mesenchymal cells but not in cells that express functional, E-cadherin-based cell-cell adhesion complexes, such as MCF7. The difference is particularly evident when the organization of the actin cytoskeleton is examined (Figure 5A). Actin protrusions into filopodia are evident on all sides of the SF767 cells, including between adjacent cells at what should be areas of cell-cell contact. This unusual morphology is in contrast to the very tight areas of cell-cell contact exhibited by the MCF7 cells, in which the actin cytoskeleton is organized in junctional actin rings such that they cannot be distinguished from each other in adjacent cells. Co-staining for E-cadherin in the SF767 cells vs. the MCF7 cells revealed a similarly unusual distribution. In MCF7 cells localization of E-cadherin was restricted to the plasma membrane at areas of cell-cell contact (Figure 5A,B). In SF767 cells E-cadherin was found across the entire surface of the plasma membrane, as well as being distributed in some cytoplasmic locations, particularly in peri-nuclear areas. At areas of what should be cell-cell contact, E-cadherin staining was diffuse and cells exhibited gaps and/or filopodia protrusions. As a second means of examining areas of cell-cell contact, SF767 and MCF7 cells were co-stained for E-cadherin and b- catenin (Figure 5B). In MCF7 cells E-cadherin and b-catenin had similar distributions to each other and were restricted primarily to intercellular junctions. E-cadherin and b-catenin also co-localized in SF767 cells. However their distribution was not restricted to areas of cell-cell contact, with E-cadherin and b-catenin also found PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13665