E-Cadherin Expression in GBM Figure 1. E-cadherin expression correlates with worse outcome for patients with glioblastomas exhibiting epithelial appearance. A. E-cadherin protein in 8 of the 9 positive cases was focally restricted to discrete nests of tumor cells, reflecting areas of epithelial-like differentiation. In these positive cells E-cadherin was found primarily on the plasma membrane. However, unlike in normal epithelial cells, the localization of E-cadherin was not concentrated at areas of homotypic cell-cell contact, but rather localized uniformly along the plasma membrane (indicated by arrows in Ai). Left: Immunostain for E-cadherin (magnified in Ai to show detail); Right: H&E. The scale bar is 20 mm and applies to all Figure 1 images except Ai and Bi. B. 1 of the 9 E-cadherin positive cases showed an unusually high expression of E-cadherin which was located both on the membrane and cytoplasmically. Left: Immunostain for E-cadherin (magnified in Bi to show detail); Right: H&E of the tumor’s epithelial component. C. Two independent examples of b-catenin localization by immunostain. Unlike E-cadherin, b-catenin is distributed throughout the tumor samples. D. Individual cases of glioblastoma exhibiting epithelial or pseudoepithelial differentiation were analyzed by immunohistochemistry for the absence (Negative) or presence (Membraneous/cytoplasmic) of E-cadherin protein. This data was correlated with overall survival using Kaplan-Meier analysis. There is a statistically significant survival difference (p = 0.021). doi:10.1371/journal.pone.0013665.g001 primary patient specimens. Importantly, this GBM model frequent- ly recapitulates several key features of the human disease that are lost in conventionally-cultured glioma cell lines, including retention of markers such as EGFR overexpression and a tendency for the orthotopically-implanted cells to invade throughout the mouse brain [38–40]. Whole cell lysates were made from 19 of these tumor lines using flank tumor specimens. These lysates were analyzed by Western blot for the expression of various human cadherins and catenins (Figure 2). All of the lines expressed b-catenin and p120 catenin, both important components of the cadherin/catenin cell- cell adhesion signaling complex. Most of the lines expressed N- cadherin and/or cadherin-11, which are frequently found in brain tumors and normal brain. Surprisingly, 5 of the 19 lines examined (GBMs 6, 8, 16, 26, and 34) expressed substantial amounts of E- cadherin. The relative amount of E-cadherin protein expression was low in the GBM xenograft lines compared to its expression in the E- cadherin positive MCF7 breast cancer cell line, which was used in this and later experiments as an example of a typical epithelial cell. The difference in E-cadherin protein levels among these cell lines was also reflected in E-cadherin mRNA levels (Lewis-Tuffin, data not shown) and may be due to a focal rather than general expression of E-cadherin in these tumors (similar to Figure 1A). Eighteen of these xenograft lines were used to establish orthotopic xenografts in mice, and mice were observed until reaching a moribund state. The brains were then sectioned and relative invasiveness was determined using H&E staining on up to 10 mice per cell line. These xenograft lines were categorized according to whether they were highly, moderately, minimally, or non invasive (Figure S2). For subsequent analysis the highly and moderately invasive lines were combined into one category containing 8 lines (GBMs 6, 8, 15, 16, 26, 34, 38, and 44) and the minimally and non invasive lines were combined into a second category containing 10 lines (GBMs 5, 10, 12, 14, 22, 28, 36, 43, 46, and 59). The expression of E-cadherin in the corresponding flank lysates was quantified relative to the amount of actin protein in each lysate. This quantification was then plotted versus the relative invasiveness of the xenograft lines in mouse brain (Figure 3). Five of the eight highly/moderately invasive lines expressed substantial amounts of E-cadherin (GBMs 6, 8, 16, 26, 34) while none of the ten minimally/non- invasive lines did. The data revealed that E-cadherin expression was higher in the more invasive xenograft lines, suggesting that E-cadherin expression and/or signaling may contribute to GBM tumor aggressiveness. PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13665