Figure 1.
Network analysis of dynamically-regulated adhesion molecules reveal that cortical cells express Cdh11 as they transition from polarized epithelial to multipolar migratory cells and exit the VZ.
(A) VZ cells electroporated with GFP expression plasmid reveal both polarized epithelial morphology “E” and multipolar migratory morphology “M” in the developing cortex. Immunostaining for the intermediate progenitor marker Tbr2 (Eomes) highlight cells undergoing the transition from E (arrowhead) to M (arrow). (B) Images extracted from the GFP image in (A) showing cells with epithelial “E” and multipolar “M” morphology. (C, D) Section of E12.5 Eomes:GFP reporter mouse. Dotted lines show how the cortex was separated, then dissociated into single cells for FACS. (D) Higher power image through E12.5 Eomes:GFP reporter mouse. (E) >3-fold up and >3-fold down regulated adhesion molecules were imported with β-catenin to populate the IPA network. Green colors indicate genes with higher expression in VZ and red indicate higher expression outside VZ. Solid lines indicate direct protein-protein interactions. (F) Immunoreactivity for CDH11 in E14.5 mouse cerebral cortex showing enriched expression in SVZ/IZ and deeper CP. Bar = 50 µm.
Figure 2.
Overexpression of CDH11 in cortical VZ precursors causes premature exit from the VZ and neuronal differentiation.
(A) E13.5 cortical precursors were electroporated in utero using either pCDNA control (N = 4) (top) or with pCAG-Cdh11 expression plasmid along with pCAG-EGFP plasmid (N = 4) and analyzed at E14.5. Electroporated cells were identified with antibody staining against GFP and sections counterstained with the DNA dye DAPI (pseudocolored blue). To quantify changes in cortical positioning of electroporated cells, ten equal sized bins were drawn over each image. Each white dot corresponds with the soma of an electroporated cell. Bar = 100 µm. (B) The fraction of the total GFP+ cells in each of the ten bins was then graphed for the two experimental conditions. Brackets indicate 1 SEM. N = 4 brains (PCDNA), 3 brains (Cdh11). (C) Sections were stained for radial glial marker Pax6, intermediate progenitor marker Tbr2, and neuronal marker Tbr1. Electroporated cells are pseudocolored green, and the respective antigens, red. Bar = 50 µm. The dot plots highlight the cell bodies of electroporated cells, with red representing electroporated cells that express the marker of interest and green indicating electroporated cells that do not express the marker. (D) Histograms represent fraction of total electroporated cells found in each brain region, showing the fraction of cells that express each marker after electroporation (red/(red+green)), showing premature neuronal differentiation. For Pax 6, Cdh11 vs. control (N = 4 brains for each, **P = 0.0072), Tbr2 (N = 3 for Cdh11, N = 4 for pcDNA control, *P = 0.077), and Tbr1 (N = 4 for each, *P = 0.0071). Unpaired T-test.
Figure 3.
CDH11 is differentially expressed in human glioma vs. normal brain, and overexpression confers worsened prognosis.
(A) Kaplan-Meier Survival Plot for samples with differential CDH11 gene expression reveal that glioma patients with overexpression (red curve) have a worse prognosis than patients with intermediate expression. Log-rank P-value for Up-Regulated vs. Intermediate = 3.8535E-6. Data from NCI 2005<http://rembrandt.nci.nih.gov>. (B) CDH11 expression is predictive for survival in GBM; fold-change 3.6, P = 0.005 by T-test (Data from [38]). (C) CDH11 expression is higher in GBM vs. mixed glioma or oligodendroglial tumors; fold-change = 1.8, P = 5.83×10−4 by T-Test (Data from [39]). (D) CDH11 expression is higher in GBM vs. neural stem cells. Fold change = 7.8, P = 8.92×10−4 (Data from [40]). (E) CDH11 is relatively overexpressed in GBM vs. normal brain tissue. TCGA glioblastoma dataset analysis shows 2.9 fold overexpression of CDH11 (P = 1.14×10−10, T-Test). GBM vs. Normal in (Bredel [41]): 2.2 fold increase; P = 0.018 (T-test). GBM vs. Normal (Sun [42]): 2.1 fold increase (P = 1.14×10−16, T-test). GBM vs. normal (Liang [39]): 3.6 fold increase (P = 0.005, T-test). GBM vs. WM (Shai [43]): 2.5 fold overexpression compared to white matter (P = 9.75×10−8; T-test). (F–I) Relative expression of cadherin genes in GBM vs. normal from [42] (F), [39] (G), [41] (H), and [40] (I) reveals that CDH11 (boxed in red) is differentially regulated in GBM vs. normal across multiple datasets. (J–M) Immunoperoxidase staining for CDH11 expression in human GBM tissue show heterogenous expression patterns, with enrichment of staining in tumor cells adjacent to tumor vessels (arrows). Cells surrounding vessels of varying sizes from small (J) to large vessels (K) express Cdh11. As typical for GBM, the tumor histoarchitecture is highly varied, with tissue showing variable necrosis and marked heterogeneity of cellularity (notably (J) and (K)). Bar = 100 µm.
Figure 4.
CDH11 expression is upregulated by endothelial cells and TGFβ.
(A) Primary human GBM cells are labeled with GFP and co-cultured with either unlabeled GBM cells or endothelial cells. GFP expressing cells are then purified by fluorescence activated cell sorting and CDH11 expression is measured by qRT-PCR. (B) Coculture with endothelial cells (HUVEC, mBend) causes increased expression of CDH11 in primary human GBM cells (GBM line 83 P = 0.0164 by ANOVA (n = 5); Line 77, P = 0.0284 by ANOVA (n = 6)). (C) CDH11 mRNA is increased in a dose-dependent fashion by TGFβ. (N = 3 for each cell line. Line 18 P<0.0001; Line 69A P = 0.0001; Line 71A P = 0.0005; all by Repeated Measures ANOVA). (D) Endothelial cell co-culture induces p-SMAD3 expression in GBM cells (top row: GBM co-cultured upon GBM; bottom row: GBM cultured with endothelial cells). Bar = 50 µm. (E) Western blots confirm induction of SMAD3 phosphorylation by co-culture of GBM with endothelial cells. (F) TGFβ transcriptional targets SMAD7 (Line 83, P = 0.0275 by ANOVA (n = 4); Line 77 n = 4) and Serpine-1 (Line 77, P = 0.0082 by Repeated Measures ANOVA (n = 3); Line 83 n = 4) are upregulated in GBM co-cultured with endothelial cells (primary HUVEC or mouse brain endothelial cell line mBend). (G) CD44 mRNA (Line 83 P = 0.0196 by ANOVA (n = 5); line 77 P = 0.0017 by ANOVA (n = 4)) and (H) cell surface expression (measured by flow cytometry) is upregulated after endothelial co-culture. For all pairwise comparisons, Newman Keuls posthoc tests were used, * P<0.05, ** P<0.01, *** P<0.001 and refer to comparison with control (GBM-GBM) unless otherwise noted.
Figure 5.
CDH11 knockdown reduces TGFβ-induced glioma cell motility.
(A) Equal numbers of GBM cells expressing non-silencing shRNA and mCherry are seeded on the top of a porous polycarbonate membrane with GBM cells expressing shRNA to CDH11 and GFP and allowed to migrate towards bottom chamber with 3T3-conditioned. (B) Western blot confirming protein knockdown in shRNA treatments. (C) Images from the bottom of the polycarbonate filter showing control NS cells and CDH11 knockdown cells, quantified in (D) N = 3 biological replicates for each cell line; repeated measures ANOVA P = 0.01 (Line 83); 0.0308 (Line 77); * P<0.05 (Newman-Keuls posthoc test).