Figure 1.
(A) Tissue specimen opened in a book-wise fashion before sampling; white asterisk: tumor; black asterisk: BAT. Sampling-grid: each tissue specimen was generally divided into eight parts, and BAT samples for molecular analyses were contiguous to those used for histology; in this way, a higher probability of homogeneity between samples used for histology and gene expression analysis, quantitative real-time PCR, western blot analysis and array-CGH was obtained. (B) H&E staining of BAT with absence of morphologically neoplastic cells. (C) GFAP staining, showing reactive astrocytes, with stellar morphology. (D) Ki67/MIB-1 was always <1%. (E) Gene-microarray analysis.
Table 1.
Primer sequences.
Figure 2.
PCA 3D view for gene expression profiles of samples of the three experimental conditions (red: CTRL; yellow: ET; blue: BAT).
Every dot represents a sample. PCA was based on log2 ratios and the expression profiles were performed across the 14,500 genes of the human HG-U133A array. The first 3 principal components are plotted. PCA representation shows samples segregation according to their tissue origin.
Table 2.
Selection of the genes significantly different between ET and BAT with a 10-fold difference in expression levels.
Figure 3.
Hierarchical cluster analysis.
Hierarchical cluster analysis based on expression of 63 transcripts (57 genes) that differed between BAT and CTRL samples (P<0.05) and had greater than 2-fold change between the means of the two tissue types. Each row represents a specific transcript; each column represents a tissue sample harvested from independent patients (R:technical replicate).
Table 3.
Selection of the genes significantly different between BAT samples and control white matter samples, with a 2-fold difference in expression levels.
Figure 4.
Validation of gene-expression.
(a) qPCR validation of a subset of genes differentially expressed between BAT and CTRL. White bars represent the fold change in the expression level between BAT and CTRL as indicated by microarray analysis, while grey bars represent the mean fold change of gene expression calculated by qPCR method. The expression of each gene was normalized to that of 18S ribosomal RNA in the same sample and fold change represents the mean signal of 5 independent samples. Each grey bar is the mean ± SEM of duplicate determinations for each gene in biological replicates. P-values (t-test) for ID3, TAZ, EGFR, IGFBP5, USH1C, SERPINI1, KLRC1 were 0.043; 0.12; 0.09; 0.02; 0.005; 0.0004; 0.003; respectively. (b) KLRC1 western blot analysis in BAT and CTRL white matter samples. β-actin protein level was used as an endogenous control for loading.
Table 4.
Consistent anomalies observed in ET and BAT by a-CGH.
Figure 5.
Immunohistochemical staining for GFAP and TAZ in ET and BAT.
The majority of GBM cells showed intense diffuse cytoplasmic staining for GFAP (A). In the BAT, only apparently normal and reactive astrocytes (black arrow) expressed GFAP protein (B). TAZ immunoreactivity, mainly nuclear, was uniformly expressed in the cells of the ET (C). In the BAT, TAZ positive cells were observed very infrequently (D). Original magnification, ×630 (A–D). Hematoxylin counterstain.
Figure 6.
Confocal microscopy images of GFAP (red) and TAZ (green) expression in ET and BAT.
In the ET samples, GFAP was highly expressed in the cytoplasm of neoplastic cells (A; C). In the BAT, it was present in the body and cytoplasmic extensions of astrocytes (D; F). In the ET, TAZ was expressed predominantly in the nucleus (white arrowheads) and few cells also showed a cytoplasmic localization (yellow arrowhead) (B; C). Rarely, the GBM cells were negative for TAZ (arrow) (B; C). In the BAT, TAZ was almost undetectable. In the photograph shown, no expression of TAZ was observed (E; F). Scale bar: 20 µm.
Figure 7.
Immunohistochemical staining for EGFR and CD99 in ET, BAT and CTRL.
In the ET samples, tumor cells showed intense staining for EGFR, mainly n the cell membrane (A). In the BAT, black arrow points to EGFR immunopositive reactive astrocytes (B). In the ET, CD99-immunoreactivity can be observed at the membrane level and in the cytoplasm (D). In the BAT, CD99 immunopositivity was found in reactive astrocytes (black arrow), in some normal glial cells (black arrowhead) (E). In the CTRL, immunoreactivity for EGFR or CD99 was rarely observed (C; F). Original magnification: x630 (A-B, D-E), x200 (C; F). Hematoxylin counterstain.
Figure 8.
Immunofluorescence of CD133 expression in ET, BAT and CTRL.
CD133 cytoplasmic immunopositivity (red) was observed in a moderate number of cells in the ET (A). In the BAT, the signal was present in reactive astrocytes (white arrows) and apparently normal cells (green arrow) (B). In CTRL samples, CD133-positive cells are extremely rare. In the field showed no CD133-positive cells are detected (C). Cell nuclei were marked with DAPI (blue). Original magnification: x400 (A, B, C).