Gene expression profile of glioblastoma multiforme invasive phenotype points to new therapeutic targets

Abstract

The invasive phenotype of glioblastoma multiforme (GBM) is a hallmark of malignant process, yet molecular mechanisms that dictate this locally invasive behavior remain poorly understood. Gene expression profiles of human glioma cells were assessed from laser capture-microdissected GBM cells collected from paired patient tumor cores and white matter-invading cell populations. Changes in gene expression in invading GBM cells were validated by quantitative reverse transcription polymerase chain reaction (QRT-PCR) and immunohistochemistry in an independent sample set. QRT-PCR confirmed the differential expression in 19 of 21 genes tested. Immunohistochemical analyses of autotaxin (ATX), ephrin B3, B-cell lymphoma-w (BCLW), and protein tyrosine kinase 2 beta showed them to be expressed in invasive glioma cells. The known GBM markers, insulin-like growth factor binding protein 2 and vimentin, were robustly expressed in the tumor core. A glioma invasion tissue microarray confirmed the expression of ATX and BCLW in invasive cells of tumors of various grades. GBM phenotypic and genotypic heterogeneity is well documented. In this study, we show an additional layer of complexity: transcriptional differences between cells of tumor core and invasive cells located in the brain parenchyma. Gene products supporting invasion may be novel targets for manipulation of brain tumor behavior with consequences on treatment outcome.

Figure 1

QRT-PCR validation of gene candidates differentially expressed between invasive rim cells and tumor core cells in cDNA microarray analysis. Names of transcripts analyzed are on the x-axis and the mean fold differential regulation (difference in relative copy number, where 1 represents equal expression in both populations) is on the y-axis. Grey bars represent the mean gene expression levels of invasive tumor cells over tumor core cells seen in the cDNA microarray analysis. Black bars represent the mean gene expression levels of invasive tumor cells over tumor core cells as evaluated by QRT-PCR using seven matched tumor samples. White bars indicate the levels of gene expression (evaluated by QRT-PCR) in the diffusely invaded white matter from one of the samples used in the cDNA microarray analysis divided by the expression levels from either invasive cells (left side, where candidates genes are upregulated) or tumor core cells (right side, where candidate genes are downregulated) of the same sample. *Denotes genes with a significance of P ≤ .05. **Denotes genes with a differential expression that reaches a significance of P ≤ .025 as calculated using the Wilcoxon signed-rank test.

Figure 2

Immunohistochemical analysis of six candidate genes ATX (C), BCLW (D), EFNB3 (E), PYK2 (F), IGFBP2 (G), and VIM (H); four overexpressed in the invasive rim cells and validated by QRT-PCR; and two underexpressed in the rim, respectively. (A and B) H&E stains of the glioblastoma invasion front. (I) Representative negative control. Large bold arrows point to the tumor core, and smaller arrows point at individually invasive glioblastoma cells. Original magnification, x400, except (A) at x200.

Figure 3

Summary of immunohistochemical evaluation of ATX and BCLW in a glioma invasion tissue microarray. The tissue type examined and the number of samples in each category are listed on the y-axis. The percentage of cases with positively staining cells within each category is on the x-axis. The shading scale represents the percentage of positively stained cells within a sample. ATX TMA (A) evaluation showing a high degree of positive cells in gliomas of different grades, but also in some neurons and reactive astrocytes. The TMA stained for BCLW (B) reveals its presence in gliomas of all grades.