Differential analysis of glioblastoma multiforme proteome by a 2D-DIGE approach

Abstract

Background: Genomics, transcriptomics and proteomics of glioblastoma multiforme (GBM) have recently emerged as possible tools to discover therapeutic targets and biomarkers for new therapies including immunotherapy. It is well known that macroscopically complete surgical excision, radiotherapy and chemotherapy have therapeutic limitations to improve survival in these patients. In this study, we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify proteins that may serve as brain tumor antigens in new therapeutic assays. Five samples of patients presenting a GBM and five samples of microscopically normal brain tissues derived from brain epileptic surgery specimen were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis) with an internal pool sample on each gel. Five gels were matched and compared with DIA (Difference In-gel Analysis) software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained.

Results: From 51 protein-spots significantly up-regulated in GBM samples, mass spectrometry (MS) identified twenty-two proteins. The differential expression of a selected protein set was first validated by western-blotting, then tested on large cohorts of GBM specimens and non-tumor tissues, using immunohistochemistry and real-time RT-PCR.

Conclusions: Our results confirmed the importance of previously described proteins in glioma pathology and their potential usefulness as biological markers but also revealed some new interesting targets for future therapies.

Figure 1

DIGE analysis on a series of 5 GBM samples (T) and 5 non tumorous samples (Nt). A) Experimental design. B) Examples of two DIGE gels: a) Gel 1: the GBM sample (T6) was labeled with Cy5 Dye (red spots) and the control sample (Nt1) with Cy3 Dye (green spots). b) Gel 2: the GBM sample (T7) was labeled with Cy3 Dye (green spots) and control sample (Nt2) with Cy5 Dye (red spots). Merge spots appeared in yellow. In all gels, internal standard was labeled with Cy-2 Dye (not visible in these images).

Figure 2

Selected proteins expression analyzed by western-blot. GBM (T) and non tumoral control (Nt) protein extracts were loaded on 1D SDS-PAGE gels together with biotinylated molecular weight markers and transferred on Hybond-P membranes, then stained with primary and secondary-HRP antibodies or Streptavidin-HRP. Immunoreaction was revealed using ECL. Protein expression was tested for HSP 27 (A), ALDH (B), Mn-SOD (C), DRP-2 (D) and DRP-3 (E).

Figure 3

Selected proteins expression analyzed by immunohistochemistry. GBM (a) and non tumor control (b) paraffin-embedded sections were stained with anti-HSP 27 (A), -ALDH (B), -Mn-SOD (C), -DRP-2 (D) and -DRP-3 (E) antibodies. Normal brain staining and one representative sample are shown for each antigen. In control samples, the major cell types are indicated with arrows: the thickest arrows designate oligodendrocytes, the intermediate arrows astrocytes and the narrow ones neurons. Magnifications for GBM samples were x 400 with a window (I, II, III, IV,V) showing magnified small region (x 1000). Control samples were magnified x 1000.

Figure 4

In situ validation of the protein expression data obtained by immunohistochemistry. For each antibody, 1000 tumor cells were counted and results were expressed as a percentage of positive cytoplasmic staining in two different and most expressive areas. Bars represent standard deviation of results obtained with the 25 GBM and the three populations (astrocytes and oligodendrocytes and neurons) of 3 non-tumorous samples (Nt samples). The expression of four proteins tested (HSP 27, ALDH, DRP-2, DRP-3) was higher in GBM cells than in normal astrocyte or oligodendrocyte populations. All neurons were positive (100%) except for HSP 27 (0%).

Figure 5

mRNA expression in 50 GBM samples analyzed by real-time RT-PCR. Results are expressed as the relative HSP 27 (A), Mn-SOD (B) and DRP-3 (C) mRNA expression compared with the same pool of non-tumorous samples.