Subventricular zone involvement in Glioblastoma - A proteomic evaluation and clinicoradiological correlation

Abstract

Glioblastoma multiforme (GBM), the most malignant of all gliomas is characterized by a high degree of heterogeneity and poor response to treatment. The sub-ventricular zone (SVZ) is the major site of neurogenesis in the brain and is rich in neural stem cells. Based on the proximity of the GBM tumors to the SVZ, the tumors can be further classified into SVZ+ and SVZ-. The tumors located in close contact with the SVZ are classified as SVZ+, while the tumors located distantly from the SVZ are classified as SVZ-. To gain an insight into the increased aggressiveness of SVZ+ over SVZ- tumors, we have used proteomics techniques like 2D-DIGE and LC-MS/MS to investigate any possible proteomic differences between the two subtypes. Serum proteomic analysis revealed significant alterations of various acute phase proteins and lipid carrying proteins, while tissue proteomic analysis revealed significant alterations in cytoskeletal, lipid binding, chaperone and cell cycle regulating proteins, which are already known to be associated with disease pathobiology. These findings provide cues to molecular basis behind increased aggressiveness of SVZ+ GBM tumors over SVZ- GBM tumors and plausible therapeutic targets to improve treatment modalities for these highly invasive tumors.

Figure 1

Representative overlapped 2D-DIGE gel image of SVZ+ and SVZ- GBM patient’s serum proteome and 3D-views of significantly altered proteins. Extracted serum proteome from SVZ+ (n = 8) and SVZ- (n = 8) GBM patients were labeled with respective CyDyes and separated on IPG strips of pH 4–7; 18 cm followed by separation on 12.5% SDS polyacrylamide gels. 2D-DIGE analysis was performed using DeCyder 2D software version 7.0 and the identities of significantly altered protein spots were established using MALDI-TOF/TOF analysis (Green colour indicates Cy3 labeled protein and red colour indicates Cy5 labeled protein).

Figure 2

Quantitative serum and tissue proteomic analysis of SVZ+ and SVZ- GBMs. Schematic of iTRAQ based proteomic analysis of (a) serum and (b) tissue specimens for the identification of quantitative proteomic alterations. (c) Serum proteomic analysis of SVZ+ (n = 12) and SVZ- (n = 12) GBMs was performed using iTRAQ method. Representative mass spectra for one of the highly up-regulated (apolipoprotein E) and down-regulated (hemoglobin subunit beta) proteins in SVZ+ GBMs (w.r.t SVZ− GBMs). (d) Tissue proteomic analysis of SVZ+ (n = 6) and SVZ- (n = 6) GBMs was performed using iTRAQ approach. Representative mass spectra of two differentially expressed proteins, thymosin beta-4-like protein 3 and annexin A2 in SVZ+ GBM tumors (w.r.t SVZ− GBMs). Relative abundance of thymosin beta-4-like protein 3 and annexin A2 protein are shown in the inset as the intensities of iTRAQ reporter ions.

Figure 3

Tissue proteomic analysis of SVZ+ (n = 6) and SVZ- (n = 6) GBMs using 2D-DIGE method. Cy3 and Cy5 overlapped 2D-DIGE gel image of SVZ+ and SVZ- GBM brain tumor tissue proteome and the 3D-views for the significantly altered proteins. The tissue proteins were separated based on their pI using IPG strips of pH 4–7; 18 cm length, followed by separation based on molecular weights using 12.5% SDS-polyacrylamide gels and image analysis was performed using DeCyder 2D software. Significantly altered protein spots were subjected to MALDI-TOF/TOF analysis to establish their identity. Dot plots and 3D-views represent the relative abundance of significantly altered proteins in the SVZ+ and SVZ- GBMs (Green colour indicates Cy3 labeled protein and red colour indicates Cy5 labeled protein).

Figure 4

Validation of significantly altered serum (hemopexin-HPX) and tissue (annexin A2-ANXA2) proteins in SVZ+ and SVZ- GBMs. (a) Box plots representing the serum levels of HPX in SVZ− (n = 10) and SVZ+ (n = 10) GBMs estimated using ELISA and (b) western blotting results representing the serum levels of HPX in SVZ− (n = 8) and SVZ+ (n = 8) GBMs. (c) Validation of ANXA2 levels in normal brain tissue (n = 2), SVZ+ (n = 3) and SVZ- (n = 3) GBM tumors using western blotting method.