iTRAQ-based Proteomics Profiling Reveals Increased Metabolic Activity and Cellular Cross-talk in Angiogenic Compared with Invasive Glioblastoma Phenotype

Abstract

Malignant gliomas (glioblastoma multiforme) have a poor prognosis with an average patient survival under current treatment regimens ranging between 12 and 14 months. The tumors are characterized by rapid cell growth, extensive neovascularization, and diffuse cellular infiltration of normal brain structures. We have developed a human glioblastoma xenograft model in nude rats that is characterized by a highly infiltrative non-angiogenic phenotype. Upon serial transplantation this phenotype will develop into a highly angiogenic tumor. Thus, we have developed an animal model where we are able to establish two characteristic tumor phenotypes that define human glioblastoma (i.e. diffuse infiltration and high neovascularization). Here we aimed at identifying potential biomarkers expressed by the non-angiogenic and the angiogenic phenotypes and elucidating the molecular pathways involved in the switch from invasive to angiogenic growth. Focusing on membrane-associated proteins, we profiled protein expression during the progression from an invasive to an angiogenic phenotype by analyzing serially transplanted glioma xenografts in rats. Applying isobaric peptide tagging chemistry (iTRAQ) combined with two-dimensional LC and MALDI-TOF/TOF mass spectrometry, we were able to identify several thousand proteins in membrane-enriched fractions of which 1460 were extracted as quantifiable proteins (isoform- and species-specific and present in more than one sample). Known and novel candidate proteins were identified that characterize the switch from a non-angiogenic to a highly angiogenic phenotype. The robustness of the data was corroborated by extensive bioinformatics analysis and by validation of selected proteins on tissue microarrays from xenograft and clinical gliomas. The data point to enhanced intercellular cross-talk and metabolic activity adopted by tumor cells in the angiogenic compared with the non-angiogenic phenotype. In conclusion, we describe molecular profiles that reflect the change from an invasive to an angiogenic brain tumor phenotype. The identified proteins could be further exploited as biomarkers or therapeutic targets for malignant gliomas.

Comprehensive characterization of glioblastoma tumor tissues for biomarker identification using mass spectrometry-based label-free quantitative proteomics

“…We have identified a number of annexins in our study to be overexpressed in GBM tumors including ANXA1 (log 2 ratio 3.91), ANXA2 (log 2 ratio 2.78), and ANXA5 (log 2 ratio 1.35). Previous studies have shown that ANXA2 and ANXA5 promote glioma cell invasion and tumor progression and are shown to be overexpressed in angiogenic compared with invasive GBM tumors (57,70). ANXA1, the annexin with the greatest magnitude of GBM overexpression in our study, has been found to be a major component in necrotic tumor cell-derived stimulants of the growth of GBM via the activation of formyl peptide receptor (FPR1) (69).…”

Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma

“…We have developed a clinically highly relevant human GBM model in rats that fully reflects the growth pattern of human tumors in situ, including extensive infiltration into the brain parenchyma (major pattern of invasion along fiber tracts and blood vessels), prominent angiogenesis, and necrosis (13)(14)(15). Comparative genomic hybridization studies have confirmed the genetic similarity between xenografted tumors and the corresponding human tumors (Fig.…”

“…To visualize human tumor cells in the rat brain and to determine their distribution outside the tumor core, we used a human-specific nestin staining, a protein expressed by the vast majority of GBM cells (15,22,23). Interestingly, treated tumors revealed a more homogeneous morphology compared with the controls (Fig.…”

LC-MS/MS Analysis of Differentially Expressed Glioblastoma Membrane Proteome Reveals Altered Calcium Signaling and Other Protein Groups of Regulatory Functions

“…Some earlier studies have focused on membrane proteome but using GBM cell lines and xenograft models (16,17). The aim of the current study was to build a high confidence, tumor-associated, differentially expressed membrane protein panel from clinical specimens of GBM, as a first step toward their targeted validation in a clinical setting by us and others in the community.…”