GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity

Abstract

Abnormal activation of the epidermal growth factor receptor (EGFR) due to a deletion of exons 2-7 of EGFR (EGFRvIII) is a common alteration in glioblastoma (GBM). While this alteration can drive gliomagenesis, tumors harboring EGFRvIII are heterogeneous. To investigate the role for EGFRvIII activation in tumor phenotype we used a neural progenitor cell-based murine model of GBM driven by EGFR signaling and generated tumor progenitor cells with high and low EGFRvIII activation, pEGFRHi and pEGFRLo. In vivo, ex vivo, and in vitro studies suggested a direct association between EGFRvIII activity and increased tumor cell proliferation, decreased tumor cell adhesion to the extracellular matrix, and altered progenitor cell phenotype. Time-lapse confocal imaging of tumor cells in brain slice cultures demonstrated blood vessel co-option by tumor cells and highlighted differences in invasive pattern. Inhibition of EGFR signaling in pEGFRHi promoted cell differentiation and increased cell-matrix adhesion. Conversely, increased EGFRvIII activation in pEGFRLo reduced cell-matrix adhesion. Our study using a murine model for GBM driven by a single genetic driver, suggests differences in EGFR activation contribute to tumor heterogeneity and aggressiveness.

Epidermal Growth Factor Receptor Variant III (EGFRvIII) Positivity in EGFR-Amplified Glioblastomas: Prognostic Role and Comparison between Primary and Recurrent Tumors

“…However, previous studies reported that EGFR amplification may be restricted to subpopulations of tumor cells, as determined in cases of glioblastomas with amplification of EGFR and PDGFRA (52,53). With respect to EGFRvIII expression, our data demonstrate that EGFRvIII immunopositivity shows marked regional heterogeneity and is often restricted to subpopulations of tumor cells in glioblastomas, thus confirming previous findings in the GGN patient cohort (17) and in several independent studies (39,42,43,51,54).…”

“…Accumulating preclinical evidence has attributed an important function of EGFRvIII-expressing glioblastoma cells in driving tumor heterogeneity and progression by promoting glioma cell proliferation, invasion, angiogenesis, stemness, and therapy resistance in different model systems (36)(37)(38)(39)(40)(41)(42)(43). In addition, several therapeutic approaches targeting overexpressed wild type EGFR protein or specifically EGFRvIII have already entered, or are about to enter clinical evaluation, including peptide-based vaccines (44)(45)(46), chimeric antigen receptor (CAR) T cells (47,48), as well as anti-EGFR antibodybased approaches (22,49,50).…”

Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion

“…Murine GBM tumorspheres established and demonstrated to have tumor-generating and differentiation capacity in Lindberg O. et al (32) were cultured and maintained as described previously (21,32,33). GBM43, GBM6, and GBM5 are tumorsphere cultures of patient-derived xenografts (PDX).…”

“…Gene expression data from human GBM were downloaded from http://www.cbioportal.org/index.do. Gene expression analysis of mRNA isolated from FACs sorted murine tumors was previously reported (32) and is available in the GEO database (accession number GSE87332). To assess enrichment of GBM subtype genes in murine tumors Gene Set Enrichment Analysis (GSEA) was implemented using the Broad Institute GSEA v2.07 software (http://www.broadinstitute.org/gsea) and subtype signature genes (2).…”

“…Cells were treated with necuparanib (Necu) or PBS (control) for 24 h, dissociated to single cells, counted ×3, diluted to 2.5×10 4 cells per 100 μL of full growth media, and transferred to the laminin coated wells. Cells were allowed to adhere for 2 hours at 37°C, after which media and non-adherent cells were gently removed, wells were gently washed with ice-cold PBS, and adherent cells were fixed with 4% paraformaldehyde (PFA), incubated on ice for 10 min, and stained with crystal violet as previously described (32,39). Transwell invasion assays were performed using BD BioCoat Growth Factor Reduced Matrigel Invasion chambers (BD Biosciences, San Jose, CA) as described previously (39) (see Supplemental Materials and Methods).…”