Glioblastoma Stem–like Cell Lines with Either Maintenance or Loss of High-Level EGFR Amplification, Generated via Modulation of Ligand Concentration

Abstract

Purpose: Despite the high incidence of epidermal growth factor receptor (EGFR) gene amplification and rearrangement in glioblastomas, no suitable cell line exists that preserves these alterations in vitro and is tumorigenic in immunocompromised mice. On the basis of previous observations that glioblastoma cells cultured with serum lose the EGFR amplification rapidly and that EGF can inhibit the growth of EGFRamplified tumor cells, we hypothesized that serum-free and EGF-free culture conditions could promote maintenance of the EGFR amplification.Experimental Design: Cells from EGFR-amplified glioblastomas were taken into culture using neural stem cell conditions with modifications, including varying oxygen concentrations and omission of routine EGF supplementation.Results: High-level EGFR amplification was rapidly lost in 5 glioblastoma cultures supplemented with EGF, whereas it was preserved in cultures from the same tumors established without EGF. Cultures from 2 glioblastomas developed into pairs of cell lines, with either stable maintenance or irreversible loss of highlevel EGFR amplification in the majority of cells. One EGFR-amplified cell line preserved expression of the receptor variant EGFRvIII. Cell lines with high-level EGFR amplification/EGFRvIII expression formed highly aggressive tumors in nude mice, whereas nonamplified cell lines were either nontumorigenic or grew significantly more slowly. In contrast, nonamplified cell lines proliferated faster in vitro. All cell lines responded to erlotinib, with inhibition of receptor activation and proliferation but partly different effects on downstream signaling and migration.Conclusions: Isogenic glioblastoma cell lines maintaining stable differences in EGFR/EGFRvIII status can be derived by varying exposure to EGF ligand and reflect the intratumoral genetic heterogeneity. Clin Cancer Res; 18(7); 1901-13. Ó2012 AACR.

The MET Oncogene Is a Functional Marker of a Glioblastoma Stem Cell Subtype

“…Notably, neurospheres derived from EGFR amp/vIII tumors displayed a decreased-or in a few cases an even normalnumber of EGFR gene copies, consistent with previous and recent data (17,18). As EGFR amplification is usually detected only in a fraction of glioblastoma cells (data not shown and ref.…”

“…39), these findings can be explained by in vitro negative selection of clones harboring EGFR amplification and positive selection of clones with a normal EGFR gene, coexisting in the same tumor. Growth of clones with normal/low number of EGFR gene copies might be favored by concentrations of exogenous EGF (20 ng/mL), likely exceeding those in brain tissues (18).…”

Comparative Expression Analysis Reveals Lineage Relationships between Human and Murine Gliomas and a Dominance of Glial Signatures during Tumor PropagationIn Vitro

“…To generate xenografts, primary tumors are commonly subjected to a number of in vitro passages, which have been shown to render them to a potential selection bias (45), whereas for example, MGMT methylation was found to be similar in primary tumors and glioma spheres (46). We compared the transcriptome and protein expression in corresponding genotypes in genetically defined cells, and a controlled and reproducible environment.…”

The Definition of Primary and Secondary Glioblastoma

“…Glioblastomas may show significant intertumoral and intratumoral heterogeneity, both histologically and genetically (80)(81)(82) and this may also apply to glioma-initiating cells (82,83). This heterogeneity reflects genomic instability and, occasionally, focal new clones arising as a result of additional genetic alterations can be seen histologically (84,85).…”