Glioblastoma stem-like cell lines with either maintenance or loss of high-level EGFR amplification, generated via modulation of ligand concentration.
OBJECTIVE
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 EGFR-amplified tumor cells, we hypothesized that serum-free and EGF-free culture conditions could promote maintenance of the EGFR amplification.


METHODS
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 high-level 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.
Departments of Neurosurgery and Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Alexander
A
Schulte
Hauke S
HS
Günther
Tobias
T
Martens
Svenja
S
Zapf
Sabine
S
Riethdorf
Clemens
C
Wülfing
Malgorzata
M
Stoupiec
Manfred
M
Westphal
Katrin
K
Lamszus
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D008024:Ligands; D051379:Mice; D008815:Mice, Inbred Strains; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D008024:Ligands; C410723:epidermal growth factor receptor VIII; D004815:Epidermal Growth Factor; D066246:ErbB Receptors