Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies.
Glioblastoma cell cultures in vitro are frequently used for investigations on the biology of tumors or new therapeutic approaches. Recent reports have emphasized the importance of cell culture type for maintenance of tumor original features. Nevertheless, the ability of GBM cells to preserve EGFR overdosage in vitro remains controversial. Our experimental approach was based on quantitative analysis of EGFR gene dosage in vitro both at DNA and mRNA level. Real-time PCR data were verified with a FISH method allowing for a distinction between EGFR amplification and polysomy 7. We demonstrated that EGFR amplification accompanied by EGFRwt overexpression was maintained in spheroids, but these phenomena were gradually lost in adherent culture. We noticed a rapid decrease of EGFR overdosage already at the initial stage of cell culture establishment. In contrast to EGFR amplification, the maintenance of polysomy 7 resulted in EGFR locus gain and stabilization even in long-term adherent culture in serum presence. Surprisingly, the EGFRwt expression pattern did not reflect the latter phenomenon and we observed no overexpression of the tested gene. Moreover, quantitative analysis demonstrated that expression of the truncated variant of receptor-EGFRvIII was preserved in GBM-derived spheroids at a level comparable to the initial tumor tissue. Our findings are especially important in the light of research using glioblastoma culture as the experimental model for testing novel EGFR-targeted therapeutics in vitro, with special emphasis on the most common mutated form of receptor-EGFRvIII.
Witusik-Perkowska
Monika
M
Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Czechoslowacka 8/10 str, 92-216 Lodz, Poland. monika.witusik-perkowska@umed.lodz.pl
Rieske
Piotr
P
Hułas-Bigoszewska
Krystyna
K
Zakrzewska
Magdalena
M
Stawski
Robert
R
Kulczycka-Wojdala
Dominika
D
Bieńkowski
Michał
M
Stoczyńska-Fidelus
Ewelina
E
Grešner
Sylwia M
SM
Piaskowski
Sylwester
S
Jaskólski
Dariusz J
DJ
Papierz
Wielisław
W
Zakrzewski
Krzysztof
K
Kolasa
Maciej
M
Ironside
James W
JW
Liberski
Paweł P
PP
D000818:Animals; D001932:Brain Neoplasms / Q000473:pathology*; D001973:Bromodeoxyuridine / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002453:Cell Cycle / Q000502:physiology; D049109:Cell Proliferation; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D005909:Glioblastoma / Q000473:pathology*; D006801:Humans; D023421:Models, Animal; D012333:RNA, Messenger / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D018874:Spheroids, Cellular / Q000473:pathology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; C410723:epidermal growth factor receptor VIII; C512478:EGFR protein, human; D066246:ErbB Receptors; D001973:Bromodeoxyuridine
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