Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies

Abstract

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.

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes

“…Those results reflect findings reported in the literature on the loss of EGFR vIII and EGFR WT amplicons [26, 29, 48-51]. The neurospheres made of glioblastoma cells proved difficult to maintain over prolonged periods of time (more than 15 passages), with senescence described as the predominant cause, irrespective of the culture conditions [30, 52, 53]. Fact that spheroids can not to be cultured infinitely in vitro forced Johnson and colleagues to use immunocompromised mice as neurosphere “incubators,” with serial xenografting of the neurospheres between animals [28].…”

Intratumoral heterogeneity of receptor tyrosine kinases EGFR and PDGFRA amplification in glioblastoma defines subpopulations with distinct growth factor response

“…For the most commonly altered RTKs, EGFR and PDGFRA, amplification is associated with gene rearrangements that produce constitutively active receptor forms often expressed together with wild-type alleles (16,19,26). Unequal segregation of amplified alleles and regionally variable selection pressure provide one mechanism to explain the intratumoral heterogeneity of RTK copy number and expression of mutant forms seen in GBM (19,27,28).…”

Targeting EGF receptor variant III: tumor-specific peptide vaccination for malignant gliomas

“…Tumor sphere culture is an in vitro method of culturing cells modeled after the neurosphere method utilized to propagate neural stem cells. It is thought to enrich for cells that are tumorigenic and capable of self-renewal [119]. Furthermore, our own work has shown that in vitro cultured stem cells derived from GBM are able to retain EGFRvIII expression, that EGFRvIII expression is restricted to a small subpopulation within the tumor and strongly correlates with CD133 expression, and that CD133 + /EGFRvIII + cells show enhanced tumor-sphere formation, secondary sphere formation and tumor initiation in NOD/SCID mice [120].…”