Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR

Abstract

Despite the high incidence of EGFR amplification in patient glioblastoma multiforme (GBM) tissues, only a single GBM cell line, of the many described in the literature, is known to contain and maintain amplified EGFR. Because EGFR mutations in GBM manifest primarily, if not exclusively, in amplified form, it follows that the availability of cell lines with mutation of endogenous EGFR would also be in short supply. In fact, there are no GBM cell lines harboring the common EGFR mutants described in patient GBMs. These observations suggest that in vivo environments select for EGFR amplification, whereas in vitro environments, specifically cell cultures, select against this gene alteration. To contrast directly the fates of EGFR amplification in vivo and in vitro, as well as to examine potential relationships between EGFR amplification and mutation, we have established and maintained GBM explants as xenografts by serial passaging in nude mice. Analysis of EGFR copy number and EGFR mutation status in 11 patient tumors and their corresponding xenografts, as well as the monitoring of EGFR copy number during the establishment of a GBM cell line from a xenograft with amplified EGFR, indicated that selection for EGFR amplification is an in vivo phenomenon. Furthermore, our data indicated that EGFR mutation occurs only in tumors with EGFR amplification and showed that the selection of amplified mutant EGFR over amplified wild-type EGFR as a xenograft occurred rapidly and completely during tumor propagation.

Characterization of ABT-806, a Humanized Tumor-Specific Anti-EGFR Monoclonal Antibody

“…PDX models may more accurately recapitulate the cellular heterogeneity, architectural and molecular characteristics of the primary human tumor compared with standard cell line-passaged xenograft models (26). PDX models are particularly relevant for assessing EGFRvIII-expressing tumors because standard GBM tumor cell lines show loss of EGFRvIII expression during passage in cell culture (17)(18)(19). In contrast with ABT-806, cetuximab binds to EGFRvIII with similar affinity but does not inhibit signaling and showed little or no activity in the EGFRvIII-expressing xenograft and PDX models.…”

“…Because glioblastoma multiforme (GBM) has a high frequency of EGFRvIII expression, the activity ABT-806 was compared with cetuximab in the U87MGde2-7 GBM model. This tumor model was engineered to express EGFRvIII as cell lines do not maintain expression of endogenous EGFRvIII (17,18). Growth of U87MGde2-7 tumors was significantly inhibited by ABT-806 treatment ( Fig.…”

Sequence survey of receptor tyrosine kinases reveals mutations in glioblastomas

“…All primers were synthesized by Integrated DNA Technology (Coralville, IA). To ensure the primers were working effectively, we tested the newly designed primers on glioblastomas with known wild-type EGFR amplification (tumor 15), EGFRvIII amplification (tumor 6), and no EGFR amplification (tumor 16) as assessed by Pandita et al (10).…”

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

“…Additionally, primary orthotopic xenografts grow at a significantly slower rate than heterotopic xenografts. Thus investigators may prefer to transplant cells into the flank of an immunocompromised mouse, where important histological and molecular features may also be retained 4 .…”

“…Human cancer cell lines represent an important first step for in vitro manipulations and in vivo xenografting studies (secondary xenografts) 1 . However, standard cancer cell cultures undergo phenotypic and genotypic transformation [2][3][4] that may not be restored in secondary xenografts 5 . Furthermore, genetic alterations such as isocitrate dehydrogenase (IDH) mutations 6 , distinct stem cell populations 7 and dependency on key signaling pathways 8 can be lost in cancer cell cultures.…”