Malignancy-promoting EGFR overexpression in glioblastoma: in vitro inhibitor susceptibility and establishment of an in vivo model

Introduction:

Glioblastoma multiforme (GBM) is the most aggressive solid brain tumor known. In spite of the current standard therapy consisting of surgical resection and radiochemotherapy the prognosis remains poor primarily due to the infiltrative growth behaviour of the tumor. This invasive capacity of glioblastoma cells as well as their proliferative capacity are influenced by the amplified and overexpressed epidermal growth factor receptor (EGFR). Within the frame of a PhD project the phenotypic output of sustained EGFR activation in glioblastoma will be traced back to the underlying EGFR-based signaling network status. For the in vitro dissection of the EGFR signaling network, a model cell line directly derived from surgical resection of a glioblastoma patient is available. Subsequently, new approaches will be developed to pharmacologically shut down or attenuate oncogenic signaling activity. Finally, with a view to translating the in vitro results to the clinic promising therapy schemes will be tested in an orthotopic mouse glioblastoma model to evaluate if individual therapy schemes are apt to reach their blood brain barrier (BBB)-protected target sites in humans. Summary of project work fields:

1. Mechanistic studies of EGFR-driven glioblastoma (Activation state analysis of crucial signaling pathways with consideration of mutation status, relative contribution of kinase-dependent and -independent EGFR functions)

2. Targeting of the EGFR signaling network (Scrutiny of causal relationships between pathways and development of minimum drug schedules for individual tumors, network behaviour pattern analysis for available cell line models)

3. in vivo transfer of in vitro results (Correlation of and feasibility analysis of drug doses, estimation of required human application doses)

Material and methods:

A cell line displaying amplification of both the wt EGFR and the EGFR vIII deletion variant (HROG33, low-passage in-house material) is used. This cell line has been shown to maintain original tumor-like membrane EGFR expression when cultured under low EGF conditions (e.g. 0.5 ng/ml). In serum-free medium these cells grow as spheroids thus providing a more realistic model compared to adherent cultures. Here the cell line is cultured with three different EGF concentrations (0.5/10/30 ng/ml) to represent different levels of EGFR amplification. For determination of IC values of a panel of inhibitors targeting various effectors of the EGFR network equal volumes of cell suspension were seeded into 96 well plates without prior dissociation of the spheroids. Cells were incubated with the respective inhibitors at different concentrations for six days with one medium exchange after three days. The number of live cells was assessed using a Calcein assay.

Results:

A test run with cell line HROG33 cultured with inhibitor concentrations in the range of published values and those exceeding previously used ones suggests possible decreases in cell survival for some of the single agents mostly at concentrations above those reported using similar systems. This implies relative resistance of the cell line against single-agent approaches. Besides limited statistical power due to technical standardization issues raises the need for further experiments (e.g. direct measurement of drug-induced cell death) to determine reliable IC values for this and additional cell lines cultured with the various EGF concentrations. Pending results of a colony formation assay might underpin successful inhibition of EGFR downstream pathways especially from the viewpoint of the migration-promoting effect of EGFR overactivity.