Exploring Novel Therapeutic Opportunities for Glioblastoma Using Patient-Derived Cell Cultures

SIMPLE SUMMARY: Glioblastomas (GBM) are aggressive brain tumors with poor prognosis that need effective treatment. GBMs are characterized by extensive cellular and molecular heterogeneity which are reflected in patient-derived cell cultures, frequently used in testing potential therapeutics. Here, we established GBM-derived cell cultures from fresh tumor specimens and characterized them at the protein and molecular levels. We confirmed the considerable intertumor heterogeneity of GBMs. As the epidermal growth factor receptor (EGFR) is a subject of common oncogenic alterations in GBM, we tested anti-EGFR therapy combined with temozolomide (first-choice medication for GBM) or with doxorubicin (common therapeutic for various solid and blood cancers). We found that GBM-derived cells were more sensitive to a combined therapy than to monotherapy, particularly cells with inactive DNA repair mechanisms. ABSTRACT: Glioblastomas (GBM) are the most common, primary brain tumors in adults. Despite advances in neurosurgery and radio- and chemotherapy, the median survival of GBM patients is 15 months. Recent large-scale genomic, transcriptomic and epigenetic analyses have shown the cellular and molecular heterogeneity of GBMs, which hampers the outcomes of standard therapies. We have established 13 GBM-derived cell cultures from fresh tumor specimens and characterized them molecularly using RNA-seq, immunoblotting and immunocytochemistry. Evaluation of proneural (OLIG2, IDH1(R132H), TP53 and PDGFRα), classical (EGFR) and mesenchymal markers (CHI3L1/YKL40, CD44 and phospho-STAT3), and the expression of pluripotency (SOX2, OLIG2, NESTIN) and differentiation (GFAP, MAP2, β-Tubulin III) markers revealed the striking intertumor heterogeneity of primary GBM cell cultures. Upregulated expression of VIMENTIN, N-CADHERIN and CD44 at the mRNA/protein levels suggested increased epithelial-to-mesenchymal transition (EMT) in most studied cell cultures. The effects of temozolomide (TMZ) or doxorubicin (DOX) were tested in three GBM-derived cell cultures with different methylation status of the MGMT promoter. Amongst TMZ- or DOX-treated cultures, the strongest accumulation of the apoptotic markers caspase 7 and PARP were found in WG4 cells with methylated MGMT, suggesting that its methylation status predicts vulnerability to both drugs. As many GBM-derived cells showed high EGFR levels, we tested the effects of AG1478, an EGFR inhibitor, on downstream signaling pathways. AG1478 caused decreased levels of phospho-STAT3, and thus inhibition of active STAT3 augmented antitumor effects of DOX and TMZ in cells with methylated and intermediate status of MGMT. Altogether, our findings show that GBM-derived cell cultures mimic the considerable tumor heterogeneity, and that identifying patient-specific signaling vulnerabilities can assist in overcoming therapy resistance, by providing personalized combinatorial treatment recommendations.