Exploring Responses of Glioblastoma Patient-Derived Cell Cultures to Drugs Reveals New Therapeutic Opportunities

Abstract

Glioblastomas (GBM) are most common, primary brain tumors in adults. Despite advances in neurosurgery, 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 RNAseq, immunoblotting and immunocytochemistry. Evaluation of proneural (OLIG2, IDH1R132H, TP53 and PDGFRα), classical (EGFR) and mesenchymal markers (CHI3L1/YKL40, CD44 and phospho-STAT3), as well as expression of pluripotency (SOX2, OLIG2, NESTIN) and differentiation (GFAP, MAP2, β-Tubulin III) markers revealed the striking inter-tumor heterogeneity of primary GBM cell cultures. Upregulated expression of VIMENTIN, N-CADHERIN and CD44 at 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 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, 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 identifying patient-specific signaling vulnerabilities can assist in overcoming therapy resistance, by providing personalized combinatorial treatment recommendations.

BLM helicase overexpressed in human gliomas contributes to diverse responses of human glioma cells to chemotherapy

“…Cell Culture and treatments LN18, LN229, T98, U87-MG and U251 cell lines were purchased from the ATCC, USA, and cultured in DMEM medium (Dulbecco's modi ed Eagle medium, Thermo Fisher Scienti c, USA). Primary glioblastoma cell lines (WG4 and WG9) were derived as described (24) and cultured in DMEM/F12 GlutaMAX media (Thermo Fisher Scienti c, USA). Normal human astrocytes (NHA, Lonza) were cultured in medium containing ABM TM Basal Medium (CC-3187) and AGM TM SingleQuots TM Supplements (CC-4123) from Lonza.. All culture media were supplemented with 10% FBS (Gibco, USA), antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin) and cultured in a humidi ed atmosphere of CO 2 /air (5%/95%) at 37 °C.…”

Regulatory networks driving expression of genes critical for glioblastoma are controlled by the transcription factor c-Jun and the pre-existing epigenetic modifications

“…Each patient signed a written consent form for the use of tumor tissues, and the samples were then anonymized. Human GBM patient-derived glioma primary cultures WG12 were generated and cultured as described [58] in DMEM/Nutrient Mixture F-12, GlutaMAX ™ medium, supplemented with 10% FBS (Gibco Life Technologies, Rockville, MD, USA) and antibiotics. See Additional file 2: Fig.…”