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

Iwona A Ciechomska; Kamil Wojnicki; Bartosz Wojtas; Paulina Szadkowska; Katarzyna Poleszak; Beata Kaza; Kinga Jaskula; Wiktoria Dawidczyk; Ryszard Czepko; Mariusz Banach; Bartosz Czapski; Pawel Nauman; Katarzyna Kotulska; Wieslawa Grajkowska; Marcin Roszkowski; Tomasz Czernicki; Andrzej Marchel; Bozena Kaminska

doi:10.3390/cancers15051562

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

Cancers (Basel). 2023 Mar 2;15(5):1562. doi: 10.3390/cancers15051562.

Authors

Iwona A Ciechomska¹, Kamil Wojnicki¹, Bartosz Wojtas¹, Paulina Szadkowska^{1

2}, Katarzyna Poleszak¹, Beata Kaza¹, Kinga Jaskula¹, Wiktoria Dawidczyk¹, Ryszard Czepko³, Mariusz Banach³, Bartosz Czapski^{1

2}, Pawel Nauman⁴, Katarzyna Kotulska⁵, Wieslawa Grajkowska⁵, Marcin Roszkowski⁵, Tomasz Czernicki⁶, Andrzej Marchel⁶, Bozena Kaminska¹

Affiliations

¹ Laboratory of Molecular Neurobiology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw, Poland.
² Postgraduate School of Molecular Medicine, Medical University of Warsaw, 02-091 Warsaw, Poland.
³ Department of Neurosurgery, Scanmed S.A. St. Raphael Hospital, 30-693 Cracow, Poland.
⁴ Institute of Psychiatry and Neurology, 02-957 Warsaw, Poland.
⁵ Department of Pathology, The Children's Memorial Health Institute, 04-736 Warsaw, Poland.
⁶ Neurosurgery Department and Clinic, Medical University of Warsaw, 02-091 Warsaw, Poland.

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.

Keywords: EGFR inhibitor (AG1478); EMT; MGMT; STAT3; cancer stem cells; doxorubicin; glioblastoma; temozolomide.

Abstract

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