P2X7 receptor antagonism by AZ10606120 significantly depletes glioblastoma cancer stem cells in vitro.

Glioblastoma is the most aggressive and lethal primary brain malignancy with limited treatment options and poor prognosis. Self-renewing glioblastoma cancer stem cells (GSCs) facilitate tumour progression, resistance to conventional treatment and tumour recurrence. GSCs are resistant to standard treatments. There is a need for novel treatment alternatives that effectively target GSCs. The purinergic P2X receptor 7 (P2X7R) is expressed in glioblastomas and has been implicated in disease pathogenesis. However, the roles of P2X7R have not been comprehensively elucidated in conventional treatment-resistant GSCs. This study characterised P2X7R channel and pore function and investigated the effect of pharmacological P2X7R inhibition in GSCs. Immunofluorescence and live cell fluorescent dye uptake experiments revealed P2X7R expression, and channel and pore function in GSCs. Treatment of GSCs with the P2X7R antagonist, AZ10606120 (AZ), for 72hours significantly reduced GSC numbers, compared to untreated cells. When compared with the effect of the first-line conventional chemotherapy, temozolomide (TMZ), GSCs treated with AZ had significantly lower cell numbers than TMZ-treated cultures, while TMZ treatment alone did not significantly deplete GSC numbers compared to the control. AZ treatment also induced significant lactate dehydrogenase release by GSCs, indicative of treatment-induced cytotoxic cell death. There were no significant differences in the expression of apoptotic markers, Annexin V and cleaved caspase-3, between AZ-treated cells and the control. Collectively, this study reveals for the first time functional P2X7R channel and pore in GSCs and significant GSC depletion following P2X7R inhibition by AZ. These results indicate that P2X7R inhibition may be a novel therapeutic alternative for glioblastoma, with effectiveness against GSCs resistant to conventional chemotherapy.

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Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

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Declaration of Competing Interest TJO has received support from the National Health and Medical Research Council (APP1176426), The Medical Research Future Fund, The National Institute of Neurological Disorders and Stroke and Monash University. He has been supported by research grants and consultancies to his institution from Eisai, UCB Pharma, Praxis Precision Medicines, BioGen and Supernus. MM has served on advisory board for Merck and has received speaker honoraria from Merch and Biogen. Her institution receives funding from Merck, Australian National Health Medical Research Council, Brain Foundation, Charles and Sylvia Viertel Foundation, and MS Research Australia.