Synergistic GSC Radiosensitization by PARP1 Inhibitor

PARP1 inhibitor Olaparib/Lynparza/AZD2281 (Adooq Bioscience, A10111; PARPi) and TMZ (Sigma Aldrich, T2577) was diluted with DMSO to make 10 mM stocks, which were stored at −20 °C. DMSO was added as a vehicular control to maintain consistent DMSO concentrations. DMSO, Olaparib and TMZ were diluted in GSC culture media to the final intended concentrations and 1 mL of the drug/DMSO dilutions was added to the desired wells. To firstly determine the inhibitory dose of Olaparib, 3D GSCs were incubated with Olaparib for 1 h followed by ionizing radiation (IR) or sham irradiated using a Caesium-137 (¹³⁷Cs) Irradiator (CIS IBL437c) to a total dose of 5 Gy. SDS-PAGE analysis of subsequent lysates determined inhibition through loss of αPARylation (500 nM) in response to IR. For combination chemosensitisation studies, 3D GSCs were treated with DMSO or Olaparib (500 nM) and incubated for one hour before TMZ (5 mM) was added (where required) followed by exposure to TTFields (72 h, 200 kHz, 1.33 V/cm RMS), or incubated at 37 °C for the untreated ‘sham irradiated’ control. Cell survival was determined as detailed above.