The aim of this study was to determine the effects of a pin-to-plate cold atmospheric plasma (CAP) on U-251 MG three-dimensional (3D) glioblastoma spheroids under different conditions. 3D tumorspheres showed higher resistance to the CAP treatment compared to 2D monolayer cells. A single CAP treatment was able to induce cytotoxicity, while multiple CAP treatments augmented this effect. CAP was also able to induce cytotoxicity throughout the tumoursphere, and we identified that reactive oxygen species (ROS) plays a major role, while H 2 O 2 plays a partial role in CAP-induced cytotoxicity in tumourspheres. We conclude that ROS-dependent cytotoxicity is induced uniformly throughout glioblastoma

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