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The combined application of ultrasound (US) and plasma microbubble (PMB) is a promising technology against glioblastoma (GBM). Here, we determined the effects of US and PMB (alone and combined) on U‐251 MG GBM and A431 epidermoid carcinoma spheroids under different conditions. The synergistic treatment of US‐ and PMB‐induced tumor sphere cell death in a time‐, dose‐, treatment frequency‐, and reactive oxygen species dependent manner, while multiple treatments augmented this effect. Synergistic treatments significantly reduced three‐dimensional GBM spheroid regrowth and cell proliferation while inducing cytotoxicity, spheroid shrinkage, and damage to the tumor microenvironment. PMB‐induced cytotoxicity throughout the tumor sphere, likely via long‐lived reactive oxygen and nitrogen species (H2O2, NO2−, and NO3−) and other reactive species. [ABSTRACT FROM AUTHOR]

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 H2O2 plays a partial role in CAP‐induced cytotoxicity in tumourspheres. We conclude that ROS‐dependent cytotoxicity is induced uniformly throughout glioblastoma and epidermoid tumourspheres by direct CAP treatment. [ABSTRACT FROM AUTHOR]