Synergistic cytotoxicity effect of ultrasound and plasma microbubble in glioblastoma 3D tumor sphere model

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.