Abstract In-vitro studies show that low frequency sound waves can interact with hemo-porphyrin to produce reactive oxygen species (ROS). This phenomenon induces tumor cell death in various cell types in-vitro and in xenograft models. However, reproducible in-vitro models with adequate parameterization in sonodynamic therapy remain scarce. This study evaluated ROS production and apoptotic marker expression in medulloblastoma cells under varying treatment durations and ultrasound power using a bench-top focused ultrasound (FUS) device. UW426 medulloblastoma cells were exposed to 5-aminolevulinic acid (5-ALA) at different concentrations for up to 24 hours. The incubation time yielding peak protoporphyrin IX (PPIX) fluorescence was identified via fluorescence-activated cell sorting (FACS). A 1.5 MHz transducer operated at 0.91 MHz was targeted to the bottom of a 96-well plate, and pressure wave delivery was verified by a hydrophone. Cells were treated with 5-ALA alone, FUS alone, or 5-ALA + FUS, then incubated for 24 hours before measuring ROS and cell death markers. ROS levels were measured with the CellROX Green Kit and Annexin V expression with the Dead Cell Stain Kit, both by FACS. Peak PPIX fluorescence in UW426 cells occurred after 12 hours of incubation with 200 µg/mL 5-ALA. The highest ROS levels were detected using 0.91 MHz at a total sonication time of 120s and an average power of 6 W. Significant cell death occurred at 3 W and 6 W with 90 s and 120 s sonication. This bench-top setup successfully induced ROS and cell death in UW426 cells. Establishing this robust in-vitro model allows further exploration of sonication parameters and supports future translation to clinical models.
HOVEY et al. (Fri,) studied this question.