Time-lapse microscopy studies of bystander effects induced by photosensitization

Reactive oxygen species (ROS) are involved in the pathogenesis of many critical diseases and are also utilized as cytotoxic agents in a variety of treatments for eradication of diseased tissue, including cancer. Oxidative stress ensues when the level of ROS in a system exceeds the antioxidant capacity. Oxidative stress can have local (direct) and long-range (bystander) effects in cells and tissue and this research was carried out to determine the spatial and temporal nature of the photosensitized bystander effect using time-lapse fluorescence microscopy. By initiating photosensitization in only a portion of the microscopic imaging field it was possible to differentiate direct from bystander effects in EMT-6 murine breast cancer cells in 6-well plates. Elevated ROS levels are seen immediately following photodynamic treatment in direct cells with a delayed increase in oxidative stress observed in bystander cells. Cytotoxicity is also seen at earlier times in direct cells and occurs in bystander cells in a delayed fashion. These studies confirm the existence of a bystander effect following photosensitization and implicate mediators capable of diffusing in an intercellular manner from directly photosensitized cells to bystander cells and also implicate increased oxidative stress as a mechanistic factor in generating damage in bystander cells.