Spatial and temporal dynamics of in vitro photodynamic cell killing: extracellular hydrogen peroxide mediates neighbouring cell death.

Photodynamic killing of a cell population is generally considered to result from direct effects that occur in each cell. In some scenarios this may be an over-simplification and the potential for cell-cell signaling processes to contribute to the response of a population to photodynamic stress is addressed in this paper. Photodynamic killing of EMT6 cells in culture was studied in time and space using computerized time-lapse microscopy. The rate of cell killing was dependent on the fluence with both rapid and slower processes evident, the proportion of the former increasing with fluence. The spatial distribution of cell death was non-random and for the slow cell killing process was found to occur preferentially in the vicinity of dead or dying cells, suggesting a local signaling process. An inhibitory effect of extracellular catalase indicated the involvement of hydrogen peroxide in the spread of cell death and NADPH oxidase was determined as the principal source of hydrogen peroxide. This cell signaling pathway was observed for membrane-bound and mitochondrial photosensitizers but not for a nuclear photosensitizer. These secondary cell signalling pathways extend the oxidative damage to cells in space and time.