EMT6/Ro spheroids approximately 500 µm in diameter were subjected to photodynamic therapy administered at various incident radiation fluence rates. Following 24 h incubation with 10 µg/ml Photofrin, groups of spheroids were irradiated at 630 nm with an identical fluence of 60 J/cm2, delivered at fluence rates ranging from 25 to 200 mW/cm2. After treatment, spheroids were dissociated, cell yields were determined, and surviving cells were assayed for their colony-forming ability. A surviving fraction was calculated for each treatment group by computing the product of the fractional cell yield and the plating efficiency. The results exhibit a strong dependence on the fluence rate, with surviving fractions decreasing from approximately 0.5 to 0.07 as the incident fluence rate was lowered from 200 to 25 mW/cm2. These data were analyzed using a mathematical model of photochemical oxygen consumption in spheroids undergoing photodynamic therapy. Calculations showed that therapy-induced oxygen consumption creates hypoxic volumes within which cells would be protected from singlet oxygen-mediated damage and that the magnitude of these hypoxic volumes depends on the radiation fluence rate. The fluence rate dependence of the spheroid cell survival was consistent with such an interpretation.
Research was supported by USPHS Grants CA36856 and S07RR05403-30.