The objective of the present study was to evaluate the sensitivity of the bladder transitional cell carcinoma (TCC) to hypericin PDT in a 3-D system using multicellular tumor spheroids. The photodynamic response in RT-112 human bladder TCC spheroids was also compared to 2-D cultured monolayer cells. Following a 2-4 h incubation with 8-30 μM hypericin, spheroids or monolayer cells were irradiated at the light dose of 12 J/cm2, delivered at a fluence rate of 10-100 mW/cm2. The PDT effects were evaluated using a clonogenic assay. The results show that compared with the cells in a monolayer, cells in spheroids were dramatically less sensitive to hypericin PDT (<2000-fold). Studies of fluorescence microphotographs of centrally cut frozen sections of hypericin-exposed spheroids showed a gradient in hypericin concentration from the peripheral to the central region of the spheroid. Although it can be suggested that heterogeneity of drug uptake might be responsible for the observed resistance of spheroid to hypericin PDT, hypericin sensitized spheroids that were dissociated prior to light irradiation were as sensitive as the monolayer cells to hypericin PDT, suggesting that other factors such as oxygen depletion might be responsible for the resistance of spheroids to hypericin PDT.

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