AN IN-VITRO MODEL TO STUDY CELLULAR PHOTOSENSITIZER UPTAKE AND PHOTODYNAMIC DOSE-RESPONSE RELATIONSHIPS OF TUMOR-CELLS

Cellular fluorescence intensity (CFI) after incubation with varying co ncentrations of the photosensitizer Photofrin and the photodynamically induced dose-response relationships of hamster melanoma cells (A-MEL- 3) were studied in a recently developed in vitro model. After administ ration of Photofrin to the extracellular serum-free medium, CFI was ev aluated by flow cytometry together with constantly fluorescing latex p articles used as a reference. After 5 min, 50% of maximal CFI was foun d, and after 60 min CFI was maximal. No further increase was obtained during the exposure to Photofrin over the incubation period of 4 h. Du ring this plateau phase, CFI was significantly related to the concentr ation of Photofrin in the extracellular medium (r = 0.94; P<0.001). Su bsequent to increasing intervals of Photofrin exposure, cells were irr adiated with laser light at 630 nm (40 mW/cm(2), 4 J). Cell viability as evaluated by trypan blue exclusion was significantly decreased with increasing concentrations of Photofrin in the medium, and significant ly correlated with CFI during the plateau phase. After photodynamic tr eatment (PDT) cell fluorescence was reduced by about 15%. This was nei ther dose- nor time-dependent. On the basis of these findings we propo se that CFI indicates photosensitizer uptake. This is also supported b y the relation between CFI and phototoxicity. The latter also suggests that CFI might be useful to predict the PDT in vivo efficacy by this in vitro model. Besides measurements of photosensitizer uptake and cel l photoxicity, the model demonstrates an excellent opportunity to stud y the molecular mechanisms of action associated with PTD.