EFFECT OF PHOTODYNAMIC TREATMENT OF HUMAN ENDOTHELIAL-CELLS ON CELL-VOLUME AND CELL VIABILITY

Photodynamic therapy (PDT) has yielded promising results in the treatm ent of malignant tumors. However, the mechanisms leading to tumor dest ruction during PDT are still not completely understood. In addition to effects on the microcirculation, damage to cellular structures has be en observed following exposure of cells to PDT. A phenomenon preceding these events might possibly be cell swelling. We therefore studied th e influence of treatment with Photofrin(R) (PF) and laser light on vol ume changes and cell viability of endothelial cells. Endothelial cells were obtained from human umbilical cord veins (HUVEC) by an adaption of the method of Maruyama. After subcultivation the cells were harvest ed and transferred as a cell suspension into a specially designed incu bation chamber. Cells received either PF in concentrations of 1.5 or 3 .0 mu g/ml and laser illumination 60 min post incubation (630 nm; 40 m W/cm(2), 4 Joule), PF alone, or laser treatment only. Following start of PF incubation and after phototreatment cell samples were taken for volume measurements using flow cytometry, and for studies of cellular morphology using scanning electron microscopy. Simultaneously, cell vi ability was monitored by the trypan blue exclusion test and the colori metric MTT assay. Both control groups, HUVEC receiving PF or laser tre atment alone, revealed constant cell volumes and cell viability during the entire course of the experiment. After PDT (60 min post-incubatio n) with 1.5 and 3.0 mu g PF/ml cell volume of HUVEC was increased at 1 5 min to 122%+/-6% and 140%+/-10% of baseline (100%), at 60 min to 152 %+/-9% and 134%+/-18%, respectively (p<0.01). The number of viable cel ls was significantly reduced of samples treated with 1.5 and 3.0 mu g PF/ml at 15 min after PDT to 81%+/-3% and 76%+/-10% of baseline (100%) , at 60 min after PDT to 32%+/-14% and 20%+/-15%, respectively (p<0.01 ). Scanning electron microscopy of cells exposed to PDT following 60 m in incubation with Photofrin (3.0 mu g/ml) revealed significant cell d amage. At the highest PF concentration HUVEC showed loss of microvilli and formation of blebs on the cellular surface. Our study demonstrate s that PDT induces a significant increase in endothelial cell volume a nd a loss of cell viability. We suggest that swelling and damage of en dothelial cells following PDT is a primary event finally contributing to cessation of blood flow and subsequent necrosis of tumors.