Aspects related to the response of cells to photodynamic therapy (PDT) have been well studied in cell cultures, which often grow in monolayers. In this work, we propose a spheroidal model of U87MG and SKBR3 cells designed to mimic superficial tumor tissue, small spheroids (<500 µm) suitable for confocal fluorescence microscopy, and larger spheroids (>500 µm) that can be xenografted onto quail chorioallantoic membrane (CAM) to study the effects of PDT in real time. Hypericin was used as a model molecule for a hydrophobic photosensitizer that can produce singlet oxygen (1O2). 1O2 production by hypericin was detected in SKBR3 and U87MG spheroid models using a label-free technique. Vital fluorescence microscopy and flow cytometry revealed the heterogeneity of caspase-3 distribution in the cells of the spheroids. The levels of caspase-3 and apoptosis increased in the cells of spheroids 24 h after PDT. Lactate dehydrogenase activity was evaluated in the spheroids as the most reliable assay to detect differences in phototoxicity. Finally, we demonstrated the applicability of U87MG spheroids on CAM in photodiagnostics. Overall, the variability and applicability of the prepared spheroid models were demonstrated in the PDT study.
Keywords: apoptosis; cancer; caspase-3; hypericin; imaging; photodynamic therapy; singlet oxygen; spheroid.