Morphological and histological changes of glioma cells immediately after 5-aminolevulinic acid mediated photodynamic therapy

Abstract

<title/>Objective: Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a new therapeutic modality for malignant glioma. This study is designed to evaluate acute morphological and histological changes of glioma cells after 5-ALA mediated PDT using fluorescence microscopy and three-dimensional spheroid models.Methods: Monolayer cultures and spheroids (small and large sizes) of C6 (rat glioma cell line) and U251MG (human glioma cell line) cells were co-incubated for 4 hours with 5-ALA (100 μg/ml) and irradiated using a diode laser (635±5 nm, 5-100 mW/cm2, and total light dose 2·5-50 J/cm2). Growth kinetics of the spheroids were followed for 7 days after PDT and morphological changes were assessed by phase contrast microscopy. In addition to hematoxylin and eosin and the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling, various fluorescent dyes including calcein AM, ethidium homodimer-1 (EthD-1), annexin V-FITC, propidium iodide and Hoechst 33342, were used to clarify the mechanism of cell death after PDT.Results: Although PDT spheroids of both cell lines showed growth inhibition during the first few days, resumption of spheroid growth on day 3 after irradiation was observed in most spheroids. Hematoxylin and eosin staining showed cells with condensed chromatin within the superficial layer of the PDT spheroids. In the PDT groups, monolayer cells in the irradiated area were negative for calcein AM and clearly positive for EthD-1. In C6 PDT spheroids, the outer layer of the spheroid was negative for calcein AM and positive for EthD-1. In monolayer cells, cells in the irradiation area in PDT groups were positive for propidium iodide and negative for Hoechst 33342.Conclusion: Light and fluorescence microscopic findings suggested necrotic cell death as an anti-tumor effect in the acute phase after PDT. These findings were observed in both C6 and U251MG monolayer culture cells and spheroids. Although various total light doses resulted in significant differences in the growth rate of spheroids after PDT, resumption of spheroid growth occurred in most spheroids.