Acute morphological sequelae of photodynamic therapy with 5-aminolevulinic acid in the C6 spheroid model
- 206 Downloads
Aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) may represent a treatment option for malignant brain tumors. We used a three-dimensional cell culture system, the C6 glioma spheroid model, to study acute effects of PDT and how they might be influenced by treatment conditions.
Spheroids were incubated for 4 h in 100 μg/ml ALA in 5% CO2 in room air or 95% O2 with subsequent irradiation using a diode laser (λ = 635 nm, 40 mW/cm2, total fluence 25 J/cm2). Control groups were “laser only”, “ALA only”, and “no drug no light”. Annexin V-FITC, a marker used for detection of apoptosis, propidium iodide (PI), a marker for necrotic cells and H 33342, a chromatin stain, were used for morphological characterization of PDT effects by confocal laser scanning and fluorescence microscopy. Hematoxylin–eosin staining and TdT-FragEL (TUNEL) assay were used on cryosections. Growth kinetics were followed for 8 days after PDT.
PDT after incubation in 5% CO2 provided incomplete cell death and growth delay in spheroids of >350 μm diameter. However, complete cell death and growth arrest occurred in smaller spheroids (<350 μm). Incubation in 95% O2 with subsequent PDT resulted in complete cell death and growth arrest regardless of spheroid size. In incompletely damaged spheroids viable cells were restricted to spheroid centers. The rate of cell death in all control groups was negligible. Cell death was accompanied by annexin/PI costaining, but there was also evidence for annexin V-FITC staining without PI uptake.
PDT of experimental glioma results in rapid and significant cell death that could be verified as acute necrosis immediately after irradiation. This effect depended on O2 concentration and spheroid size.
KeywordsMalignant glioma 5-ALA photodynamic therapy C6-Spheroids
This work was supported by Deutsche Krebshilfe e.V. Projekt-Nr. 70–2864. We gratefully acknowledge advice from Jorg Cristian Tonn, Prof., MD, Department of Neurosurgery, Herbert Stepp, Ph.D., Tobias Beck, Dipl. phys., Laser Research Laboratory, Ludwig-Maximilians University, Munich, Germany.
- 12.Georgakoudi I, Nichols MG, Foster TH (1999) The mechanism of Photofrin photobleaching and its consequences for photodynamic dosimetry. Photochem Photobiol 65:135–144Google Scholar
- 15.Henderson BW, Dougherty TJ (1992) How does photodynamic therapy work? Photochem Photobiol 55:777–779Google Scholar
- 21.Macecek J, Kolarova H, Psotova J, Bajgar R, Huf M, Nevrelova P, Tomeeka M, Mosinger J (2004) Assessment of cellular damage by comet assay after photodynamic therapy in vitro. Acta Medica (Hradec Kralove) 47:327–329Google Scholar
- 26.Mahaley MS Jr, Mettlin C, Natarajan N, Laws ER Jr, Peace BB (1998) National survey of patterns of care for brain-tumor patients. J Neurosurg 71:826–836Google Scholar
- 45.Wallner KE, Galicich JH, Krol G, Arbit E, Malkin MG (1989) Patterns of failure following treatment for glioblastoma-multiforme and anaplastic astocytoma. Int J Rad Oncol Biol Phys 16:1405–1409Google Scholar