Artikel
Phototherapy of C6 glioma spheroids with 5-aminolevulinic acid using a novel technique for the evaluation of phototherapy effects
Phototherapie von C6-Spheroiden nach Gabe von 5-Aminolävulinsäure unter Verwendung einer neuartigen Technik zur Wirkungserfassung der Phototherapie
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Autoren
Veröffentlicht: | 23. April 2004 |
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Gliederung
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Objective
5-ALA-mediated phototherapy (ALA-PT) is under investigation as an adjunct for malignant glioma therapy. However, little is known on the variables involved in this modality. For further insight, we have used a three-dimensional cell culture - the C6 glioma spheroid model, to study the effects of ALA-PT.
Methods
Spheroids were incubated for 4 hours in 100 mcg/ml 5-ALA in 5% CO2 or 100% O2 and where then irradiated using a 633 nm diode laser with 40 mW/cm2 to a total dose of 25 J/cm2. Necrotic, apoptotic and vital cells were assessed using a newly developed method of fluorescence staining with Annexin-FITC, Propidium Jodid and Hoechst 33342 (Oncogene, Germany). Fluorescence images were acquired by confocal laser scanning microscopy and fluorescence microscopy. In order to confirm the reliability of the new method, we performed hematoxylin-eosin staining and TUNEL assays (Oncogene, Germany) on cryosections. Cell death was estimated in the first hour after ALA-PT and one week later. Spheroid's growth kinetics was observed during 8 days following ALA-PT.
Results
ALA-PT after incubation in 5% CO2 provides 70% cell death and growth delay in spheroids of 400 mcm diameter. However, 100% cell death and a complete stop of growth occurs in smaller spheroids (300 mcm). Incubation in 100% O2 with following ALA-PT resulted in 100% cell death and a complete stop of growth regardless of spheroid size. In all cases, cell death after ALA-PT was established to be an apoptosis-resembling process. With 70% cell death, vital cells were restricted to the centre of spheroids. Cell death in control groups, i.e. "laser only", "5-ALA only" and "intact group" did not exceed 10%, and permanent growth was recorded.
Conclusions
ALA-PT of experimental glioma results in rapid and significant cell death, which depends on O2 concentration and spheroid size. We established a modern, easy and time-saving method for estimation of PT effects, that can be used experimentally and will be tested clinically.