Acute morphological sequelae of photodynamic therapy with 5-aminolevulinic acid in the C6 spheroid model.
OBJECTIVE
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.


METHODS
Spheroids were incubated for 4 h in 100 microg/ml ALA in 5% CO(2) in room air or 95% O(2) with subsequent irradiation using a diode laser (lambda = 635 nm, 40 mW/cm(2), total fluence 25 J/cm(2)). 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.


RESULTS
PDT after incubation in 5% CO(2) provided incomplete cell death and growth delay in spheroids of >350 microm diameter. However, complete cell death and growth arrest occurred in smaller spheroids (<350 microm). Incubation in 95% O(2) 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.


CONCLUSIONS
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 O(2) concentration and spheroid size.
Zelenkov
Pitr
P
Department of Neurosurgery, Ludwig-Maximilians University, Munich, Germany.
Baumgartner
Reinhold
R
Bise
Karl
K
Heide
Michael
M
Meier
Richard
R
Stocker
Susanne
S
Sroka
Ronald
R
Goldbrunner
Roland
R
Stummer
Walter
W
D000622:Aminolevulinic Acid / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000473:pathology; D005910:Glioma / Q000188:drug therapy* / Q000473:pathology; D051379:Mice; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology*; D018874:Spheroids, Cellular / Q000187:drug effects* / Q000473:pathology; D014407:Tumor Cells, Cultured
D017319:Photosensitizing Agents; D000622:Aminolevulinic Acid