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

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Abstract

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 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.

Results

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.

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 O2 concentration and spheroid size.

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Acknowledgments

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.

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Authors and Affiliations

  1. Department of Neurosurgery, Ludwig-Maximilians University, Munich, Germany

    Pitr Zelenkov & Roland Goldbrunner

  2. Laser Research Laboratory, Ludwig-Maximilians University, Munich, Germany

    Pitr Zelenkov, Reinhold Baumgartner, Michael Heide, Richard Meier, Susanne Stocker & Ronald Sroka

  3. Institute for Neuropathology, Ludwig-Maximilians University, Munich, Germany

    Karl Bise

  4. Department of Neurosurgery, Heinrich-Heine University, Moorenstr. 5, 40225, Düsseldorf, Germany

    Walter Stummer

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  1. Pitr Zelenkov
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Correspondence to Walter Stummer.

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Zelenkov, P., Baumgartner, R., Bise, K. et al. Acute morphological sequelae of photodynamic therapy with 5-aminolevulinic acid in the C6 spheroid model. J Neurooncol 82, 49–60 (2007). https://doi.org/10.1007/s11060-006-9252-8

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