Experimental Glioma Simulated by C6 Spheroids: Photodynamic Therapy with 5-ALA

P.V. Zelenkov1,4, R. Baumgartner2, S. Stoker2, R. Sroka2, R. Meier2, K. Wise3, M. Heide2, V. Stummer1,5

(1 Grosshadern Neurosurgical Clinic, Germany;
2 Laboratory of Laser Studies, Germany;
3 Institute of Neuropathology, Ludwig-Maximilian University, Munich, Germany;
4 Moscow Medical Academy named after I.M. Sechenov, Moscow, Russia;
5 Neurosurgical Clinic of the Heinrich-Heins University Dusseldorf, Germany.)

Photodynamic therapy (PDT) is a prospective method of treatment of cerebral malignant tumors. An effect of its combined use with 5-aminolevulinic acid (5-ALA) on glioma was studies with the help of a 3-D cellular structure, known as C6 spheroids. They were incubated in 100 g/ml of 5-ALA under conditions of 5% CO2 or 100% O2 during 4 hours. It was followed by their irradiation with a diode laser (A=635 pulses, a dose of 25 J/cm2 in 40mW/cm). Fluorescent staining of unfixed spheroids with Annexin-FITC, Propidium Jodid, Hoechst 33342 (Oncogene, Germany) was developed for estimating the PDT effect and revealing necrosis, apoptosis and living cells. Optic sections were obtained by means of using a confocal laser scanning microscope and a fluorescent microscope. The results of application of the above method were confirmed by frozen sections, stained with hematoxylin-eosin, as well as by the TUNEL immunohistochemical method (Oncogene, Germany) for revealing apoptosis. A rate of the spheroid growth was estimated during a week after irradiation. A level of cell death (CD) was assessed immediately after irradiation and in a week.

Results. In case of incubation in 5% CO2 PDT resulted in CD of 70% and a slower growth of spheroids with a diameter of 400 , whereas spheroids of a smaller diameter (about 300 m) were characterized by CD of 100% and complete discontinuation of growth. PDT, carried out after incubation in 100% O2, led to CT of 100% and complete discontinuation of growth, which were not dependent on a size of spheroids. An apoptosis-like process accounted for 90% of CD in all the cases. When cell death was incomplete, living cells were watched only in a spheroid center. There were CD of 10% and a constant growth in control groups.

Conclusions. PDT of experimental glioma causes marked CD during a short period of time. The CD level and the spheroid growth rate depend on O2 concentration and a size of spheroids at the moment of irradiation. The new method allows to carry out a reliable and easy estimation of an effect of different PDT regimens.