Abstract 568: Effects of irradiation on glioblastoma spheroids cultured in stem cell medium
Authors: Jakobsen Ida P. , Aaberg-Jessen Charlotte , Jensen Stine S. , Nielsen Morten , Halle Dorte , Kristensen Bjarne W. 
Published: June 2012

Abstract
Radiotherapy is an important part of the treatment of glioblastomas – the most frequent and malignant primary brain tumor. However, the current treatment remains palliative and new treatments are urgently needed. Since a subpopulation of cells – cancer stem cells – is believed to be particularly resistant to the current treatment and is most likely the cause of tumor recurrence, the use of models preserving cancer stem cell features is of increasing importance. Recent reports have suggested that glioblastoma spheroids derived from short term cultures in stem cell medium preserve the pheno- and genotype better than when cultured in serum containing medium. With this background, the aim of this study was to investigate the response of irradiation of such spheroids as well as spheroids implanted into organotypic corticostriatal slice cultures in order to have a model preserving the invasive feature of glioblastomas. The response to irradiation (2, 5, 10 and 50 Gy) was investigated in spheroids from a short term culture with cancer stem cell-like properties established in our laboratory (C76) and the U87 glioblastoma cell line for comparison. Cell death was measured by uptake of propidium iodide (PI) and release of lactate dehydrogenase (LDH) and moreover, expression of p53 and the proliferation marker (MIB-1) was assessed by immunohistochemical staining. The formation of secondary spheroids after irradiation was evaluated using a limiting dilution assay as well as a spheroid proliferation assay at non-clonal density. Finally, fluorescently labeled C76 spheroids implanted into slice cultures were irradiated and the response on invasion was evaluated by confocal microscopy and anti-human vimentin immunohistochemical staining. For both C76 and U87 spheroids, the results showed a minor increase in PI uptake with increasing irradiation dose but no increase in LDH release. The expression of p53 also increased, whereas MIB-1 was reduced. The spheroid formation capacity in the limiting dilution assay was higher for C76 than U87 but decreased for both C76 and U87 with increasing irradiation dose along with a decrease in the number and size of secondary formed spheroids in the spheroid proliferation assay. The spheroid formation capacity was clearly best preserved for C76, but otherwise the results did not show major differences for C76 and U87. Using the invasion assay, the invasion of C76 spheroids into brain tissue was not affected by irradiation. In conclusion, irradiation of glioblastoma spheroids derived from short term cultures with cancer stem cell-like properties is feasible and easy to use as an in vitro model. The results might suggest that irradiation also affects tumor cells with a more stem-like phenotype although the effects on the unique tumor stem cells need to be further investigated.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 568. doi:10.1158/1538-7445.AM2011-568