gms | German Medical Science

Objective: Glioblastoma multiforme (GBM) is characterized by high rates of relapse (~90 %) after surgery despite patients receiving radio- and chemotherapy. The main reason for relapse is the survival of glioblastoma stem cells (GSCs) which is characterized by increased expression of specific resistance genes. Our aim is to identify and target those genes in personalized medicine.

Methods: GSCs were isolated from GBM patients via neurosphere formation assay and characterized by IF-staining for established markers (GFAP, Nestin) and their ability to differentiate. Their sensitivity towards radio- and/or chemotherapy was compared with patient-derived GBM cell lines (n=4), using WST-1 after 5 days or 3D Cell Viability Assay after 7 days, respectively. To determine gene expression correlated with resistance mRNA expression of various genes were analyzed by qPCR, GeneChip microarrays, and RNAseq analysis.

Results: For combined treatment of patient-derived GBM cell lines, semi-lethal doses of 5 Gy and 100 µM temozolomide (TMZ) were used. TMZ and radiation showed an additive effect and reduced cell viability by 78 ± 1 %. In contrast to the primary GBM cell lines, GSCs showed significantly reduced sensitivity towards radio- and chemotherapy. Sensitivities were determined by spheroid size and Cell Titer Glo^® Assays. After 5 days following radiation with 10 Gy, viability of primary GBM cells was reduced by 96 ± 0.02 %, whilst viability and spheroid size of GSCs was reduced by only 37 ± 14 % after treatment applying the same radiation dose. Resistant GSCs were treated with 350 µM TMZ and 30 Gy irradiation. Compared to control cells, the combination therapy reduced cell viability and spheroid size by 69 ± 8 % according to both methods, while single treatment affected cell viability significantly less. Despite a relatively harsh treatment, GSC spheroid sizes still increased slightly (≈1.5-fold) over 7 days of cultivation. Differential expression of genes induced by the latter treatment involve genes for ABC transporters as well as HAS2, CD44, and ITGA6. In addition, results of differential analysis using microarray and NGS techniques will be presented.

Conclusion: To minimize occurrence of GBM relapse it is essential to eliminate GSCs, which are particularly resistant to standard therapy. A workflow for the systematic determination of relevant genes in GBM patient material has been developed and provides a platform for further clinical use in order to optimize the existing therapy options.