Preservation of the Hypoxic Transcriptome in Glioblastoma Patient-Derived Cell Lines Maintained at Lowered Oxygen Tension

SIMPLE SUMMARY: The extent of tumour oxygenation is crucial for glioblastoma progression and the effectiveness of radio- and chemotherapy. Patient-derived in vitro cell cultures are the mainstay of molecular biology research, the discovery of new therapeutic targets, and drug testing. Therefore, mirroring as many aspects of in vivo settings as possible, including oxygen concentration, is desired. Here we analyse the effect of oxygen tension on the transcriptome of numerous patient-derived GBM cells and demonstrate that cells cultured in lowered oxygen tension express more genes indicative of higher levels of hypoxia, metabolic adaptation, stemness and tumour progression than cells growing in standard, atmospheric oxygen concentration. The same transcriptomic pattern was also found in primary GBM samples. Its specificity for GBMs was confirmed using the public TCGA dataset. Our data strongly argue for the benefit of lower oxygen tension during culturing of patient-derived GBM cells to preserve oxygen-sensitive pathways in GBM. The proposed approach better mimics certain aspects of GBM pathophysiology than traditional cultures and may advance GBM research in finding a cure. ABSTRACT: Despite numerous efforts aiming to characterise glioblastoma pathology (GBM) and discover new therapeutic strategies, GBM remains one of the most challenging tumours to treat. Here we propose the optimisation of in vitro culturing of GBM patient-derived cells, namely the establishment of GBM-derived cultures and their maintenance at oxygen tension mimicking oxygenation conditions occurring within the tumour. To globally analyse cell states, we performed the transcriptome analysis of GBM patient-derived cells kept as spheroids in serum-free conditions at the reduced oxygen tension (5% O(2)), cells cultured at atmospheric oxygen (20% O(2)), and parental tumour. Immune cells present in the tumour were depleted, resulting in the decreased expression of the immune system and inflammation-related genes. The expression of genes promoting cell proliferation and DNA repair was higher in GBM cell cultures when compared to the relevant tumour sample. However, lowering oxygen tension to 5% did not affect the proliferation rate and expression of cell cycle and DNA repair genes in GBM cell cultures. Culturing GBM cells at 5% oxygen was sufficient to increase the expression of specific stemness markers, particularly the PROM1 gene, without affecting neural cell differentiation markers. GBM spheroids cultured at 5% oxygen expressed higher levels of hypoxia-inducible genes, including those encoding glycolytic enzymes and pro-angiogenic factors. The genes up-regulated in cells cultured at 5% oxygen had higher expression in parental GBMs compared to that observed in 20% cell cultures, suggesting the preservation of the hypoxic component of GBM transcriptome at 5% oxygen and its loss in standard culture conditions. Evaluation of expression of those genes in The Cancer Genome Atlas dataset comprising samples of normal brain tissue, lower-grade gliomas and GBMs indicated the expression pattern of the indicated genes was specific for GBM. Moreover, GBM cells cultured at 5% oxygen were more resistant to temozolomide, the chemotherapeutic used in GBM therapy. The presented comparison of GBM cultures maintained at high and low oxygen tension together with analysis of tumour transcriptome indicates that lowering oxygen tension during cell culture may more allegedly reproduce tumour cell behaviour within GBM than standard culture conditions (e.g., atmospheric oxygen tension). Low oxygen culture conditions should be considered as a more appropriate model for further studies on glioblastoma pathology and therapy.