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

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% O₂), cells cultured at atmospheric oxygen (20% O₂), 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.

Keywords: 5% oxygen; DNA repair; RNA sequencing; cell cycle; glioblastoma; glioblastoma stem cells; hypoxia; inflammation; spheroid; temozolomide.

Figure 1

Efficiency and time required for developing GBM-derived cell cultures. Cell cultures derived from 28 freshly resected GBM fragments were maintained at 5% and 20% oxygen. (A) Graph depicts a number of cultures obtained at a single oxygen condition (5% or 20% oxygen), both conditions (5% and 20% oxygen) and a number of failed attempts. (B) The time required for cell line establishment is calculated in days, from a day of tumour isolation till the first biobanking (details in Methods and Materials section). Twenty cultures were grown as spheroids, and four cultures (SOS19K6, WUM17, WUM18 and WUM33) were grown as adherent cells. (C) Heatmap depicting genes characteristic for four GBM subtypes in 10 GBMs, 5% oxygen culture and 20% oxygen culture.

Figure 2

Expression of stemness and differentiation marker and EMT genes in GBMs and cells cultured at 5% and 20% oxygen. (A) Heatmap shows an expression of selected stemness (pink) and differentiation (grey) genes in tumour and its derivative cell lines at 5% oxygen and 20% oxygen, with row clustering applied. (B,C) Volcano plots depict the expression of a group of EMT genes and mesenchymal GBM subtype genes in cells grown at 5% and 20% oxygen. Genes differentially expressed between conditions (expression higher than log2 Fold equal to 0.5, and adjusted p lower than 0.05) are annotated in red.

Figure 3

Most molecular pathways and genes differentiating transcriptomic profiles in cell cultures and corresponding GBMs. KEGG pathways enrichment analysis of genes differentially expressed in culture and a corresponding tumour. (A) Genes most down-regulated in 20% cell cultures vs. tumours. (B) Genes most down-regulated in 5% cell cultures vs. tumours. (C) Genes most up-regulated in 20% cell cultures vs. tumour. (D) Genes most up-regulated in 5% cell cultures vs. tumours. Black circle diameter reflects a number of up- or down-regulated genes belonging to the particular KEGG pathway. Volcano plots showing the global difference in gene expression between 20% culture and tumour (E) and 5% culture and tumour (F). Genes with the lowest and highest difference in the particular cell lines set and tumour are annotated on the plots, e.g., genes for which log2 fold change was below −10.5 and above 2.25, adjusted p-value < 1 × 10⁻²⁰ for 20% oxygen cultures vs. tumour, and genes for which log2 fold change was below -10 and above 2 and adjusted p-value < 1 × 10⁻²⁰ for 5% oxygen cultures vs. tumour).

Figure 4

Expression of cell cycle and DNA repair genes in GBM cell cultures and parental tumours. Cnetplots of selected KEGG pathways genes comparing gene expression in GBMs and 20% (A) or 5% (B) cell cultures. Black dots represent a number of statistically differentially expressed genes in the given pathway in the particular comparison. (C) Flow cytometry analysis of DNA content in GCW04, GCW21 and GCW22 cell cultures maintained at 5% or 20% oxygen. Cell cycle distribution is shown as a percentage of cells at the indicated cell cycle phase. (D) Exemplary histograms showing DNA content in three cell cultures kept at 20% or 5% oxygen.

Figure 5

Most distinct molecular pathways in cell cultures maintained at 5% and 20% oxygen. (A) KEGG pathways most up-regulated in 5% vs. 20% cell cultures. (B) Cnetplot showing genes from most differentiating KEGG pathways. Black dots represent a number of genes falling into the pathway category from those statistically differentially expressed between 5% and 20% cell cultures.

Figure 6

Identification of 30 most differentially expressed genes (DEGs) in tumours, cell cultures, and the TCGA dataset. (A) Volcano plots showing a global difference in gene expression between 20% culture and 5% culture. Genes with the lowest and highest expression. Cut-off just for the colour of significance is adjusted p-value < 0.05. For the genes annotated on the plot log2 fold change was below-0.5 and above 2.25 for genes down-regulated and up-regulated in 5% oxygen cultures, respectively. (B) Heatmap created for 30 most differentially expressed genes in the tumour, 5% and 20% cell cultures. (C) Heatmap depicting expression levels of top 30 DEGs in the TCGA dataset comprising normal brain, and gliomas of WHO grades I–IV.

Figure 7

Effects of oxygen tension on temozolomide toxicity. GBM cells cultured at 5% or 20% oxygen were treated for 72 hr with TMZ at increasing concentrations or a relevant amount of DMSO as a solvent. Cell viability was calculated as a percentage of the viability of vehicle-treated cultures. (A–C) GBM cell cultures s GCW04, GCW20 and GCW21 maintained at 5% or 20% oxygen were seeded and treated as spheres. (D,E) GCW20 and GCW21 cell lines were seeded and treated as adherent cultures. (F) GCW20a cells were cultured from the beginning as a monolayer. All experiments were repeated two or three times. Data are presented as mean, error bars represent S.E. Statistical significance was evaluated with 2-way ANOVA with Sidak’s correction, * p < 0.05, ** p < 0.01, *** p < 0.001.