Simple SummaryGlioblastoma multiforme (GBM) is a deadly brain tumour with little progression in the way of improved quality of life in patients. The possible root cause for this dilemma stems from the inefficiency of traditional two-dimensional (2D) cell-based models used in evaluating potential anticancer agents. In this paper, we investigated the proximity with which three-dimensional (3D) GBM models encapsulate key aspects of clinical GBM samples that have been made available in many genetic data banks. The analysis identified a number of key genes highly expressed in both 3D GBM cell-based models and GBM clinical samples that may play a role in GBM therapy resistance. In conclusion, the findings suggest that 3D GBM cell-based models serve as reliable models for clinical GBM samples.AbstractA paradigm shift in preclinical evaluations of new anticancer GBM drugs should occur in favour of 3D cultures. This study leveraged the vast genomic data banks to investigate the suitability of 3D cultures as cell-based models for GBM. We hypothesised that correlating genes that are highly upregulated in 3D GBM models will have an impact in GBM patients, which will support 3D cultures as more reliable preclinical models for GBM. Using clinical samples of brain tissue from healthy individuals and GBM patients from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Chinese Glioma Genome Atlas (CGGA), and Genotype-Tissue Expression (GTEx) databases, several genes related to pathways such as epithelial-to-mesenchymal transition (EMT)-related genes (CD44,TWIST1,SNAI1,CDH2,FN1,VIM), angiogenesis/migration-related genes (MMP1,MMP2,MMP9,VEGFA), hypoxia-related genes (HIF1A,PLAT), stemness-related genes (SOX2,PROM1,NES,FOS), and genes involved in the Wnt signalling pathway (DKK1,FZD7) were found to be upregulated in brain samples from GBM patients, and the expression of these genes were also enhanced in 3D GBM cells. Additionally, EMT-related genes were upregulated in GBM archetypes (wild-typeIDH1R132) that historically have poorer treatment responses, with said genes being significant predictors of poorer survival in the TCGA cohort. These findings reinforced the hypothesis that 3D GBM cultures can be used as reliable models to study increased epithelial-to-mesenchymal transitions in clinical GBM samples.