Polysome profiling of a human glioblastoma reveals intratumoral heterogeneity

Glioblastoma (GBM) is one of the most aggressive cancers, with median survival of less than 2 years. Despite of considerable advance in molecular classification of GBMs, no improvements in therapy have been described. The scenario is further complicated by tumor heterogeneity and the relationship among genetic, transcriptional and functional findings. Classically, gene expression has been evaluated by steady-state mRNA, however, this does not take translational control into consideration, which contributes considerably to the composition of the proteome. In this study, we evaluated the transcriptomic and translatomic signature of a GBM obtained from a single patient focusing in tumor heterogeneity. In a sampling of eight fragments, we investigated the translation rates, mTORC1 and ERK1/2 pathways and identified both total and polysome associated mRNAs. An increased translation rate was observed in fragments with high-grade histological features. High-grade histology was also associated with the expression of genes related to extracellular matrix (ECM) and angiogenesis, in both transcriptomes and translatomes. However, genes associated with epithelial to mesenchymal transition and stress response, were observed only in translatomes from high-grade fragments. Overall, our results demonstrate that isolation of translated mRNA can be used to identify biomarkers and reveal previously unrecognized determinants of heterogeneity in GBMs. Six histologically distinct fragments from a secondary glioblastoma, which were classified as high- or low-grade, by an experienced pathologist according to the 2016 World Health Organization Classification of Tumors of the Central Nervous System. Total mRNA and polysome-associated (poly) mRNA were purified using Trizol (Life Technologies). RNA integrity was verified by Agilent 2100 Bioanalyzer with an RNA Pico 6000 microfluidics kit (Agilent Technologies). Gene expression in total and poly mRNA was determined using a two-color microarray-based gene expression (Whole Human Genome 4x44K microarray platform) and the Quick Amp Labeling Kit (Agilent Technologies, CA, USA) according to the manufacturer’s recommendations. Microarrays for total and poly mRNA from each of the six fragments were run in duplicates. RNA from the GBM cell line LN-229 (ATCC® CRL-2611™) was used as reference in all reactions (Cy5 channel).