Glioblastoma-derived extracellular vesicles facilitate transformation of astrocytes via reprogramming oncogenic metabolism

Background: Glioblastoma (GBM) may arise from brain astrocytes through a multistep process that occurs by the temporal accumulation of genetic mutations. Here, we explore whether GBM-derived extracellular vesicles (EVs) may facilitate neoplastic transformation and malignant growth of astrocytes. Methods: We utilized conditioned medium (CM) of glioma cell and stem cell, its sequential filtration, diverse cell-based assays, RNA sequencing, and metabolic assays to compare the effects of EV-containing and EV-depleted CM on the transformation of human and mouse astrocytes in vitro and tumor growth in vivo. Results: GBM EVs facilitated the neoplastic growth of astrocytes pre-transformed by oncogenic mutations or viruses but were unable to transform normal human and mouse astrocytes. GBM EVs induced proliferation, self-renewal, and colony formation of pre-transformed astrocytes, and enhanced astrocytoma growth in a mouse allograft model. Analysis of extracellular RNAs (exRNAs) in GBM identified multiple full-length mRNAs encoding ribosomal proteins, oxidative phosphorylation, and glycolytic factors. GBM EVs appear to reprogram astrocyte metabolism by inducing a shift in gene expression that may be partly associated with EV-mediated mRNA transfer from glioma cells. Conclusions: Our study suggests a novel EV/exRNA-mediated mechanism contributing to astrocyte transformation via metabolic reprograming and implicates horizontal mRNA transfer in this process. Human transformed astrocytes (HTAs), mouse transgenic EGFRVIII; CDKN2-/-; PTENlox/lox astrocytes (EC), and mouse transgenic EGFRVIII; CDKN2-/-; PTEN-/- astrocytes (ECP) were treated with either 10X complete glioblastoma conditioned medium (10x EV(+) CM) or the one with extracellular vesicles depleted (10X EV(-) CM). Six days later, RNA was purified with Norgen BioTek total RNA purification kit with on-column DNase treatment. The libraries were prepared and sequenced on Illumina HiSeq X with PE150 mode to produce approximately 20 M reads per sample by Novogene Co., Ltd. The reads were quality controlled with FastQC, trimmed with Trimmomatic, aligned with HiSat2 to hg38 or mm10, and quantified with HTSeq-count using the Galaxy platform.