Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity.

Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma.

A novel patient stratification strategy to enhance the therapeutic efficacy of dasatinib in glioblastoma.

BACKGROUND: Glioblastoma is the most common primary malignancy of the central nervous system with a dismal prognosis. Genomic signatures classify isocitrate dehydrogenase 1 (IDH)-wildtype glioblastoma into three subtypes: proneural, mesenchymal, and classical. Dasatinib, an inhibitor of proto-oncogene kinase Src (SRC), is one of many therapeutics which, despite promising preclinical results, have failed to improve overall survival in glioblastoma patients in clinical trials. We examined whether glioblastoma subtypes differ in their response to dasatinib and could hence be evaluated for patient enrichment strategies in clinical trials. METHODS: We carried out in silico analyses on glioblastoma gene expression (TCGA) and single-cell RNA-Seq data. In addition, in vitro experiments using glioblastoma stem-like cells (GSCs) derived from primary patient tumors were performed, with complementary gene expression profiling and immunohistochemistry analysis of tumor samples. RESULTS: Patients with the mesenchymal subtype of glioblastoma showed higher SRC pathway activation based on gene expression profiling. Accordingly, mesenchymal GSCs were more sensitive to SRC inhibition by dasatinib compared to proneural and classical GSCs. Notably, SRC phosphorylation status did not predict response to dasatinib treatment. Furthermore, serpin peptidase inhibitor clade H member 1 (SERPINH1), a collagen-related heat-shock protein associated with cancer progression, was shown to correlate with dasatinib response and with the mesenchymal subtype. CONCLUSION: This work highlights further molecular-based patient selection strategies in clinical trials and suggests the mesenchymal subtype as well as SERPINH1 to be associated with response to dasatinib. Our findings indicate that stratification based on gene expression subtyping should be considered in future dasatinib trials.

The pro-apoptotic Bcl-2 family member Harakiri (HRK) induces cell death in glioblastoma multiforme.

Harakiri (HRK) is a BH3-only protein of the Bcl-2 family and regulates apoptosis by interfering with anti-apoptotic Bcl-2 and Bcl-xL proteins. While its function is mainly characterized in the nervous system, its role in tumors is ill-defined with few studies demonstrating HRK silencing in tumors. In this study, we investigated the role of HRK in the most aggressive primary brain tumor, glioblastoma multiforme (GBM). We showed that HRK is differentially expressed among established GBM cell lines and that HRK overexpression can induce apoptosis in GBM cells at different levels. This phenotype can be blocked by forced expression of Bcl-2 and Bcl-xL, suggesting the functional interaction of Bcl-2/Bcl-xL and HRK in tumor cells. Moreover, HRK overexpression cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a known tumor-specific pro-apoptotic agent. Besides, secondary agents that augment TRAIL response, such as the histone deacetylase inhibitor MS-275, significantly increases HRK expression. In addition, GBM cell response to TRAIL and MS-275 can be partly abolished by HRK silencing. Finally, we showed that HRK induction suppresses tumor growth in orthotopic GBM models in vivo, leading to increased survival. Taken together, our results suggest that HRK expression is associated with GBM cell apoptosis and increasing HRK activity in GBM tumors might offer new therapeutic approaches.