ABROGATION OF EXOSOME BIOGENESIS SIGNIFICANTLY AFFECTS CELL MOTILITY IN HETEROGENOUS SUB-POPULATIONS OF PAEDIATRIC-TYPE DIFFUSE HIGH-GRADE GLIOMA

Abstract Paediatric-type diffuse High-Grade Gliomas (PDHGG) are highly heterogenous tumours comprised of distinct cell sub-populations co-existing within the same tumour mass. We have shown that primary patient-derived sub-clones, as well as optical-barcoded sub-clones, function as an interconnected network conferring an aggressive phenotype. Here, we explored the role of exosomes in mediating PDHGG inter-clonal communication. A comprehensive characterization of 7 optical-barcoded single cell-derived clones obtained from two patient-derived cell lines (one DMGH3K27-altered and one diffuse high-grade paediatric-type glioma H3WT), confirmed extensive genomic and phenotypic heterogeneity. Live single-cell tracking in 3D migration and invasion assays demonstrated the key role of the inter-clonal crosstalk in driving a more aggressive phenotype. To determine the exosome role in this crosstalk, we first characterised them in terms of size, marker expression and cargo. Moreover, we demonstrated that exosomes were actively internalized by the sub-clones. Exosomal proteomic analysis showed differential protein contents implicated in the regulation of biological processes such as focal adhesion and extracellular matrix organization. The analysis of exosomal miRNome did not show differentially expressed miRNAs between sub-clones, however, specific and distinct exosomal miRNAs were found uniquely expressed by each sub-clone. The abrogation of the exosome biogenesis by GW4869 phospholipase inhibitor did not affect sub-clones viability, but significantly inhibited their motility, when cultured individually and more prominently in co-culture condition. Analysis of the exo-miRNAs uniquely expressed by the sub-clones highlighted a set of target genes regulating cell motility/invasion/migration. These target genes were differentially expressed when sub-clones were co-cultured compared to mono-culture. Moreover, the expression levels of these genes (e.g. CD44, PTRZ, GLI3, NTRK2) were significantly modulated upon GW4869 treatments. In conclusion, our study highlights the importance of the exosomes in the inter-clonal communication and suggests that interfering with the exosome biogenesis may be a valuable strategy to inhibit cell motility in PDHGG.