Background: Extracellular vesicles (EVs) are critical mediators of intercellular communication in development, physiology, and disease. In vivo models such as Drosophila melanogaster, Caenorhabditis elegans, and Danio rerio (zebrafish) now provide powerful platforms to visualize EV dynamics in real time. However, the full potential of these models remains underutilized due to the lack of reliable, cell-specific EV labelling tools and robust EV isolation protocols. Here, we present an optimized workflow for the isolation of EVs from zebrafish larvae and the in vivo labelling of EVs in a cell-type-specific manner. Methods: To isolate EVs from larval zebrafish, we used size exclusion chromatography (SEC). By comparing different tissue digestion methods and performing step-by-step optimisation of sample preparation prior to SEC, we established a novel protocol that enables EV isolation without compromising cell viability. EV size and concentration were assessed by nanoparticle tracking analysis (NTA), with subsequent characterization by transmission electron microscopy (TEM) and Western blotting. To evaluate the sensitivity of our protocol, we treated zebrafish larvae with GW4869, a known inhibitor of EV biogenesis, and assessed the dose-dependent effects on EV release. To specifically label EVs from distinct cell types, we have generated a UAS: CD63-GFP construct which can be expressed under control of the Gal4 transcriptional activator. Results: Through a systematic comparison of tissue dissociation techniques, we identify Bacillus licheniformis protease as a superior alternative to conventional collagenase treatment, which compromises cell integrity. Treatment with GW4869 confirmed that EV biogenesis and release can be inhibited in a dose-dependent manner and demonstrated that our protocol is sensitive enough to detect and quantify changes in EV levels. To enable cell-specific EV tracking in vivo, we combined the UAS: CD63-GFP construct with a radial glia-specific Gal4 driver line, providing a proof-of-concept for targeted EV imaging in intact tissues. Conclusions: These advances provide a versatile toolkit for mechanistic studies of EV function in vivo. The broad availability of cell-type-specific Gal4 driver lines in zebrafish and Drosophila will now allow researchers to trace EV dynamics from virtually any cell type, while our isolation protocol enables rigorous, quantitative EV analyses across developmental and pathological contexts.

Background: Glioblastoma (GBM) is the most malignant and the fastest-progressing type of primary brain tumours. Temozolomide (TMZ) is a chemotherapeutic drug for the treatment of GBM. Extracellular vesicles (EVs) have been recently confirmed to have a substantial role in the GBM, and their contents released from GBM cells have been considered a target for treatment. The purpose of this study is to evaluate the impact of TMZ on heat shock proteins (HSPs) derived from EVs originated from GBM cell lines (U87-MG and LN229) and the significance of EVs in response to chemotherapy in GBM. Methods and Results: NTA, ELISA, and immunoblotting were used to characterization studies of EVs and results showed that U87-MG cells released many EVs compared to LN229 cells. The effect of TMZ treatments on HSPs expression levels were assessed with immunoblotting and was found to be led to increases in HSF-1, Hsp90, Hsp70, Hsp60 and Hsp27 expression in GBM cells and their EV contents, which these increases are related to therapeutic resistance. What is more, in Real-time PCR studies showing which signalling pathways might be associated with these increases, it was observed that TMZ triggered the expression of RAD51 and MDM2 genes in cells and EV contents. More strikingly, we discover a correlation between EV and parental cells in regard of mRNA and protein level in both cell lines as a result of TMZ treatment. Conclusions: Our data suggest of EVs in the treatment of GBM may have potential biomarkers that can be used to investigate the treatment response.