Search

The zebrafish (Danio rerio) is a valuable model organism for studying human biology due to its easy genetic manipulation and small size. It is optically transparent and shares genetic similarities with humans, making it ideal for studying developmental processes, diseases, and drug screening via imaging-based approaches. Solid malignant tumors often contain hypoxic areas that stimulate the release of extracellular vesicles (EVs), lipid-bound structures released by cells into the extracellular space, that facilitate short- and long-range intercellular communication and metastatization. Here we investigate the effects of EVs derived from neuroblastoma (NB), a pediatric solid tumor, on metastatic niche formation using the zebrafish as an in vivo model. Intravascular injection in zebrafish embryos allows a non-invasive visualization of EVs dispersion, uptake, and interactions with host cells. To improve repeatability of our results and ease the injection steps, we used an agarose device replica molded from a custom designed micromilled aluminum mold. We first demonstrated that EVs released under hypoxic conditions promote angiogenesis and are more easily internalized by endothelial cells than those purified from normoxic cells. We also showed that injection of with hypoxic EVs increased macrophages mobilization. We then focused on the caudal hematopoietic tissue (CHT) region of the embryo as a potential metastatic site. After hypoxic EVs injection, we highlighted changes in the expression of mmp-9 and cxcl8b genes. Furthermore, we investigated the ability of NB-derived EVs to prime a metastatic niche by a two-step injection of EVs first, followed by NB cells. Interestingly, we found that embryos injected with hypoxic EVs had more proliferating NB cells than those injected with normoxic EVs. Our findings suggest that EVs released by hypoxic NB cells alter the behavior of recipient cells in the zebrafish embryo and promote metastatic outgrowth. In addition, we demonstrated the ability of the zebrafish embryo to be a suitable model for studying the interactions between EVs and recipient cells in the metastatic process. [ABSTRACT FROM AUTHOR]

Despite contributing to cancer progression, extracellular vesicles (EVs) could serve as potential drug delivery systems in cancer treatment, having the ability to dissolve water-insoluble drugs and facilitate targeted delivery. However, the clinical translation of EVs is still in its infancy. While traditional methods for EV modifications will remain relevant, microfluidic approaches are expected to replace benchtop methods. Taking advantage of lab-on-chip devices, passive cargo loading through microfluidic mixing and incubation may be an important strategy to produce functional engineered EVs. This study focuses on developing a microfluidic device to generate EVs loaded with verteporfin (VP), a hydrophobic porphyrin with potential applications in neuroblastoma (NB) therapy, aiming to enhance its therapeutic effectiveness. The platform ensures perfect mixing and tunable incubation time for mesenchymal stem cell-derived EVs and VP, demonstrating a significantly higher loading efficiency than traditional methods, while operating under gentle conditions that preserve EV integrity and functionality, unlike other microfluidic techniques that involve harsh mechanical or chemical treatments. The VP-loaded EVs (VP-EVs) can then be easily recovered, making them available for subsequent analysis and use. MTT assay confirmed that VP-EVs are more efficient than free VP in reducing the viability of a NB cell line. Finally, immunofluorescence assay and western blot demonstrated a greater reduction in YAP expression after treatment with VP-EVs in an NB cell line when compared to free VP. Being both non-destructive and straightforward, this microfluidic loading technique facilitates its adaptability to a wide spectrum of therapeutic compounds. As a versatile tool, microfluidic technology will help to fully unlock the potential of EVs for speeding up precision medicine and disease treatment.