The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment.

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E2 (PGE2) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E2 synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE2 biosynthesis. PGE2 in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE2 dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE2 biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action.

Novel insights into the isolation of extracellular vesicles by anion exchange chromatography.

Extracellular vesicles (EVs) are membrane structures enclosed by a lipid bilayer that are released into the extracellular space by all types of cells. EVs are involved in many physiological processes by transporting biologically active substances. Interest in EVs for diagnostic biomarker research and therapeutic drug delivery applications has increased in recent years. The realization of the full therapeutic potential of EVs is currently hampered by the lack of a suitable technology for the isolation and purification of EVs for downstream pharmaceutical applications. Anion Exchange Chromatography (AEX) is an established method in which specific charges on the AEX matrix can exploit charges on the surface of EVs and their interactions to provide a productive and scalable separation and purification method. The established AEX method using Eshmuno® Q, a strong tentacle anion exchange resin, was used to demonstrate the principal feasibility of AEX-based isolation and gain insight into isolated EV properties. Using several EV analysis techniques to provide a more detailed insight into EV populations during AEX isolation, we demonstrated that although the composition of CD9/63/81 remained constant for tetraspanin positive EVs, the size distribution and purity changed during elution. Higher salt concentrations eluted larger tetraspanin negative vesicles.

Detailed Characterization of Small Extracellular Vesicles from Different Cell Types Based on Tetraspanin Composition by ExoView R100 Platform.

Small extracellular vesicles (sEV) hold enormous potential as biomarkers, drug carriers, and therapeutic agents. However, due to previous limitations in the phenotypic characterization of sEV at the single vesicle level, knowledge of cell type-specific sEV signatures remains sparse. With the introduction of next-generation sEV analysis devices, such as the single-particle interferometric reflectance imaging sensor (SP-IRIS)-based ExoView R100 platform, single sEV analyses are now possible. While the tetraspanins CD9, CD63, and CD81 were generally considered pan-sEV markers, it became clear that sEV of different cell types contain several combinations and amounts of these proteins on their surfaces. To gain better insight into the complexity and heterogeneity of sEV, we used the ExoView R100 platform to analyze the CD9/CD63/CD81 phenotype of sEV released by different cell types at a single sEV level. We demonstrated that these surface markers are sufficient to distinguish cell-type-specific sEV phenotypes. Furthermore, we recognized that tetraspanin composition in some sEV populations does not follow a random pattern. Notably, the tetraspanin distribution of sEV derived from mesenchymal stem cells (MSCs) alters depending on cell culture conditions. Overall, our data provide an overview of the cell-specific characteristics of sEV populations, which will increase the understanding of sEV physiology and improve the development of new sEV-based therapeutic approaches.