Proteomic Characterization of Two Extracellular Vesicle Subtypes Isolated from Human Glioblastoma Stem Cell Secretome by Sequential Centrifugal Ultrafiltration

Abstract

Extracellular vesicles (EVs) released from tumor cells are actively investigated, since molecules therein contained and likely transferred to neighboring cells, supplying them with oncogenic information/functions, may represent cancer biomarkers and/or druggable targets. Here, we characterized by a proteomic point of view two EV subtypes isolated by sequential centrifugal ultrafiltration technique from culture medium of glioblastoma (GBM)-derived stem-like cells (GSCs) obtained from surgical specimens of human GBM, the most aggressive and lethal primary brain tumor. Electron microscopy and western blot analysis distinguished them into microvesicles (MVs) and exosomes (Exos). Two-dimensional electrophoresis followed by MALDI TOF analysis allowed us to identify, besides a common pool, sets of proteins specific for each EV subtypes with peculiar differences in their molecular/biological functions. Such a diversity was confirmed by identification of some top proteins selected in MVs and Exos. They were mainly chaperone or metabolic enzymes in MVs, whereas, in Exos, molecules are involved in cell–matrix adhesion, cell migration/aggressiveness, and chemotherapy resistance. These proteins, identified by EVs from primary GSCs and not GBM cell lines, could be regarded as new possible prognostic markers/druggable targets of the human tumor, although data need to be confirmed in EVs isolated from a greater GSC number.

Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles

“…Additionally, the low number of EVs secreted by cells could be too diluted in the culture medium; or, in clinical studies, only a limited volume of biofluids is available, in which the EVs at low quantities could be masked by the highly abundant matrix components and become undetectable . Many strategies have been widely adopted for EV separation, including ultracentrifugation (UG) or differential centrifugation, ultrafiltration (UF), immuno-affinity capture (IAC), size-exclusion chromatography (SEC), and polymer-based precipitation . However, the heterogeneous EVs cannot be simply distinguished by only one property, like one surface marker, size, density, or surface hydrophobicity, and it is difficult to completely remove the abundant matrix components.…”

The Activation of Mesenchymal Stem Cells by Glioblastoma Microvesicles Alters Their Exosomal Secretion of miR-100-5p, miR-9-5p and let-7d-5p

“…EVs are particles released by cells and delimited by a lipid bilayer [ 9 ] that can be divided into 2 main populations: exosomes, which are very small (50–150 nm) membrane-derived vesicles generated through the endocytic pathway and microvesicles (MVs)(100–1000 nm) that are generated by blebbing of the plasma membrane [ 10 , 11 , 12 ]. They carry proteins, DNA, RNA as well as metabolites that constitute critical messengers contributing to tumor growth, dissemination and drug resistance [ 13 , 14 ].…”

Tumor-derived extracellular vesicles as messengers of natural products in cancer treatment

“…For example, the purity of EVs extracted from urine by ultrafiltration and SEC combined with AF4 is much higher than that obtained by a single method 91 . Continuous centrifugation and ultrafiltration were used to isolate the EVs secreted by human glioblastoma multiforme stem cells and to identify the microvesicles and exosomes subtypes 92 . Size-based EVs purification can also be designed as an automated operating system, such as Exo-pos.…”