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

Fabrizio Di Giuseppe,Roberto Pallini,Mariachiara Zuccarini,Roberta Di Pietro,Patricia Giuliani,Lucia Ricci-Vitiani,Paolo De Sanctis,Renata Ciccarelli,Marzia Carluccio,Stefania Angelucci

doi:10.3390/biomedicines9020146

Fabrizio Di Giuseppe, Roberto Pallini + Show 8 more

Open Access

https://doi.org/10.3390/biomedicines9020146

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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.

Highlights

For many decades, it has been thought that communications among cells relied on soluble molecules released from the cells themselves, which can act in autocrine/paracrine/endocrine fashions
We isolated two subtypes of extracellular vesicles (EVs) by applying the sequential centrifugal ultrafiltration (SCUF) technique to conditioned medium (CM) removed from cultured GBM stem-like cells (GSCs) derived from primary GBM of two patients
Our study has identified two main types of EVs from the CM of GSCs deriving from human primary GBM, which differed in terms of size and transported signals

Summary

Introduction

It has been thought that communications among cells relied on soluble molecules released from the cells themselves, which can act in autocrine/paracrine/endocrine fashions. Biomedicines 2021, 9, 146 endosome-derived exosomes (Exos, 30–100 nm size) and plasma membrane-derived microvesicles (MVs, 100–1000 nm size) These nanoparticles are secreted by virtually all cell types and carry a heterogeneous number of substances, deriving from intracellular compartments and including different types of nucleic acids, proteins, and lipids. In tumors, besides apoptotic bodies (1000–5000 nm) released from cells undergoing programmed cell death [3], a novel population of large EVs, named oncosomes, has been identified. These vesicles have a diameter of 1–10 μm and seem to contain oncogenic material and to be cancer-specific [4,5]

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