Extracellular vesicle-mediated transfer of CLIC1 protein is a novel mechanism for the regulation of glioblastoma growth.

Matteo Setti,Germana Rappa,Aurelio Lorico,Barbara Ortensi,Alexandre Mironov,Lorenzo Fornasari,Mario Faretta,Giuliana Pelicci,Massimiliano Del Bene,Daniela Osti,Cristina Richichi,Alessandro Cuomo,Galina Beznoussenko,Tiziana Bonaldi

doi:10.18632/oncotarget.5105

Abstract

Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and lethal among brain tumors. Recently we have demonstrated that Chloride Intracellular Channel-1 (CLIC1) is overexpressed in GBM compared to normal tissues, with highest expression in patients with poor prognosis. Moreover, CLIC1-silencing in cancer stem cells (CSCs) isolated from human GBM patients negatively influences proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential. Here we show that CLIC1 exists also as a circulating protein, secreted via extracellular vesicles (EVs) released by either cell lines or GBM-derived CSCs. Extracellular vesicles (EVs), comprising exosomes and microvesicles based on their composition and biophysical properties, have been shown to sustain tumor growth in a variety of model systems, including GBM. Interestingly, treatment of GBM cells with CLIC1-containing EVs stimulates cell growth both in vitro and in vivo in a CLIC1-dose dependent manner. EVs derived from CLIC1-overexpressing GBM cells are strong inducers of proliferation in vitro and tumor engraftment in vivo. These stimulations are significantly attenuated by treatment of GBM cells with EVs derived from CLIC1-silenced cells. However, CLIC1 modulation appears to have no direct role in EV structure, biogenesis and secretion. These findings reveal that, apart from the function of CLIC1 cellular reservoir, CLIC1 contained in EVs is a novel regulator of GBM growth.

Highlights

Glioblastoma (GBM) is the most aggressive among tumors of glial origin and represents, with a median patient survival of 14 months, a social and economic burden [1, 2]
To further confirm Chloride Intracellular Channel-1 (CLIC1) protein release by GBM cells, we employed U87 MG cells overexpressing CLIC1 protein fused to Green Fluorescent Protein (GFP) at the N-terminal end (U87 CLIC1 GFP). 24 hours after cell plating, we collected culture medium and immunoprecipitated GFP-tagged CLIC1 protein
A single band of 50 kDa corresponding to CLIC1 GFP fusion protein was detected in the medium from U87 CLIC1 GFP cells, while no band was detected in the medium from mock transfected cells, confirming that exogenous CLIC1 GFP protein was released in the medium (Fig. 1B)

Summary

Introduction

Glioblastoma (GBM) is the most aggressive among tumors of glial origin and represents, with a median patient survival of 14 months, a social and economic burden [1, 2]. GBM is composed by a subpopulation of cancer stem cells (CSCs) able to fuel and sustain tumor development and progression, as well as tumor cells and non-neoplastic parenchymal cells, comprising vascular cells, microglia, peripheral immune cells, deeply intermingled throughout the tumor mass [3,4,5]. All these cells communicate with each other via the secretion and uptake of a number of factors that play a pivotal role in controlling the course of pathology. The recent identification of CLIC1 outside the cellular environment, in biological fluids like plasma [21] and serum [22, 23], urine [24] and in the medium of different cell lines often in association with secreted EVs [25,26,27,28,29,30,31], fostered the hypothesis that a circulating CLIC1 protein is detectable in the context of brain tumors in association to EVs, and it might potentiate the function of CLIC1 intracellular reservoir as a novel regulator of GBM growth

Methods

Results

Conclusion