Elsevier

Biochimie

Volume 155, December 2018, Pages 11-15
Biochimie

Short communication
Temozolomide affects Extracellular Vesicles Released by Glioblastoma Cells

https://doi.org/10.1016/j.biochi.2018.02.007Get rights and content

Highlights

  • Blood of brain tumour patients contains more circulating extracellular vesicles.

  • Release of extracellular vesicles is enhanced in resistant tumour cells exposed to temozolomide.

  • Mass spectrometry allows the analysis of the extracellular vesicle cargo.

  • Temozolomide augments cell adhesion-related proteins in extracellular vesicles.

Abstract

Glioblastoma multiforme (GBM) is the most aggressive primary tumour within the brain as well as the most common and lethal cerebral cancer, mainly because of treatment failure. Indeed, tumour recurrence is inevitable and fatal in a short period of time. Glioblastoma stem-like cells (GSCs) are thought to participate in tumour initiation, expansion, resistance to treatments, including to the alkylating chemotherapeutic agent temozolomide, and relapse. Here, we assessed whether extracellular vesicles (EVs) released by GSCs could disseminate factors involved in resistance mechanisms. We first characterized EVs either circulating in peripheral blood from newly diagnosed patients or released by patient-derived temozolomide-resistant GSCs. We found that EVs from both sources were mainly composed of particles homogeneous in size (50–100 nm), while they were more abundant in liquid biopsies from GBM patients, as compared to healthy donors. Further, mass spectrometry analysis from GSC-derived EVs unveiled that particles from control and temozolomide-treated cells share core components of EVs, as well as ribosome- and proteasome-associated networks. More strikingly, temozolomide treatment led to the enrichment of EVs with cargoes dedicated to cell adhesion processes. Thus, while relatively inefficient in killing GSCs in vitro, temozolomide could instead increase the release of pro-tumoral information.

Introduction

Glioblastoma multiforme (GBM) is the most common and aggressive primary cerebral tumour in adults. Treatment consists of surgical resection, radiotherapy, and adjuvant chemotherapy with the alkylating agent, temozolomide (TMZ) [1]. In spite of current treatment strategies, patients invariably relapse due to resistance and have a fatal outcome after 7–10 months [1]. A subpopulation of cells of the tumour mass exhibiting stem properties, named GSCs (for Glioblastoma Stem-like Cells) are thought to participate in tumour initiation, and expansion [2,3]. They were also described with the ability to escape treatments and thus repopulate the tumour mass [2,3].

Extracellular vesicles (EVs) are stable, lipid bilayer spherical structures, ranging from 30 to 1000 nm, and release either from the cell surface or are generated through the endosomal compartments, into the surrounding microenvironment. They may access biofluids, such as blood, urine, cerebrospinal fluid, and saliva [4]. EVs were shown to carry a wide range of nucleic acids (DNA, mRNA, miRNA), proteins, lipids and other metabolites, together with some common markers reflecting their biogenesis (CD9, CD63, CD81, ALIX…). They display individualities, which allow for tracking of their cellular origin, and mirror the state of the host cell [5]. They have also emerged as particularly important means of intercellular communication within the brain and in GBM pathogenesis [[6], [7], [8]]. Of note, circulating EVs might carry tumour mutations and might be used as predictive markers [[8], [9], [10]].

To better understand the mechanisms involved in glioblastoma resistance to therapies, we seek to determine the nature of circulating EVs from newly diagnosed GBM patients and explore TMZ-resistant patient-derived GSC-released EVs.

Section snippets

Human plasma

Peripheral blood was collected from newly diagnosed GBM patients before treatment (French glioblastoma biobank #1476342v2, CHU Angers, France) and from healthy donors (Etablissement Français du Sang, Nantes, France). All signed informed consent. Plasma were stored at −20 °C.

Cell culture

Patient-derived glioblastoma stem-like cells (GSC1, GSC4, and GSC9) were obtained from primary glioblastoma resection, as previously described [7]. GSCs were maintained as spheres in NS34 medium (DMEM-F12, with N2, G5, and

Characterization of Extracellular Vesicles from glioblastoma

In order to evaluate EVs that could emanate from GSCs and disseminate throughout the body [8,12,13], we first characterized EVs from peripheral blood and compared them to the ones obtained in primary cell cultures. EVs were purified from healthy donors and newly diagnosed GBM patient liquid biopsies, using differential ultracentrifugation [12,13] and size exclusion chromatography (SEC). The number and size of the particles eluted from SEC were evaluated using tunable resistive pulse sensing

Discussion

A wide variety of cancer cells have been reported to release increased number of EVs and their components are thought to participate to tumour growth and aggressiveness. Our findings provide new insight on specific EV content released by glioblastoma cells and their possible involvement in temozolomide-mediated tumour resistance. In this study, we present a comprehensive analysis of protein cargoes in GBM-derived EVs upon temozolomide treatment in resistant cells. More than 320 proteins were

Conclusions

Overall, our results show that (i) EVs can be easily detected in peripheral blood and accumulate in GBM patient liquid biopsies, (ii) TMZ treatment modulates GSC-released EVs, and, (iii) GO and KEGG pathway analyses of protein interaction networks unveil an enriched cell adhesion pathway, which could open new perspectives about the role of EVs in chemoresistance and recurrence.

Author contributions

G.A.G designed and performed the experiments; N.B and J.G provided valuable resources used in the manuscript; G.A.G wrote the manuscript; N.B and J.G edited the manuscript. All authors approved the manuscript.

Conflict of interest

The authors declare no potential conflicts of interest.

Acknowledgements

The authors wish to thanks Hélène Rogniaux from the INRA BIBS platform at ONIRIS (INRA, Nantes, France) for expertise and excellent technical support for proteomic analysis, Kathryn Jacobs and Carolina Nicolau (SOAP, Nantes, France) for their careful reading and comments on the manuscript, Quentin Sabbagh (SOAP, Nantes, France) for his technical help, and Micropicell facility (SFR Sante Francois Bonamy, Nantes, France). This research was funded by a postdoctoral fellowship from Fondation de

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