Cancer Letters

Cancer Letters

Volume 433, 1 October 2018, Pages 210-220
Cancer Letters

Original Articles
Delivery of MGMT mRNA to glioma cells by reactive astrocyte-derived exosomes confers a temozolomide resistance phenotype

https://doi.org/10.1016/j.canlet.2018.06.041Get rights and content

Highlights

  • Glioma cells stimulate normal astrocytes into reactive astrocytes via a non-contact way.

  • Co-culture with astrocytes enhances the chemoresistance of glioma cells.

  • Astrocyte-derived exosomal MGMT mRNA can be functionally translated into MGMT protein by recipient glioma cells.

Abstract

The glioma-astrocyte interaction plays an important role in tumor microenvironment remodeling; however, the underlying mechanism has not been completely clarified. In this study, we show that glioma cells stimulate normal human astrocyte (NHA) into reactive astrocyte (RAS) in a non-contact manner. Additionally, the amount of O6-alkylguanine DNA alkyltransferase (MGMT) mRNA in exosomes (EXOs) released by RAS was significantly increased compared with that in non-reactive NHA. Importantly, MGMT-negative glioma cells can take up RAS-EXOs and acquire a temozolomide (TMZ)-resistant phenotype via the translation of exogenous exosomal MGMT mRNA both in vitro and in vivo. Our findings illuminate a novel phenomenon that may be a potent mechanism underlying glioma recurrence in which glioma-associated NHAs protect MGMT-negative glioma cells from TMZ-induced apoptosis by the functional intercellular transfer of exosomal MGMT mRNA.

Introduction

Malignant glioma (WHO grade III–IV) is the most common and most lethal primary tumor of the central nervous system (CNS) in adults [1]. Diffuse infiltration of tumor cells throughout the brain is a core characteristic of high-grade glioma and is responsible for treatment failure even if maximal surgical resection is performed [2,3]. Although temozolomide (TMZ) has been shown to prolong survival, the treatment response is frequently limited by the progression of drug resistance [4,5]. Thus, identifying potential mechanisms underlying TMZ resistance could provide prospective molecular targets for glioma therapy.

In addition to the drastic alterations occurring at the cellular level, the tumor microenvironment (TME) is also relevant for cancer progression [6]. The TME consists of various non-tumor cell types such as endothelial cells, fibroblasts, and inflammatory cells. In the context of glioma, astrocytes and microglia cells are essential components of the TME. Glioma and non-tumor cells frequently exchange information via gap junctions and tunneling nanotubes, also in contact-independent ways such as the secretion of secretory proteins and extracellular vesicles (EVs) [[7], [8], [9], [10]]. These cells interact with each other to form complicated communication networks, thereby profoundly affecting several cancer phenotypes such as neovascularization and chemo/radioresistance [11]. Recent studies have revealed that cells can mutually communicate via exosomes (EXOs), microvesicles that are released by endosomes and can be taken up by cells via endocytosis. The internalization of EXOs by target cells leads to the release of bioactive molecules including proteins, mRNAs, and non-coding RNAs [[12], [13], [14]].

Although recent advances have concentrated on exosomal microRNAs (miRNAs), some studies reported that exosomal mRNA can be translated into functional proteins in vitro and in vivo [[13], [14], [15], [16], [17], [18]]. Zomer et al. found that exosomal mRNA can be functionally translated into protein in vivo, as evidenced by transfer of CRE mRNA within EVs to a cell carrying a fluorescent reporter gene that is expressed only after CRE-mediated DNA excision of a STOP signal and can be detected by fluorescence changes [15]. Pastuzyn et al. showed that Arc EVs and capsids can mediate intercellular transfer of Arc mRNA in neurons [18]. A growing body of research has suggested that EXOs released by glioma cells modulate surrounding cells to create an optimal environment for cancer development [[19], [20], [21]]; however, the functional roles of astrocyte-derived EXOs in modulating the malignant phenotypes of recipient glioma cells have not been elucidated [14,22].

Recent reports have shown that mRNAs are functionally secreted in microvesicles.

Shao et al. showed the presence of MGMT mRNA encoding O6-alkylguanine DNA alkyltransferase in patient-derived EXOs and demonstrated that the level of the exosomal MGMT mRNA could predict the response of glioblastoma patients to TMZ [23]. Furthermore, Garnier et al. reported that the MGMT mRNA profile reflective of TMZ resistance was recapitulated in the transcriptome of EVs released by glioma stem cells [17]. Although these studies are beginning to shed light on the importance of exosomal MGMT mRNA in predicting the outcome after TMZ application, the pathological effect of EXOs meditated MGMT mRNA exchange in glioma progression remains unclear. Therefore, we hypothesized that MGMT mRNA embedded in astrocyte derived EXOs might be a potential transmitter to acquire TMZ resistance in recipient cells.

In this study, we showed that NHA (normal human astrocyte) is stimulated by glioma cells, thereby presenting a reactive condition that leads to an increased expression level of MGMT mRNA in both cell and EXOs. Delivery of reactive astrocyte (RAS) MGMT mRNA via EXOs to MGMT-negative glioma cells might transfer TMZ resistance through effective translation of MGMT mRNA.

Section snippets

Materials and methods

The Supplementary material section is referred for details on western blotting, RNA extraction, quantitative reverse transcription–PCR, methylation-specific PCR, plasmid construction, transfection, stable cell establishment, flow cytometric analysis, EdU proliferation assay (EdU), CCK-8 assay and immunofluorescence (IF) [24,25].

Invasion of glioma cells away from the original tumor results in satellite lesions and provides close interaction with surrounding astrocytes

In four surgical specimens collected from GBM patients, although the cell density was lower in the peritumoral brain zone (PBZ) than in the core region, there was still pathological mitosis and cell aggregation of disseminated glioma cells (Fig. 1A). We named these assembled glioma cells satellite lesions because the lesions were small and surrounded the main lesion. In four tumor-bearing mice, we found invasive glioma cell masses of different sizes and location (Fig. 1B, n = 20). Increasing

Discussion

In this study, we showed that reactive astrocytes express significantly higher levels of exosomal MGMT mRNA than non-reactive astrocytes and that the exosomal transfer of MGMT mRNA from reactive NHAs to neighboring glioma cells enhances the chemoresistance of the recipient cells to TMZ through repression of apoptosis.

The heterogeneity of high-grade gliomas reflects not only the composition of bona fide tumor cells but also the range of intermingling stromal cells, especially tumor-associated

Acknowledgment

We thank Mary Derry, PhD ELS, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

Conflicts of interest

The authors declare no conflicts of interest.

Funding

This work was supported by grants from the National Key Research and Development Plan (2016YFC0902500), National Natural Science Foundation of China (81772682, 81672501), Jiangsu Province's Natural Science Foundation (20170108, 20151585), the Program for Advanced Talents within Six Industries of Jiangsu Province (2015-WSN-036, 2016-WSW-013), Jiangsu Province's Key Discipline of Medicine (ZDXKA2016001), and the Priority Academic Program Development of

References (36)

  • R. Stupp et al.

    Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma

    N. Engl. J. Med.

    (2005)
  • N.A. Charles et al.

    The brain tumor microenvironment

    Glia

    (2012)
  • O. Okolie et al.

    Reactive astrocytes potentiate tumor aggressiveness in a murine glioma resection and recurrence model

    Neuro Oncol.

    (2016)
  • W.C. Sin et al.

    Astrocytes promote glioma invasion via the gap junction protein connexin43

    Oncogene

    (2016)
  • H. Valadi et al.

    Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

    Nat. Cell Biol.

    (2007)
  • J. Godlewski et al.

    Belonging to a network–microRNAs, extracellular vesicles, and the glioblastoma microenvironment

    Neuro Oncol.

    (2015)
  • A. Yokoi et al.

    Malignant extracellular vesicles carrying MMP1 mRNA facilitate peritoneal dissemination in ovarian cancer

    Nat. Commun.

    (2017)
  • J. Ashley et al.

    Retrovirus-like gag protein Arc1 binds RNA and traffics across synaptic boutons

    Cell

    (2018)
  • Cited by (63)

    • Mechanisms of multidrug resistance in cancer

      2022, Aptamers Engineered Nanocarriers for Cancer Therapy
    • Thorny ground, rocky soil: Tissue-specific mechanisms of tumor dormancy and relapse

      2022, Seminars in Cancer Biology
      Citation Excerpt :

      At homeostasis, astrocytes support the integrity of the BBB through endfoot contact with the vessels and expression of Fas ligand, which repels infiltrating lymphocytes [220]. But in response to inflammation, metastasis, or glioma-secreted exosomes, astrocytes undergo reactive gliosis and become pro-metastatic [186,209,221–223]. Brain metastatic breast and lung carcinoma cells directly transfer cGAMP to astrocytes through gap junctions which activates astrocytes to secrete paracrine IFNα and TNF back to the tumor cells, promoting survival and chemoprotection through STAT1 and NF-κB pathways [224,225].

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text