Original ArticlesDelivery of MGMT mRNA to glioma cells by reactive astrocyte-derived exosomes confers a temozolomide resistance phenotype
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)
- et al.
Glioblastoma and chemoresistance to alkylating agents: involvement of apoptosis, autophagy, and unfolded protein response
Pharmacol. Therapeut.
(2018) - et al.
Astrocytes upregulate survival genes in tumor cells and induce protection from chemotherapy
Neoplasia
(2011) - et al.
Reactive astrocytes protect melanoma cells from chemotherapy by sequestering intracellular calcium through gap junction communication channels
Neoplasia
(2010) - et al.
The microenvironmental landscape of brain tumors
Canc. Cell
(2017) - et al.
Communication by extracellular vesicles: where we are and where we need to go
Cell
(2016) - et al.
In Vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior
Cell
(2015) - et al.
Cell biology of astrocyte-synapse interactions
Neuron
(2017) - et al.
Paediatric and adult glioblastoma: multiform (epi)genomic culprits emerge
Nat. Rev. Canc.
(2014) - et al.
Glioblastoma: from molecular pathology to targeted treatment
Annu. Rev. Pathol.
(2014) - et al.
Surgical oncology for gliomas: the state of the art
Nat. Rev. Clin. Oncol.
(2018)
Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma
N. Engl. J. Med.
The brain tumor microenvironment
Glia
Reactive astrocytes potentiate tumor aggressiveness in a murine glioma resection and recurrence model
Neuro Oncol.
Astrocytes promote glioma invasion via the gap junction protein connexin43
Oncogene
Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells
Nat. Cell Biol.
Belonging to a network–microRNAs, extracellular vesicles, and the glioblastoma microenvironment
Neuro Oncol.
Malignant extracellular vesicles carrying MMP1 mRNA facilitate peritoneal dissemination in ovarian cancer
Nat. Commun.
Retrovirus-like gag protein Arc1 binds RNA and traffics across synaptic boutons
Cell
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2022, Seminars in Cancer BiologyCitation 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].
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These authors contributed equally to this work.