The emergent role of exosomes in glioma

Journal of Clinical Neuroscience xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn

Review article

The emergent role of exosomes in glioma J. Gourlay a, A.P. Morokoff a,b, R.B. Luwor a, H.-J. Zhu a, A.H. Kaye a,b, S.S. Stylli a,b,⇑ a b

Department of Surgery, The University of Melbourne, The Royal Melbourne Hospital, Parkville, VIC 3050, Australia Department of Neurosurgery, The Royal Melbourne Hospital, Parkville, VIC 3050, Australia

a r t i c l e

i n f o

Article history: Received 14 July 2016 Accepted 26 September 2016 Available online xxxx Keywords: Glioma Glioblastoma multiforme Extracellular vesicles Exosomes

a b s t r a c t Extracellular vesicles (EVs) are known mediators of intercellular communication for both normal and tumour cells. With the capability to transfer nucleic acids, proteins and lipids, EVs are able to influence numerous functional and pathological aspects of both donor and recipient cells. The tumour microenvironment possesses a high level of complex heterogeneity, particularly within the most prominent brain malignancy, glioblastoma multiforme (GBM). This complexity relies on a network-based communication between many different components of the local niche, including the various cell types, stroma, blood vessels, secreted factors and surrounding matrix. Exosomes are one type of EV which facilitates this intercellular communication and cross-talk within the tumour microenvironment. Exosomes secreted by tumour cells are increasingly recognized in a number of processes underlying tumour progression including facilitating the transport of receptors, signalling molecules, oncogenic genes and miRNA. They are emerging as a key component in the biogenesis of glioma, in addition to contributing to the modification of the surrounding microenvironment to support tumour progression. In this review we describe advancements in the understanding of the biology of exosomes, as well as their roles in tumour progression, as a tumour biomarker for tracking cancer progression, and as a potential therapeutic target/delivery system, with a contextual emphasis on GBM. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Glioma tumours that arise from glia or glial precursor cells are the most prevalent brain tumour, with an estimated 23,000 new cases and 16,000 deaths in the USA in 2016 [1]. According to the CBTRUS Statistical report (2008–2012), gliomas account for over 32% of all central nervous system (CNS) tumours and approximately 80% of malignant primary CNS tumours [2]. The most prevalent form of glioma with the most dismal prognosis is the grade IV glioblastoma multiforme with an incidence rate of 3.2 per 100,000 population [2] and evidence suggesting that it is increasing every year [3]. With a median survival rate of only 14.6 months [4], GBM is the most intractable and lethal primary brain malignancy [5]. The customary treatment protocol usually involves surgery followed by post-operative fractionated radiation therapy and concomitant chemotherapy [4,5]. However, with near universal recurrence, this approach provides only a degree of palliation [6,7]. The invasive nature of GBM prevents total resection, making them surgically incurable. In 2005, a pivotal study was published showing that ⇑ Corresponding author at: Department of Surgery, The University of Melbourne, The Royal Melbourne Hospital, Parkville, VIC 3050, Australia. E-mail address: [email protected] (S.S. Stylli).

concurrent radiotherapy and temozolomide-based chemotherapy led to a modest 2.5 month increase in the dismal median survival of GBM patients and improvement in their health-related quality of life. This combinatorial treatment also led to a significant increase from 10% to 26% of patients who survived beyond two years [8]. The treatment, now referred to as the Stupp protocol, has been universally adopted as the standard of care for them [9]. Even alternative adjuvant therapies such as photodynamic therapy have only achieved similar median survival figures (14.8 months) as the Stupp treatment protocol for GBM patients [10–12]. GBM tumours are both morphologically and molecularly complex. Not only do they display properties indicative of diverse cellular phenotypes [13–17], they are also significantly heterogeneous at the inter-tumour and intra-tumour levels [18]. As a result, the classification and subsequent treatment of GBM are made considerably more difficult as a result of this heterogeneity [19]. Regional diversity observed in molecular pathways, cellular communication and tumour stem cell signalling may also contribute to the varied therapeutic response observed in GBM patients. Such diversity has driven the efforts in refining existing classification systems focus on the sub-typing of GBM by utilizing genomic and expression data [20,21]. Categorization of GBM based on distinct gene sets and particular signalling pathways yields

http://dx.doi.org/10.1016/j.jocn.2016.09.021 0967-5868/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Gourlay J et al. The emergent role of exosomes in glioma. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j. jocn.2016.09.021

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potential for more specific and targeted therapeutics [22,23]. Further complicating the aspect involving the heterogeneous nature of GBM, single cell sequencing has recently identified that different regions within an individual GBM lesion may exhibit several subtype-specific signatures [24,25]. With this taken into consideration, selection of particular sub-populations of GBM patients in the future could enable the design of personalized molecularly– targeted therapies for them [26–28]. Tumour cells possess the ability to communicate between the different compartments of the tumour microenvironment and ultimately influence neighboring cells. This effect can be varied with respect to the different subtypes of GBM, depending on the composition of the molecules they secrete. Mesenchymal cells have been shown to promote an invasive microenvironment by manipulating surrounding cells via the involvement of miRNA [29,30]. The molecules which are secreted by tumour cells and are subsequently utilized for communicative purposes are often encapsulated in structures known as extracellular vesicles (EVs). EVs are small portions of organelle-free cytosol enclosed by a spherical lipid bilayer. EVs can be categorized further depending upon their site of origin and their size, which can range from 30 to 2000 nm. Vesicles that are derived from multi-vesicular bodies (MVBs) are referred to as exosomes, whilst those produced directly from the plasma membrane are known as microvesicles [31]. Cells can also shed contents into the microenvironment in moments of stress or cell death and this involves the process of blebbing by apoptotic bodies [31]. It has now become evident that exosomes are particularly important structures as they are involved in a variety of physiological processed including the intercellular exchange of proteins and RNA [32,33], induction of angiogenesis [34] and immune regulation [35–37]. However, given the expanding and complex research field on EVs, this review will only focus on a brief overview of the current literature that has investigated the potential roles of exosomes in brain tumours.

2. Exosomes

lated proteins into specific micro-domains [31]. ESCRT0 then binds to the ESCRTI complex [35] which in turn recruits ESCRTII subunits, initiating the reverse budding of ILVs into MVBs [40]. Cytosolic RNAs and proteins have direct access into the forming vesicles during this internalisation stage. Next, the ESCRTII complex recruits ESCRTIII subunits inside the neck of the nascent ILVs, which results in their cleavage into free vesicles [32]. The MVB (or late endosome) can then fuse with the peripheral membrane and release the exosomes into the extracellular space [41]. A schematic representation of exosome biogenesis and release is presented in Fig. 1. Several functional RNA species including miRNA, mRNA, rRNA and tRNA have been identified within exosomes [42,43]. The protection from enzymatic RNAse degradation afforded by their inclusion inside exosomes, enables safe passage through the extracellular environment and vasculature system [44–47]. Subsequent, release of cargo mRNA and microRNA into the recipient cell can modulate gene expression through translational and post-translational regulation of target mRNAs [40]. Exosomes are able to alter the transcriptome and signalling activity within recipient cells, allowing them to induce specific phenotypic changes [48–50]. Many exosome associated proteins have been identified but the core composition of exosomes remains the focus of continued research. In addition, whilst contributing to the fusion and budding processes of vesicles, lipids can play a vital role in membrane rigidity and stability [51]. The lipid composition of exosomes has not been entirely elucidated as various lipids have been reported to exist in the lipid bilayer of exosomes. These include cholesterol, sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, ganglioside GM3, prostaglandins and lysobisphosphatidic acid [52,53]. Exosomes contain many membrane-associated proteins that contribute to a variety of functions, including adhesion via tetraspanins (CD63, CD81, CD9, CD37, CD53 and CD82), ICAM-1 and integrins; intracellular transport and membrane fusion (Rab proteins and Annexins); MVB formation (Alix, TSG101); and antigen presentation (MHC-I, MHC-II, HLA-G) [54]. Other proteins may be present depending on the cell type and method of isolation as outlined in the databases – ExoCarta [55,56], Vesiclepedia [57] and EVpedia [58].

2.1. Exosomal composition Exosomes can be defined via a number of main morphological and physical characteristics. Firstly, they range in size between 40 and 120 nm in diameter, are of endocytic origin and sediment at approximately 100,000 g (sucrose density gradient of 1.13– 1.19 g/ml) [38]. Morphologically they appear as spherical structures with a well-defined lipid bilayer when viewed with an electron microscope [37]. Contained within their aqueous core or in the lipid membrane, are various proteins, nucleic acids and receptors that are reflective of the parental donor cell (2,3). The variety of proteins and receptors that are present in exosomes is largely dependent on their cell of origin. Whilst the biogenesis of exosomes and cargo regulation is complex, we know that exosomes are generated first as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs), via mechanisms that are either dependent or independent of the ‘Endosomal Sorting Complex for Transport’ (ESCRT). Proteins can be sorted into ILVs independent of ESCRT machinery, through raft-based micro-domains of endosomes which are rich in sphingolipids. Sphingolipids are formed into ceramides by sphingomyelinases which induce the union of their microdomains and trigger ILV formation [39]. ESCRT dependent mechanisms act on MVBs, allowing for selection of particular proteins and receptors into ILVs. The ESCRT family includes a variety of complexes, the first being ESCRT0 which identifies ubiquitinylated proteins protruding into the cytosolic side of the endosomal or MVB membrane. ESCRT0 has the ability to separate the ubiquitiny-

2.2. Exosomes in glioma progression Exosomes represent an important extension of the complex array of metabolites, growth factors, cytokines and ions that are secreted by tumour cells [59]. It has been established that exosomes mediate the transfer of histones [60], oncogenic species (EGFRvIII) [61], non-coding RNA (miRNA) [62] and tumour suppressors (PTEN) in glioma cells [63]. However, the ramifications of intercellular communication between cells in the GBM tumour microenvironment, facilitated by the exchange of exosomes, have not been fully elucidated. Notably, this includes the possibility of different active intracellular signalling pathways within the various GBM subtypes leading to the activation of different vesicle biogenesis pathways. The four molecular subtypes of GBM (known as mesenchymal, classical, neural and pro-neural) have been observed to differ substantially in the mRNA levels of known markers of exosome biogenesis [21]. Exosomes released from glioma cells have been implicated in the shaping of the tumour microenvironment in an array of processes including the transfer of functional RNA transcripts [64], angiogenesis, clonogenicity and heightened proliferation arising from paracrine induction [61,64], spread of pro-migratory factors [65] and influencing immune-tolerance or inducing malignancy in normal cells [66–68]. In addition, exosomes of GBM origin may also modify cell surface protein expression and cytokine secretion, as well as influence the immunity functions of the

Please cite this article in press as: Gourlay J et al. The emergent role of exosomes in glioma. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j. jocn.2016.09.021

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Fig. 1. The release and uptake of exosomes. Exosome generation begins with early endosome formation during endocytosis. The membrane proteins are internalized through clathrin-coated vesicles and delivered to early endosomes. This leads to intraluminal vesicles forming through the inward budding of the membrane and formation of multivesicular bodies (MVBs). Upon maturation, the exosome MVBs are sent to lysosomes for degradation or fused with the plasma membrane for release to the extracellular environment.

tumour microenvironment through alteration of the phagocytic capacity of immune cells (macrophages and microglia) [69,70]. As stated previously, a major contributing factor in the inevitable tumour recurrence in GBM patients following surgical intervention, is its highly invasive nature, which has made the study of the molecular mechanisms of this potent invasiveness a field of great interest [71]. It has been shown over the last 10–15 years that a property shared by several types of tumour cells with high invasive or metastatic potential is an ability to form structures known as invadopodia [72–77]. These are dynamic actin-rich protrusions on the cell membrane which adhere to and proteolytically degrade extracellular matrix (ECM) substrates via the activities of numerous transmembrane and secreted extracellular proteases [78–82]. A complex network of cell adhesion, intracellular signalling, adaptor and actin regulatory proteins coordinate their activities to drive the proteolytic function of invadopodia [72– 75]. Importantly, the role of invadopodia in glioma invasion has been shown through the presence of functional (matrixdegrading) invadopodia in glioma cell lines [83] and primary tumour cells derived from ex vivo cultured GBM specimens [84]. Notably, Tks5 (also known as SH3PXD2A), which is an adaptor protein critical for invadopodia formation and proteolysis-directed invasion of tumour cells through the ECM [85–87], has been shown to possess prognostic potential in glioma patients with increased Tks5 expression resulting in significantly reduced survival among glioma patients [88]. Recently, it has also been demonstrated that the multiple invadopodia lifecycle steps, including their formation,

stabilization and exocytosis of matrix degrading proteinases, may be mediated by the EVs known as exosomes [89]. This study identified an important role for exosomes in promoting tumour cell invasiveness through a cooperative relationship between invadopodia and exosome secretion in the modification or degradation of the surrounding ECM. Furthermore, exosomes can also facilitate change of the microenvironment by altering the surrounding support cells’ phenotype into one that is supportive of tumour progression and invasion. Incubation of cells with mesenchymal-derived exosomes can facilitate tumour progression. The mesenchymal-derived exosomes were not only observed to have an effect on the normal support cells (astrocytes), but they also produced a phenotypic change on the other molecular subtypes of GBM that were present [69]. In this regard the intercellular mechanisms of exosome transport can be seen as a potential contributor to the progression and biogenesis of glioblastoma. Datamining of the online OncomineÒ platform for the genes involved in the biogenesis of exosomes in glioma related datasets was carried out and is displayed in Table 1. Oncomine (version 4.4.4.3 – www.oncomine.org, Compendia BioscienceTM, Ann Arbor, MI, USA, part of Life Technologies) is an online tool that contains 715 mRNA and copy number expression datasets from 86,733 cancer and normal tissue samples. The information extracted from the Oncomine Compendium presented in the table shows the mRNA levels of a number of the genes of exosomal markers which are elevated in glioma tissue relative to normal brain. The exosomal markers include Alix, TSG101, CD9, CD63,

Please cite this article in press as: Gourlay J et al. The emergent role of exosomes in glioma. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j. jocn.2016.09.021

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Exosomal Marker/Gene

Glioma Type

Sample Number (normal/tumour)

Mean fold-log2 change vs. normal tissue

P-value

Total no. of measured genes

Overexpression gene rank

Ranking (%)

Platform

Alix (PDCD6IP)

GBM GBM GBM GBM GBM AA DA AO

10/542 10/5 23/81 3/22 4/80 23/19 23/7 6/4

2.254 1.907 1.396 1.377 2.101 1.251 1.314 1.881

2.16E-11 7.16E-4 2.25E-5 0.014 5.90E-4 0.009 0.040 0.004

12,624 12,624 19,574 19,574 19,574 19,574 19,574 19,574

284 470 3584 2382 2516 4301 3947 1470

2 4 19 13 13 22 21 8

Human Human Human Human Human Human Human Human

GBM GBM GBM A AO O GBM GBM GBM GBM GBM AA A A

4/27 7/27 10/542 7/5 6/23 7/3 4/27 3/22 7/2 23/81 10/542 23/19 6/45 7/5

1.507 1.433 1.115 1.400 1.519 2.453 1.303 2.313 2.499 1.392 3.178 1.477 3.059 2.469

0.007 4.18E-5 0.010 5.62E-4 6.76E-4 2.93E-7 0.039 0.015 0.004 0.011 1.69E-7 0.010 0.006 0.005

14,836 8,603 12,624 8,603 19,574 8,603 14,836 19,754 8,603 19,574 12,624 19,574 5,338 8,603

2695 592 4746 465 2608 18 4154 2429 1638 6563 1422 4440 528 1058

19 7 38 6 14 1 28 13 20 34 12 23 10 13

ND* Human Genome U95A-Av2 Array Human Genome U133A Array Human Genome U95A-Av2 Array Human Genome U133 Plus 2.0 Array Human Genome U95A-Av2 Array ND* Human Genome U133 Plus 2.0 Array Human Genome U95A-Av2 Array Human Genome U133 Plus 2.0 Array Human Genome U133A Array Human Genome U133 Plus 2.0 Array HumanGeneFL Array Human Genome U95A-Av2 Array

Bredel Brain 2 Shai Brain TCGA Brain Shai Brain French Brain Shai Brain Bredel Brain 2 Lee Brain Shai Brain Sun Brain TCGA Brain Sun Brain Rickman Brain Shai Brain

CD63

GBM GBM GBM GBM AA A A DA PA O O

4/80 7/27 23/81 10/542 23/19 6/45 7/5 23/7 3/8 7/3 23/50

2.119 3.259 1.931 2.558 1.450 3.510 2.433 1.542 2.772 2.400 1.232

0.016 8.37E-9 7.67E-13 1.71E-9 3.25E-4 3.09E-12 1.36E-4 0.034 0.007 2.10E-4 0.005

19,574 8,603 19,574 12,624 19,574 5,338 8,603 19,574 8,603 8,603 19,574

5625 60 797 596 2336 8 242 3661 394 506 4241

29 1 5 5 12 1 3 19 5 6 22

Human Genome U133 Plus 2.0 Array Human Genome U95A-Av2 Array Human Genome U133 Plus 2.0 Array Human Genome U133A Array Human Genome U133 Plus 2.0 Array HumanGeneFL Array Human Genome U95A-Av2 Array Human Genome U133 Plus 2.0 Array Human Genome U95A-Av2 Array Human Genome U95A-Av2 Array Human Genome U133 Plus 2.0 Array

Murat Brain Shai Brain Sun Brain TCGA Brain Sun Brain Rickman Brain Shai Brain Sun Brain Gutmann Brain Shai Brain Sun Brain

CD81

GBM GBM GBM A A AO AO

4/27 7/2 10/524 6/45 7/2 6/23 6/4

1.315 1.250 1.543 2.668 1.414 1.710 1.504

8.79E-4 0.043 4.41E-8 6.30E-4 0.010 2.17E-4 0.032

14,836 8,603 12,624 5,338 8,603 19,574 19,574

1759 2598 1109 243 1403 1996 3994

12 31 9 5 17 11 21

ND* Human Genome U95A-Av2 Array Human Genome U133A Array HumanGeneFL Array Human Genome U95A-Av2 Array Human Genome U133 Plus 2.0 Array Human Genome U133 Plus 2.0 Array

Bredel Brain 2 Shai Brain TCGA Brain Rickman Brain Shai Brain French Brain French Brain

CD151

GBM GBM GBM GBM GBM GBM AA A AO AO

4/27 3/30 4/80 7/27 23/81 10/542 23/19 6/45 4/6 6/4

3.531 1.806 1.643 1.810 4.264 2.547 1.657 1.558 1.573 2.128

6.50E-11 1.03E-6 0.011 1.70E-7 1.55E-15 1.25E-7 0.020 0.003 0.002 0.034

14,836 9,957 19,574 8,603 19,574 12,624 19,574 5,338 14,836 19,574

140 88 5159 134 457 1338 5035 394 364 4121

1 1 27 2 3 11 26 8 3 22

ND* ND* Human Genome U133 Plus 2.0 Array Human Genome U95A-Av2 Array Human Genome U133 Plus 2.0 Array Human Genome U133A Array Human Genome U133 Plus 2.0 Array HumanGeneFL Array ND* Human Genome U133 Plus 2.0 Array

Bredel Brain 2 Liang Brain Murat Brain Shai Brain Sun Brain TCGA Brain Sun Brain Rickman Brain Bredel Brain 2 French Brain

TSG101

CD9

Genome Genome Genome Genome Genome Genome Genome Genome

Oncomine Dataset U133A Array U133A Array U133 Plus 2.0 U133 Plus 2.0 U133 Plus 2.0 U133 Plus 2.0 U133 Plus 2.0 U133 Plus 2.0

Array Array Array Array Array Array

TCGA Brain TCGA Brain Sun Brain Sun Brain Murat Brain Sun Brain Sun Brain French Brain

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Please cite this article in press as: Gourlay J et al. The emergent role of exosomes in glioma. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j. jocn.2016.09.021

Table 1 Exosomal marker gene expression is elevated in glioma tissue relative to normal brain tissue.

A – Astrocytoma; AA – Anaplastic Astrocytoma; AOA – Anaplastic Oligoastrocytoma; AOD – Anaplastic Oligodendroglioma; DA – Diffuse Astrocytoma; GBM – Glioblastoma multiforme; O- Oligodendroglioma; PA – Pilocytic Astrocytoma. * Platform not defined.

AO 6/23 1.570 7.47E-5 19,574 1555 8 Human Genome U133 Plus 2.0 Array French Brain O 23/50 1.700 3.69E-4 19,574 2900 15 Human Genome U133 Plus 2.0 Array Sun Brain PA 3/8 12.179 0.001 8,603 158 2 Human Genome U95A-Av2 Array Gutmann Array mRNA expression of the nominated exosomal markers was examined in various glioma types contained within the Oncomine database. Displayed in this table are the mean fold changes vs. corresponding normal tissue in each study, overall p-value, overexpression gene rank and percentage ranking for the relative mRNA in that dataset. Gene expression data are log transformed and normalized as previously described [158].

Exosomal Marker/Gene

Table 1 (continued)

Glioma Type

Sample Number (normal/tumour)

Mean fold-log2 change vs. normal tissue

P-value

Total no. of measured genes

Overexpression gene rank

Ranking (%)

Platform

Oncomine Dataset

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CD81 and CD151 and as observed, many of them were ranked in the top 10% of genes that were differentially expressed between tumour and normal tissue. Understanding the mechanisms by which exosomes are involved in the progression of GBM has provided the foundation for investigating a variety of potential clinical applications, ranging from prognostic biomarkers to targeted therapeutics. GBMoriented exosome research is progressing in at least four major therapeutic directions, with preclinical studies for brain tumour specific applications in: (i) utilization of exosomes as biomarkers and in disease diagnostics; (ii) disruption of intercellular communication by targeting exosomes; (iii) exploiting the targeting and uptake mechanisms of exosomes to enable delivery of molecular or pharmacological therapeutics; and (iv) the development of cancer vaccines or immunomodulation by the manipulation of the exosomes’ role in immunoregulation. 2.3. Exosomes as a diagnostic biomarker for GBM patients It is understood that the heterogeneous nature of GBM may contribute to the variation in survival observed in GBM patients with regards to response to the current clinical treatments. Patients may be tracked through the histological analysis of sequential surgical biopsies of the tumour, or post-operative magnetic resonance imaging (MRI), however, additional avenues of monitoring disease progression or response to therapy are urgently required. One potential approach is through the utilization of tumour-secreted exosomes as functional biomarkers. Obtaining longitudinal ‘liquid biopsies’ of bio-fluids from the patients, containing exosomes, may provide a minimally invasive means of following disease progress, and enabling profound modifications to the diagnosis and treatment strategies in personalizing treatments available to GBM patients [90]. The ideal biomarker has the potential for providing vital information for a variety of applications such as allowing for early detection of disease progression, the molecular sub-typing of tumours and potentially predicting response to therapy. Identification of such a biomarker in GBM would potentially enable a molecularly based method of detection, complementing the current approaches of histological analysis of tissue biopsies and MRI evaluation of the patient’s tumour progression. The specificity of MRI ranges between 50 and 80% for correctly distinguishing GBM tumours from other intracranial lesions or metastases [91] and has been documented to miss early lesions with a resolution limit of approximately 2–3 mm [92]. MRI has significant limitations in assessing the response of the tumour to therapies in determining early progression of the tumour. Tumour biopsies are about a single time point in the biogenesis of GBM and it may not entirely be representative of the evolving and heterogeneous nature of the tumour and importantly, interaction and response to its microenvironment [93–95]. As such, assessments of resected or biopsied samples also possess restricted capabilities for predicting chemotherapeutic resistance, and in clearly differentiating between tumour progression and post-treatment necrosis [96,97]. The strength of the ‘fluid-based’ exosomal biomarker approach is that it would be minimally invasive but reliant on the enrichment of molecules isolated from tumour cell secreted exosomes relative to the pool of healthy human circulating exosomes, which have been shown to contain over 60 proteins of cell trafficking and sorting origin [98]. Exosomes containing brain tumour cell-derived proteins, lipids, RNA and DNA have been isolated from blood samples and the cerebrospinal fluid (CSF) of glioma patients. This highlights that glioma-based exosomes are capable of leaving the brain parenchyma and transiting into peripheral circulation [99]. They can be isolated from patient bio-fluid samples via various methodologies including ultracentrifugation and filtration, or antibody aggregation.

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Compared to highly invasive tumour biopsies or resections, exosome isolation from biological fluids could be performed multiple times throughout the course of a patient’s treatment [99]. Exosomes secreted by tumour cells are also enriched with tumour-specific molecules, with levels nearly 100-fold more than those found in the tumour cell of origin. CSF is the most appealing source of exosomes as it avoids the blood brain barrier, an obstacle which may limit the concentration of extracellular vesicles that are able to pass through into the peripheral circulation. CSF also has the benefit of less ‘noise’ from non-tumour based exosomes or platelet derived particles circulating in the blood [100]. A number of exosomal RNA have already been identified as glioma-specific biomarkers including a mutant form of EGFR (EGFRvIII), IDH1 and miR-21 [64,101]. Categorized by unique mutations or expression levels, these biomarkers can be associated with molecular subtypes, prognosis, and have the capacity for offering individualized therapeutic targets derived from these specific signatures. In addition, many mutations associated with GBM are not expressed by healthy tissue and are highly likely to be specific for their tumour of origin [102]. Exosomal EGFR wildtype expression levels in patient CSF has also been linked to chemotherapeutic response and can potentially act as a marker for drug sensitivity in GBM patients, as EGFR over-expression is present in up to 70% of GBM cases [103,104]. In addition, as EGFRvIII is known to be associated with the ‘classical’ molecular subtype of GBM [105], the downstream signalling pathways will differ from that of wild-type EGFR and detection of exosomal EGFRvIII may allow for a tailored therapeutic approach and a potential improvement in patient survival outcome [21]. Importantly, exosomal EGFRvIII RNA has been isolated from the serum of GBM patients and it is not detectable in exosomes isolated from healthy individuals not bearing a GBM tumour [64]. Furthermore, utilization of an iMER (immune-magnetic exosome RNA) assay approach can correctly identify GBM tissue at an 84.4% accuracy with the marker EPHA2 and at 78.1% using EGFR. Combination of these two markers increases the overall accuracy to 90% for correctly identifying GBM tissue [102]. MiRNA based analyses is also extremely useful, as aberrant miRNA expression has been linked to gliomagenesis. MiR-21 is a known regulator of EGFR expression and has been detected in the CSF of 100% of GBM patients studied, indicating a highly sensitive marker for GBM detection. In addition, the importance of miR-21 expression has also been linked to the tumourinduced manipulation of the microenvironment during glioma cell invasion [30,106]. Nucleic acids are of great value as biomarkers because of the high sensitivity of detection using PCR technology, but the detection of critical single-point mutations (SPMs) that may drive gliomagenesis is more difficult to detect. The use of high resolution methods including BEAMing PCR and droplet digital PCR [23] has allowed for the detection of SPMs (IDH1 – IDH.132) from RNA extracted from exosomes isolated from the CSF of GBM patients. IDH1 is a mutation associated with the ‘proneural’ molecular subtype and has been shown to correlate with a more favorable clinical prognosis [21,64,101,107]. The detection of SPMs and other molecules within tumour derived exosomes is minimally evasive, as sampling can occur during current clinical practice and ultimately this approach has the potential to provide a greater insight into the predominant molecular subtype of the tumour and subsequently allow for a personalized treatment approach for the patient. 2.4. Exosomes as biomarkers of chemotherapeutic resistance Exosomes have the capacity to provide an insight into the sensitivity of the tumour to clinical treatment such as chemotherapy. O-6-methylguanine-DNA-methyltransferase (MGMT) is an enzyme

capable of repairing DNA damage arising from TMZ treatment. Elevated promoter DNA methylation of MGMT has been shown to enhance TMZ response in GBM patients by reducing the expression of specific nuclear proteins in cells. Methylation of the promoter region of MGMT is observed in 22–57% of GBM patients, whilst iMER analysis of patient serum found that exosomal mRNA expression levels are increased following TMZ treatment within cohorts of patients exhibiting levels of drug resistance [102,108]. The expression of other molecules can also provide an indirect quantification of markers of interest, including miR-603 and miR181d, whose expression have been linked directly with MGMT expression levels [109]. In addition, the expression levels of alkylpurine-DNA-N-glycosylase (ADNG) detected within the serum of GBM patients has also been correlated with the degree of drug resistance [110,27]. Therefore, the exosomal mRNA levels of these enzymes are detectable, can be monitored during treatment in tracking the patient response to therapy.

3. Exosomal based therapy 3.1. Inhibiting exosome biogenesis, uptake and secretion It is evident that exosomes may play a role in a range of biological processes within the progression of GBM and therefore targeting exosome-mediated cellular interactions is an area of interest for therapeutics. Studies are revealing the mechanisms which are involved in the trafficking, targeting and secretion of exosomes, however, many aspects still remain unclear and additional research is required to strengthen the knowledge base in the field. Nonetheless, areas of potential exosomal intervention have been proposed including the exosome-mediated interaction between brain tumour stem-like cells and their progeny [111], tumour angiogenesis [112] and inflammatory processes within the brain tumour microenvironment [113]. Inhibitors of exosome biogenesis have already been shown to affect the response of inflammatory cells and attenuate cellular vesiculation. Agents such as the tricyclic antidepressants (despiramine, imipiramine and amitryptiline), S1P receptor antagonist, FTY720, and hydrochloride hydrate (GW4869) have been shown to induce functional loss of acid sphingomyelinase (aSMase) activity [114,115]. aSMase is a soluble hydrolase that is critical in generating ceramide which triggers budding of exosomes into multivesicular endosomes, a vital step in the biogenesis pathway [116]. The phosphorylation product of ceramide, C1P (a bioactive lipid), is not only associated with intracellular trafficking, but is also involved in the regulation of apoptosis, proliferation, differentiation and migration. These are all key elements of both cell regulation and tumour biogenesis, indicating the complexity of interactions and cross-talk involved between cellular functions and the activity of exosomes [117,118]. Rab GTPases are already well-recognized as targets for human disease, as mutations or deregulated expression is known to contribute to a number of diseases [119]. They play a pivotal role in a cell’s response to signalling and external stimuli, through the regulation of membrane trafficking via exosomes. Rab27a and Rab27b are GTPases that regulate exosomes through a number of different functions, including the coordination of cargo selection, acting as scaffolds for vesicle budding and aiding in target membrane fusion. Inhibition of Rab27a has been shown to reduce the emission of cancer-promoting exosomes both in vitro and in vivo, as well as reducing primary tumour growth and the dissemination of metastatic colonies [120,121]. Targeting the biogenesis of exosomes is one potential therapeutic approach, however another avenue, could involve manipulation of the exosomal cargo. For example, a recently identified family of miRNAs (miR-103/107) identified

Please cite this article in press as: Gourlay J et al. The emergent role of exosomes in glioma. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j. jocn.2016.09.021

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as inducers of epithelial–mesenchymal transitions (EMT) are also inhibitors of the expression of the miRNA regulator, DICER. Treatment with an antisense oligonucleotide, antagomiR-103/107, is able to reduce metastatic colonization and restore the levels of mature miRNAs [122,123]. Several agents currently used in the clinic are postulated to block exosome uptake by various cellular mechanisms. These FDA-approved drugs include the commonly prescribed blood thinner heparin [124,125], malarial medication chloroquine [126], diuretic amiloride [126], antipsychotic drug chlorpromazine used to treat schizophrenia [127], and a lipid lowering medication commercially known as zocor or simvastatin [128]. As our knowledge of the activity and pathways of tumour-derived exosomes improves, utilization of these FDA-approved drugs may prove to be successful in treating stratified patient populations based on their tumour-related exosome profile.

surface, which facilitates drug resistance by promoting the efflux of drugs back into the extracellular space. The delivery of exosomal-anti-miR-9 to the resistant GBM cells reversed the expression of the multidrug transporter and re-sensitized the GBM cells to TMZ [136]. It would be extremely beneficial if TMZ, as the current therapeutic of choice for GBM patients could be delivered directly to the tumour via an exosomal-based approach as this may reduce chemoresistance whilst minimizing off-target system side-effects. In addition, radiation treatment of GBM patients is used in conjunction with TMZ as part of the Stupp protocol [8]. Radiation has been shown to increase the abundance of exosomes released by GBM cells and surrounding normal astrocytes, which are more readily taken up by acceptor cells as a result of the radiation treatment [65]. Radiation pre-treatment may provide an avenue for enhancing the uptake of exosome-encased therapeutics for the treatment of GBM in the future.

3.2. Exosomes as a therapeutic delivery system

3.3. Exosomes as a trigger for immunotherapy

Exosomes possess a number of desirable features that can form the foundation of an ideal drug delivery system. With a long circulating half-life, the intrinsic ability to target acceptor tumour cells, biocompatibility, and minimal toxicity issues, exosomes appear to be a viable carrier, overcoming the limitations observed with the majority of polymeric drug delivery systems [129]. As exosomes are structures encased in a lipid bilayer membrane with an aqueous core, they are capable of housing both hydrophobic and lipophilic drugs. It is only recently that exosomes have been proposed as potential delivery systems for pharmacological drugs. Exosomes derived from endothelial cells of the brain were used to deliver the drugs, paclitaxel and doxorubicin across the blood brain barrier (BBB) [130]. When the drugs were administered in the absence of exosomes as the carriers, they remained within the vasculature circulation and were unable to penetrate the BBB. As exosome encapsulation allowed for the delivery of the therapeutics across the BBB, a reduction of tumour progression was also observed. This highlights a very useful feature for exosomalbased therapy, allowing the delivery of anti-tumour agents through the blood brain barrier which is otherwise highly impermeable to many chemotherapeutic agents. Building on the concept of exosomal-based drug delivery, it is possible to further direct this approach by priming exosomes for a particular cell target. Genetically engineering cells to express ligands on their cell surface that will be transferred to the surface of the exosome can determine binding to specific receptors on the target acceptor cells. A proof of principle study showed that engineered exosomes derived from dendritic cells expressing a select number of membrane proteins have been successful in delivering siRNA across the BBB in a murine animal model [131]. This was achieved by the fusion of rabies viral glycoprotein (RVG) which specifically targets acetylcholine receptors on neurons, to the N-terminal of the Lamp2b protein, which is an endogenous constituent of the exosomal membrane. This fusion construct was transfected into murine dendritic cells and the purified exosomes from these cells displayed RVG-Lamp2b particles on the outer surface. SiRNA were loaded into these exosomes which were then successfully delivered to the target neurons [131–133]. Exosome profiling experiments have also revealed an inverse correlation between tumour cell chemosensitivity and the expression of genes related to vesicle secretion. It is known that exosomes play a potential role in the export of cytotoxic substances impairing the localization of drug within the tumour cell, ultimately leading to an increase in chemoresistance [134,135]. In 2013, Munoz et al. reported an increase in miR-9 expression within temozolomide (TMZ) resistant GBM cells. MiR-9 is involved in the increased expression of drug efflux transporter, P-glycoprotein on the cell

The immune system can be manipulated to recognize selected tumour-associated mutations or antigens, forming the basis of an immunotherapeutic approach [137], although the glioma cells may be at least partially protected from the immune response by the blood brain barrier. Immunotherapy poses an attractive treatment for GBM patients as they tend to be immune suppressed, and this approach whilst targeting specific populations of tumour cells, can also boost their immune system. Unlike chemotherapy or radiation, immunotherapy is able to potentially target tumour cells whilst sparing non-tumour cells. A study by Zitvogel [138] observed that dendritic cells that were incubated in the presence of tumour peptides released exosomes, and subsequently had the ability to stimulate cytotoxic T lymphocytes CD8+ cells and suppress tumour growth in a murine model. These findings have been further extended to reveal that dendritic cell-derived exosomes (DCDE) express both MHC class I and II, which allows the exosome to display peptide fragments of non-self proteins to cytotoxic T cells (Fig. 2). DCDE also contain CD63 within their lipid bilayer, which is a co-stimulatory protein that assists in the priming of T cells during antigen presentation [139]. As focal mediators of intercellular communication and immunological function, exosomes can therefore exert effects on immune cells including B and T lymphocytes and monocytes (microglia). This provides exosomes with the ability to enhance opsonisation, regulate antigen presentation, and induce immune activation and modify immune suppression [140]. Current FDA-approved immunotherapy-based treatments include high doses of cytokines (IL-2), monoclonal antibodies or treatment with stimulated immune effector cells. The application of immunotherapy utilizing exosomes for brain tumour treatment is still in its infancy [141], with DCDE being tested primarily in the treatment of metastatic melanoma and non-small cell lung cancer models [141,142]. 3.4. Exosomes and miRNA It was reported in 2007 that exosomes arising from human mast cells contained over 1300 mRNA and 120 non-coding RNA/miRNA [40], which has since been extended as it is now known that most cells produce exosomes containing miRNA [143]. Measurements of miRNA levels in unfractionated whole serum, urine, saliva, cerebrospinal fluid and exosomes have indicated that the majority of extracellular miRNA is actually contained within exosomes [144,145]. Exosomal packaging of RNA serves as protection from extracellular RNAases, enabling exosomes to act as intercellular traffickers of miRNA. Such cell-to-cell transfer of miRNA can ultimately influence the intracellular regulation of mRNA translation as a result of the donated functional miRNA [146]. It is not known

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Fig. 2. The role of exosomes in modulating immune response. Tumour derived exosomes are endocytosed and bind with antigen presenting cells, priming CD8+ and CD4+ cells. Major Histone Compatibility complexes I and II (MHC I and II) are also expressed in exosomes. (B) Exosomes secreted by APC (antigen presenting cells) have been shown to directly modulate an antigen specific response with CD8+ and CD4+ cells, whilst also activating natural killer (NK) cells. (C) Exosomes also carry and transfer opsonins and complement factors which enhance extracellular surveillance, apoptotic cells phagocytosis/clearance and antigen presentation.

if all exosomes contain RNA, but several studies have indicated that tumour cell-derived exosomes possess a different RNA composition when compared with those from the parental cell [147– 150]. This variation in exosome composition has also been observed in the differential levels of glioma-associated exosomes when compared with normal tissue-derived exosomes [151]. However, it still remains unclear which mechanism determine the RNA that is selected and packaged into exosomes for release into the extracellular environment [152]. Therapeutics involving the exosomal-based delivery of miRNA have been conceptualized as a result of the efficient transfer of functional miRNAs within exosomes. Proof of concept was observed with the stable over-expression of miR-335 in leukemic derived human T cell that do not naturally express miR-335. This miRNA was not only found to be contained within T-cell exosomes, but T cells were able to transfer functional miR-335 to surrounding recipient cells which also did not naturally express miR-335 [153]. These findings established that cultured ‘donor’ cells could be induced to package miRNAs into exosomes and subsequently transfer these functional miRNA into recipient cells, ultimately setting the foundation for exosomal mediated miRNA transfer in future clinical therapy. It has also been shown that deletions on chromosome 10 are a common chromosomal alteration found within GBM. Region 10q24-26 is commonly lost which leads to a loss of miR-146b normally located at 10q24.32 [154]. Loss of miR-146b is known to facilitate migration and invasion in a number of different cancers, including GBM [155]. This miRNA reduces glioma cell motility and invasion, and EGFR mRNA is a binding-target for miR-146bmediated silencing [156]. Transfection with miR-146b reduces glioma cell invasion, migration, viability and expression of EGFR, which is normally amplified in approximately 40% of all GBM

patients. To ascertain the effect of exosomal delivery of miR146b, marrow stromal cells (MSC) treated with miR-146b were compared to the control, cel-miR-67, which has no mammalian targets. Exosomes secreted from both cell types were administered by intra-tumour injection into separate intracranial rat tumours, and 10 days post injection those treated with the miR-146b-MSC secreted exosomes showed significant reductions (up to 60%) in tumour volumes [155]. Identifying discrepancies in the levels of specific miRNA in GBM is a potential key for improving the choice and development of therapeutics. MiR-1 has been identified both as a regulator of exosomal function, in addition to GBM growth and invasion. Whilst the decreased expression of miR-1 in GBM cells is shown to increase in vivo growth, neovascularization and invasiveness, these affects can be attenuated when miR-1 levels are restored back to basal levels [106]. This restoration hints at the possibility of miRNA-based approaches having a strong therapeutic potential, which could be achieved through the use of exosomes as delivery and transfer agents. From a clinical scenario, intravenous administration of an exosomal-based therapy would form the most ideal and convenient approach for treatment. Whilst previous examples have utilized intra-tumour injection as the mode of delivery, intravenous delivery of an exosomal-based therapy has been trialled in breast cancer models. Exosomes containing miRNA let-7 were modified to express the transmembrane domain of plateletderived growth factor, fused with the GE11 peptide, a ligand for the EGF receptor. Intravenous injection of the let-7/GE11+ exosomes showed reduced growth of subcutaneous breast cancer tumours [157]. As EGFR expression is known to be increased within GBM, this approach may also be transferrable to GBM, as the potential of exosomes to cross the BBB has been previously demonstrated [62,64].

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Please cite this article in press as: Gourlay J et al. The emergent role of exosomes in glioma. J Clin Neurosci (2016), http://dx.doi.org/10.1016/j. jocn.2016.09.021