Extracellular vesicles as modulators of the cancer microenvironment

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Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

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Review

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Extracellular vesicles as modulators of the cancer microenvironment

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Jason Webber, Vincent Yeung, Aled Clayton ∗ Institute of Cancer and Genetics, School of Medicine, Cardiff University, Velindre Cancer Centre, Whitchurch, Cardiff CF14 2TL, United Kingdom

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Article history: Available online xxx

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Keywords: Exosomes Microvesicles Immune-evasion Angiogenesis Stroma Metastatic niche

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Contents

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The tumour microenvironment is a highly complex and dynamic tissue. It comprises not only neoplastic cells, but also other resident cells within the milieu such as stroma and vascular cells in addition to a variable cellular infiltrate from the periphery. A host of soluble factors such as growth factors, chemokines, eicosanoids soluble metabolites and extracellular matrix components have been extensively documented as factors which modulate this environment. However, in recent years there has also been growing interests in the potential roles of extracellular vesicles (EV) in many of the processes governing the nature of cancerous tissue. In this brief review, we have assembled evidence describing several distinct functions for extracellular vesicles in modulating the microenvironment with examples that include immune evasion, angiogenesis and stromal activation. Whilst there remains a great deal to be learnt about the interplay between vesicles and the cancerous environment, it is becoming clear that vesicle-mediated communication has a major influence on key aspects of cancer growth and progression. We conclude that the design of future therapeutics should acknowledge the existence and roles of extracellular vesicles, and seriously consider strategies for circumventing their effects in vivo. © 2015 Published by Elsevier Ltd.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunological control by EV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Immune activating EV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Lymphocyte inhibition by EV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Indirect mechanisms of EV supported immune evasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EV stimulated angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Delivery of angiogenic proteins by EV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. RNA delivery by EV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Modulation of EV secretion, cargo and function by hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer associated stroma and EV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. EV mediated activation of mesenchymal stroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Actions of EV produced by mesenchymal stromal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer EV and metastasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Like most cell types, neoplastic cells release small lipid-bounded vesicles into the extracellular space, but they may do so extensively

∗ Corresponding author. Tel.: +44 29 20 196148; fax: +44 29 20 529625. E-mail address: [email protected] (A. Clayton).

compared to their non-neoplastic counterparts [1]. Genotoxic, hypoxic, metabolic and other forms of cellular stress [2–4] lead to heightened levels of vesicle secretion, together with alterations in vesicle-cargo molecules. In cancer therefore, where such conditions are particularly rife, the vesicle secretion pathway appears to be a major feature. Cells can release different types of vesicles, which have been difficult to categorise in a definitive manner [5]. There

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are fundamentally two principal vesicle types under discussion. Microvesicles, which are considered large (>200 nm diameter) and dense, and arise from outward budding of the plasma membrane. Traditionally this process may have been related to a mode of purging regions of damaged membrane from the cell in response to sub-lethal complement attack for example, and is considered by many therefore as a form of debris associated with cellular damage [6]. Exosome vesicles are generally smaller (30–150 nm diameter) [7], have a characteristic density of 1.1–1.2 g/ml [8], and are manufactured within multivesicular endosomes of the late endocytic tract [8]. Small (∼100 nm) plasma-membrane derived vesicles have also been reported [9]. Categorising vesicles based on their size or subcellular origin therefore remains problematic. Furthermore defining them on the basis of molecular cargo is not straightforward due to the likely overlap between different types of vesicles. Methods such as nano-particle tracking that facilitate the counting of small particulate material invariably demonstrate the predominance of the smaller types of vesicles present in biological fluids or in cell-conditioned media [10]. Whether or not one type of vesicle is biologically more significant than another is simply unclear from our current understanding, hence, the term extracellular vesicles (EV) has been adopted by the field as these questions continue to be investigated. The transmission of EV from cancer cells to other cell types has been the subject of intensive studies in recent years. It is a process which offers a sophisticated form of cellular communication through the delivery of highly complex and dynamic cargo, packaged within a readily captured vesicle. Recipient cells usually uptake EV through endocytic processes [11], and/or for microvesicles through membrane fusion events [12,13]. Cells receive not only classical receptor–ligand interactions from EV, but do so in the context of co-delivered factors including proteins, lipids and RNA. Hence the biological effects of EV delivery can be profound, as well as difficult to study and characterise. Nevertheless there are many well characterised examples of EV functions in cancer, many of which may indeed become viewed as a coordinated set of mechanisms that act to promote disease.

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2. Immunological control by EV

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2.1. Immune activating EV

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A key discovery by Raposo et al. described the first biological effect of EV interaction with a recipient cell [8]. The study showed the capacity for EV to mimic the function of the parent cell, in this case B-lymphocytes, by stimulating T cell proliferation in an antigen and MHC-restricted manner. Hence the concept of EV-based vaccine therapeutics in cancer was born, and several studies followed demonstrating the potential for EV principally of dendritic cell origin to prime T cell responses against tumour cells [14–18]. In addition, EV from cancer cells harbour tumourspecific antigens, and these pulsed onto dendritic cells could also be a means of eliciting anti-tumour immunity [14,19], in a manner that is advantageous compared to soluble forms of antigen [20]. Modulating tumour cells in various ways, such as heat shock [21] or by forced expression of certain inflammatory factors [22] can render the delivery and cross-presentation of exosomal-antigen more efficient through maturation of the dendritic cells. Components of innate immunity, such as NKT cells may in addition be able to bolster the efficacy of exosome-based vaccines [23]. However, despite such translationally focussed studies which continue to evolve, there is mounting evidence pointing to a predominantly immune-suppressive function for exosomes of cancer cell origin.

2.2. Lymphocyte inhibition by EV Several diverse mechanisms have been reported by which EV directly participate in tumour immune evasion, with some dramatic effects such as the induction of T cell death. Among the earliest such observations was the description of EV of melanoma cells, which express Fas-ligand (CD95L) on the outer vesicle surface. When encountering Fas-positive (CD95) lymphocytes, these EV induce apoptotic death [24]. This was also confirmed as a phenomenon in colorectal cancer [25] and as a property of vesicles isolated from the sera of ovarian cancer patients [26]. In nasopharyngeal carcinoma, a tumour related to Epstein Barr virus (EBV) infection, circulating EV exhibit high levels of Galectin-9, which mediates interactions with CD4+ helper T cells through the Tim-3 receptor. This is again related to apoptotic death of a subset of T cells that would otherwise participate in tumour, or EBV-specific immune responses [27]. Immune-effector cells do not always undergo death in response to EV. Several examples of functionally important changes in the protein expression profiles of lymphocytes have been reported. The expression of the CD3/T cell receptor complex for example becomes perturbed, in a Fas-ligand related mechanism leading to suboptimal function of surviving T cells [26,28]. The c-type lectin NKG2D receptor, present on CD8+ T cells, NK cells and ␥␦-T cells is an important mechanism for recognising and responding to virally infected cells, and tumours [29]. However, in addition to proteolytic cleavage of the ligands from the tumour cell surface [30], the ligands are also actively secreted in the form of EV [31] which together with vesicular transforming growth factor beta-1 (TGF␤1), downregulate NKG2D expression levels, negatively impacting cytokine secretion and cytotoxic functions of CD8+ T cells and NK cells [32]. This particular mode of immune-control is also documented as a foetal protective mechanism during pregnancy [33], and exhibits both local and systemic effects. Other studies also point to impaired NK cell function following exposure to murine breast cancer EV, resulting in defective NK-cell mediated tumour clearance in vivo [34], although the molecular basis for this is not fully defined. The NK cell response can also be modulated by EV in haematological malignancies, where NK-activity against CLL was negatively impacted following dysregulated EV-expression of the ligand of NKp30 (termed BAG6/BAT3), with reduced vesicular BAG6/BAT3 leading to immune evasion in a xenograft model [35]. As well as directly impacting effector cells, cancer derived EV can also modulate the regulatory arm of the immune system. This was first demonstrated with pleural malignant mesothelioma derived EV’s which strongly inhibited the proliferative response of CD8+ T cells to interleukin-2 (IL-2), partly by activating the suppressive function and elevating the numbers of Foxp-3 positive Tregs [36]. This lead to the discovery that TGF␤1 is present at the vesicle surface of certain cancer derived EV, and was responsible for their antigen independent effects on Tregs [36], and in some cases interleukin-10 (IL-10) may also be involved [37]. This phenomenon is now acknowledged by several groups [37,38], and has been confirmed with EV isolated from malignant effusions [39]. In fact, such is the importance of vesicular-TGF␤1 in controlling immune responses that manipulating vesicles to express heightened TGF␤1 levels may be a novel strategy to control autoimmunity, impacting not only Treg functions but also countering inflammatory Th1 and Th17 T cell responses [40]. EV secreted by mesenchymal stem cells (MSC) thought to contribute to changes within the cancer stroma, may also exhibit inhibitory mechanisms involving TGF␤1, Galectin-1 and PDL-1 present on the EV surface, and hold potential for therapeutic use in autoimmune conditions [41]. EV, however, are also able to exert a more selective suppressive effect, through induction of antigen-specific tolerance. Elegant studies by Robbins et al. may contradict some of the

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aforementioned studies, showing transmission of antigen to dendritic cells by tumour EV can modulate dendritic cells towards a phenotype that is not activating, but rather tolerogenic in nature, leading to non-responsive T cells against a model antigen in vivo [42]. Several other studies support a negative impact of cancer EV on myeloid cells, where differentiation is skewed away from generating potent antigen presenting cells and instead towards a more suppressive cell phenotype [43–45]. Vesicular expression of TGF␤1 and the prostaglandin-E2 were identified as mediators of this differentiation [45], but signalling related to Toll like receptors, through MyD88 are also implicated [46]. The observed in vitro effects were reproduced using EV-isolated from melanoma patients but not control subjects [43], and are seen in tumour bearing mice pre-treated with tumour exosomes [46]. In addition, Hsp72 present on the surface of cancer derived EV may further exacerbate myeloid-mediated immune-suppression, by triggering autocrine IL-6 secretion enhancing their suppressive activity [47]. Together, tolerogenic or defective antigen presentation and enhancement of myeloid-cell suppression of immunity are brought about by cancer derived EV.

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2.3. Indirect mechanisms of EV supported immune evasion

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There are also examples where EV may influence immunity, in a manner that does not require direct interactions with immunecells. Naturally occurring, or therapeutically administered tumour specific antibodies can be a major mechanism for eliminating cancer cells. However, because the target epitopes may also be present on tumour EV, they act as decoy targets, sequestering antibodies and impairing antibody-dependent cell cytotoxicity [48]. Another example describes the presence of certain enzymes associated with EV that also impact immune function through their catalytic activities. Co-expression of CD39 and CD73 by tumour EV converts extracellular ATP into adenosine. T lymphocytes in particular are susceptible to adenosine, which elevates intracellular cAMP levels, and inhibits signalling from the TCR. Hence, adenosine generated by EV isolated from cell lines, or from malignant effusions render T lymphocytes dysfunctional in a contact-independent manner [49]. Cleary cancer derived EV encompass several mechanisms by which they can negatively influence the immune response to cancer. Whilst interfering with such exosome functions may be clinically attractive, this may risk perturbing a potential important mode of tumour antigen dissemination to professional antigen presenting cells (Fig. 1).

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3. EV stimulated angiogenesis

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3.1. Delivery of angiogenic proteins by EV

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Among the major hallmarks of cancer is the capacity for growing tumours to generate their own vasculature; an essential element in progressive disease [50–52]. It is increasingly clear that cancer derived EV can exert complex effects on endothelial cells, their progenitors and on supporting cells, contributing to vessel formation within tumours (Fig. 2). The expression of tetraspanin proteins is a characteristic feature of exosomes, where this complex family of proteins is often very highly enriched in exosomes compared to the parent cells. Such proteins support a host of biological functions such as adhesion, motility, etc. and are also important in angiogenic modulation. The tetraspanin CO-029 (also known as Tspan8) is expressed for example on pancreatic cancer cells, and exhibits striking angiogenesis promoting functions, particularly in triggering endothelial branching. It is disseminated locally and likely systemically in the form of EV, directly promoting vessel branching [53]. Cancer cell EV binding

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and internalisation by endothelial cells may also be modulated by Tspan8 participating directly in endothelial cell interactions [54]. EV internalised by endothelial cells stimulated elevated levels of von Willebrand factor, VEGF and VEGF-R2 and other factors driving endothelial cell proliferation, migration, sprouting and progenitor maturation [54]. The morphogenic patterning of endothelial cells along a growing, branching vessel is known to be tightly regulated by Notch signalling, which dictates a tip or stalk phenotype to endothelial cells [55]. Inhibition of Notch signalling is mediated by deltalike 4 (Dll4) which is elevated in angiogenesis. Whilst Dll4 is usually restricted to endothelium, it has also been found on colorectal cancer cells and in gliomas and on the surface of their EV [56,57]. Vesicular delivery of Dll4 to endothelial cells resulted in high Dll4/Notch-receptor ratio, low Notch signalling and drives formation of filopodia, and a tip-cell phenotype. Such changes supported increased vessel branching in tubule formation assays [57]. Other important observations document the transmission of lung cancer EV to endothelial cells delivering mutated EGF-receptor protein [13]. Upon assimilation by endothelial cells the acquired EGF-R continues to be active, signalling through MAPK and Akt. This drives the secretion of autocrine VEGF and elevated VEGFR2, enhancing endothelial cell responses to VEGF. Interfering with this mechanism, by addition of annexin-V homodimers which bind to the surface of EV negatively modulates the interaction with endothelial cells, resulting in reduced microvascular density in vivo [58]. This work therefore provides evidence that targeting EV therapeutically can give beneficial outcomes in model systems. The mutated EGF-R protein was also found on EV of glioblastoma origin in the serum of tumour bearing mice [13], and later protein [59] and mRNA [60] for EGF-R found on EV in human sera. Again the ability of such EV to support endothelial cell tubule formation in vitro was shown, likely driven by a mechanism involving proangiogenic factors associated with EV that include IL-6, IL-8 and angiogenin, FGF, TIMP-1, VEGF, and TIMP2. 3.2. RNA delivery by EV Notably, the study by Skog et al. [60], examined the mRNA content of glioblastoma EV demonstrating an enrichment in transcripts related to angiogenesis, and provided evidence that EV deliver mRNA that is subsequently translated to protein in recipient cells. Similar observations have also been made for colorectal cancer [61], pointing also to a pro-proliferative impact on endothelial cells, as well as enhanced tubule formation in 3D-culture systems [61]. Thus, genetic material and proteins like mutated EGF-R are disseminated by EV into the microenvironment, and into the circulation, providing access perhaps to a set of useful biomarkers [60]. In addition to mRNA, EV may also encapsulate micro-RNA (miR) [62], and potentially deliver this form of cargo to regulate the recipient cell’s transcriptome. One such study highlights that members of the miR-17–92 cluster can be transmitted to endothelial cells by EV, resulting in reduced expression of certain target genes such as integrin alpha5 [63]. Such alterations enhance endothelial cell migration and tube formation. Similarly, in a study of renal carcinoma, EV carried proangiogenic mRNA and miR, that supported vessel formation in vivo and drove heightened metastasis. This property however was associated exclusively to CD105-positive EV, a marker of MSC, isolated from a sub-population of tumour initiating cells. This EV function was not a property of CD105-negative EV. Of note, RNAse treatment of the CD105 positive EV abrogated these effects, implicating EV-associated RNA as mechanistically critical. However this challenges somewhat the established paradigm,

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Fig. 1. Mechanisms in which EV modulate immune responses: These include transmission of tumour-antigens to dendritic cells (DC), which may cross-present these to T cells, or may tolerise T cells in an antigen specific manner. EV-antigens may sequester antibodies, impacting antibody-dependent killing of tumour cells. Differentiation of myeloid (CD14+) cells to functional antigen presenting cells is inhibited by EV, whilst also supporting the generation of myeloid derived suppressor cells (MDSC). T lymphocytes may apoptose, or downregulate functionally important receptors, and a subset of CD4+ CD25-high cells may be induced into regulatory cells, that suppress proliferation to mitogenic stimuli. NK cell functions may also be impaired through loss of activating surface receptors.

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where RNA encapsulated within the vesicle is protected from nuclease damage [62]. If indeed so, these authors could be characterising the functions of extravesicular RNA in these observations [64].

3.3. Modulation of EV secretion, cargo and function by hypoxia Classically of course among the best recognised stimuli for angiogenesis is hypoxia; an often transient phenomenon present

Fig. 2. EV modulation of angiogenesis, and stromal activation. Delivery of mutated EGF-receptor by EV to endothelial cells elicit uptake of constitutively active receptor, driving the VEGF secretory and receptor machinery. Angiogenic modulators such as VEGF, FGF and certain tetraspanin proteins may also be associated with EV and directly control endothelial motility, and vessel formation. Delivery of Delta-like IV to endothelial cells downregulates Notch signalling, triggering enhanced vessel branching. TGF␤ activation of stromal fibroblasts or MSC leads to myofibroblastic differentiation with angiogenic and other tumour promoting functions. EV production by stromal cells, modulate cancer cell motility through a Wnt11 dependent mechanism.

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at microscopic sites within tumours that is associated with aggressive tumour phenotypes, treatment resistance and therefore poor outcome in a range of different cancers [65]. Hypoxia is also an important exogenous factor that impacts vesicle secretion and cargo, with a subsequent enhancement of EV angiogenic functions [2,66,67]. Hypoxia inducible factors (HIFs) regulate the GTPase Rab22a, which controls vesicle budding from the plasma membrane [68]. Overexpression or knockdown of Rab22a enhanced or impaired metastasis respectively, in a mouse orthotopic breast cancer model, implicating the HIF-EV pathway in tumour progression [68]. Other examples include a study by King et al., of breast cancer cell lines, demonstrating enhanced exosomes secretion under hypoxic compared to normoxic conditions and this was abrogated upon HIF-1␣-silencing by siRNA treatment. Conversely, pharmacological stimulation of HIF-1␣ enhanced EV production, mimicking hypoxia [69]. The vesicle cargo was also altered in this study, with hypoxia elevating miR-210, and hence putatively implicating this miR in angiogenesis modulation [69]. Other groups have also documented miR-210 is strikingly elevated in EV during hypoxia [70], and point to this particular EV cargo in the regulation of Ephrin-A3 activity. However, administering a complementary sequence to miR210, (an anti-miR), to abrogate these effects was only partially successful, pointing to other factors delivered by EV in these processes [70]. EV mRNA cargo may also be altered during hypoxia, with the repertoire of transcripts reflecting nicely the hypoxic status of the parent cell [71], and this is also paralleled by enhanced pro-angiogenic activities of such EV both in vitro and in vivo [71]. Importantly of course, the protein secretome during hypoxia is also drastically altered, and a significant proportion of such changes reside in the EV fraction [2]. Cancer cell EV therefore, are dynamic in the response to hypoxia and likely other extracellular factors and are capable through multiple mechanisms, of aiding the formation of tumour associated vasculature in vivo.

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was required for this process, and the resulting phenotype was described as more inflammatory than that generated from fibroblasts treated with soluble TGF␤ [76]. In fact the differences in the myofibroblasts generated with exosomes compared to soluble TGF␤ were manifold, with a clearly enhanced capacity to support angiogenic responses, and promote tumour growth in xenograft models [77]. Tumour cells rendered deficient in the secretion of exosomes, through selective knockdown of Rab27a [78] lost their capacity to grow larger tumours in this model, in the presence of co-administered stromal cells. This is therefore one example where vesicles in cancer to stroma communication function to promote disease. The cellular origins of cancer-altered stroma remains ambiguous however, with some suggesting a contribution from infiltrating MSC [79]. Breast cancer derived exosomes show the ability to differentiate mesenchymal stem cells of adipose tissue origin, into ␣SMA-positive myofibroblast like cells, secreting high levels of stromal derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF) and other factors [80]. Again, exosomal activation of the TGF␤/SMAD pathway was implicated in this, and similar findings have been reported for MSC of cord-blood origin [81]. In gastrointestinal stromal tumour, the transmission of oncogenic protein tyrosine kinase-(KIT) containing EV to adjacent smooth muscle rich stroma plays an important role [82]. Here, progenitor smooth muscle cells are converted to tumour-promoting stromal cells through activation of pathways downstream of KIT. Phenotypic changes were characterised by heightened release of several metalloproteinase including MMP1. Such matrix modulation factors supported tumour invasiveness, and would therefore contribute towards metastatic spread. It may well be that the programming of diverse stromal precursors is mediated by cancer derived EV and regardless of the originating cell, the stromal phenotype arising appear to exhibit tumour promoting functions. 4.2. Actions of EV produced by mesenchymal stromal cells

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4. Cancer associated stroma and EV Fibroblasts are the predominant cellular component of interstitial connective tissue (or stroma) and they play a matrix homeostasis function in health. This compartment however undergoes significant alterations in response to neoplasia in adjacent tissue, akin to a non-resolving wound healing-like response [72]. Cancer associated fibroblasts (CAFs) almost invariably accompany tumourigenesis, and are associated with differentiation towards a myofibroblastic phenotype [73]. Such cells are traditionally thought to secrete heightened growth factors including HGF, that promote tumour proliferation [71], inflammation and angiogenesis [72], and to aid remodelling of the extracellular matrix, contributing to invasion and metastasis [73]. However, some recent studies in pancreatic cancer give important contradictory examples where ablation of cancer-associated fibroblasts can exacerbate, rather than attenuate disease [74,75]. This is clearly an evolving research area, and the detailed fibroblastic-phenotype arising in cancer requires further study. The roles that EV play in modulating stromal cells and the consequences of such alterations have recently been explored. 4.1. EV mediated activation of mesenchymal stroma Stimulating normal fibroblasts with EV from some cancer cell lines triggers their differentiation into a “wound-healing” myofibroblastic phenotype [76]. This was characterised by the de-novo onset of alpha-smooth muscle actin (␣SMA) expression, organised into classical contractile stress fibres. EV-associated TGF␤ triggered signalling through the SMAD-dependent pathway which

Stromal cells can themselves also secrete vesicles, and these may contribute to pathogenic changes in the microenvironment. For example the migratory potential of breast cancer cells were promoted by fibroblast exosomes [83]. The mechanism underlying this process was quite unique, and involved the uptake of CD81-positive fibroblast exosomes by cancer cells, resulting in their acquisition of Wnt11 during their intracellular trafficking. This promoted the interaction between Wnt11 and Fzd6 at the leading edge of carcinoma cell protrusions, which was essential for heightened motility and metastasis in vivo. Notably, other researchers have also linked Wnt-family members and exosomes, revealing control of exosome secretion in melanoma cells as a Wnt5A regulated process, implicated in angiogenesis [84]. MSC also secrete vesicles with tumour promoting effects in vivo. One mechanism was attributed to the ERK1/2 dependent exosomemediated promotion of tumour derived VEGF. Co-implantation of tumour cells with MSC-exosomes resulted in elevated tumour incidence and growth, with evidence for local stromal myofibroblast involvement (␣SMA-positive staining) and angiogenesis [85]. MSC and the EV which they produce have also recently been implicated in myeloma. Exosomes from myeloma educated MSC exhibited different miR profiles, and elevated oncogenic proteins, adhesion molecules and other factors capable of promoting the growth of myeloma cells [86]. Importantly the phenotype of MSC used in such studies is critical as not all MSC exosomes exhibit diseasepromoting effects. In this study, normal bone marrow derived MSC impaired myeloma growth [86] and similar effects have been reported in other studies of MSC derived EV [87]. This certainly raises an intriguing possibility of using exosomes isolated from an appropriate MSC phenotype as a therapeutic agent. The complex

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interactions that such EV may have on physiological systems in general, however would require considerable future investigation and a suitable degree of caution. 5. Cancer EV and metastasis An important paradigm in the complexities of metastatic tumour spread is that tissues receive long distance signals from primary tumour sites in order to prepare for the eventual landing and seeding by tumour cells. This “pre-metastatic niche” formation is an aspect of considerable interest to EV researchers as cancer vesicles which are known to be present in the circulation may be an ideal modality for long-range signal dissemination. Among the earliest reports of EV playing a role in this phenomenon was by Hood et al. [88], demonstrating the priming of sentinel lymph nodes by melanoma derived EV. This resulted in micro-anatomical changes within the lymph node tissue, where melanoma cells would migrate to, and accumulate in, exosomerich sites. Subsequent changes in extracellular matrix and vascular growth factors, promoted establishment of the metastatic site. Similarly, melanoma EV can also stimulate metastasis to bone and brain, without always impacting growth at the primary site [89], and can elevate permeability of lung endothelia, facilitating metastatic spread. There is also an important role for EV in mobilising bone marrow progenitors, an aspect documented as important for metastatic progression [90]. This aspect involved the EV-transfer of MET to the progenitors, as shRNA-mediated knockdown of MET levels in EV strongly reduced metastatic spread. Acquisition of MET enhanced mobilisation of both vasculogenic and haematopoietic progenitors into the circulation, and the observations made in mice were also confirmed in patients with advanced stage melanoma demonstrating circulating progenitor cells with elevated levels of MET. Inhibiting the secretion of EV by such melanoma cells by knockdown of Rab27a lead to strikingly diminished metastasis in these models [89]. However in a breast cancer model, knockdown of EV secretion by Rab27a, impaired tumour growth and metastasis in only one of two cell lines tested [91], suggesting perhaps that the dependency for EV-functions may well be cell-type/line dependent. Furthermore in other reports, exosomes alone may not be sufficient for forming a metastatic niche, requiring in addition other factors within the cancer cell secretome such as CD44v6 [92]. Collectively these data point to a complex interplay between vesicular and non-vesicular components of the extracellular environment in driving tumour spread. 6. Conclusions and future perspectives As the EV-research field continues to mature, the evidence for the important roles of vesicles in microenvironment modulation is compelling. EV clearly exert multiple, and highly complex effects involving many distinct molecular mediators and pathways. There is currently enormous enthusiasm and research activity involving profiling the RNA content of EV, but direct evidence for a causal relationship between RNA-delivery by EV and microenvironmental alterations is not yet fully convincing. Given the technical difficulties in modulating the RNA-cargo of EV, or in reporting the consequence of acquired vesicular RNA, these gaps in our understanding are perhaps not surprising. We are able to manipulate proteins or their activities with greater success, and there are clearcut data pointing to potent effects of EV-associated oncoproteins, cytokines and growth factors and various other receptor ligands in modulating the behaviour of cells towards promoting disease. Having the capacity to inhibit the secretion of EV by cancer cells in a clinical setting would likely yield some significant advantages in the treatment of cancer. These include aspects we have

discussed herein such as alleviating immune suppression, attenuation of angiogenesis and stromal activation, limiting the capacity of cancers to form a pre-metastatic niche and their motility and invasive characteristics. In addition, however, there are yet further relevant aspects to also consider. The expulsion of cytotoxic drugs from cancer cells can occur partly through EV production [93], hence abrogating EV production would prolong the effective intracellular concentration of drugs, improving treatment efficacy. Furthermore, particularly in advanced cancers, many patients exhibit complications related to coagulation, that may also be due to elevated tissue-factor positive EV in the blood [94]. Whilst some developments attempt to achieve reduction of circulating EV using a blood-filtration approach [95], the wisdom of such a global EV-removal tool is currently unclear. Our knowledge is growing about EV in pathological situations; however there remains a major knowledge gap in terms of the roles of EV under normal, steadystate conditions. Hence removing all EV from the blood for example may present unexpected and unwanted consequences. Currently, therefore, we require small molecular inhibitors or other clinically applicable reagents that can interfere with these EV driven mechanisms, or better still, that can selectively abrogate EV production by cancer cells altogether. This area of cancer research is clearly rife for future exploration, and provides optimistic avenues to prolong the survival of people living with cancer.

Acknowledgements The Cardiff Exosome group is funded principally by a Programme Grant from Cancer Research Wales, awarded to AC, and Q3 also by the Life Science Research Network Wales, an initiative funded through the Welsh Government’s Ser Cymru programme Q4 (Studentship (to VY), by the Movember Global Action Plan-1 (AC), by Prostate Cancer UK (AC & JW), and by the British Lung Foundation (AC)).

References [1] Yu X, Harris SL, Levine AJ. The regulation of exosome secretion: a novel function of the p53 protein. Cancer Res 2006;66:4795–801. [2] Park JE, Tan HS, Datta A, Lai RC, Zhang H, Meng W, et al. Hypoxic tumor cell modulates its microenvironment to enhance angiogenic and metastatic potential by secretion of proteins and exosomes. Mol Cell Proteomics 2010;9:1085–99. [3] Clayton A, Turkes A, Navabi H, Mason MD, Tabi Z. Induction of heat shock proteins in B-cell exosomes. J Cell Sci 2005;118:3631–8. [4] de Jong OG, Verhaar MC, Chen Y, Vader P, Gremmels H, Posthuma G, et al. Cellular stress conditions are reflected in the protein and RNA content of endothelial cell-derived exosomes. J Extracell Vesicles 2012;1:18396. [5] Gould SJ, Raposo G. As we wait: coping with an imperfect nomenclature for Q5 extracellular vesicles. J Extracell Vesicles 2013. [6] Pilzer D, Gasser O, Moskovich O, Schifferli JA, Fishelson Z. Emission of membrane vesicles: roles in complement resistance, immunity and cancer. Springer Semin Immunopathol 2005;27:375–87. [7] Johnstone R, Adam M, Hammond J, Orr L, Turbide C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 1987;262:9412–20. [8] Raposo G, Nijman HW, Stoorvogel W, Leijendekker R, Harding CV, Melief CJM, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med 1996;183:1161–72. [9] Booth AM, Fang Y, Fallon JK, Yang J-M, Hildreth JEK, Gould SJ. Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol 2006;172:923–35. [10] Webber J, Clayton A. How pure are your vesicles? J Extracell Vesicles 2013:19861. [11] Svensson KJ, Christianson HC, Wittrup A, Bourseau-Guilmain E, Lindqvist E, Svensson LM, et al. Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid raft-mediated endocytosis negatively regulated by caveolin1. J Biol Chem 2013;288:17713–24. [12] del Conde I, Shrimpton CN, Thiagarajan P, López JA. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005;106:1604–11. [13] Al-Nedawi K, Meehan B, Micallef J, Lhotak V, May L, Guha A, et al. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 2008;10:619–24.

Please cite this article in press as: Webber J, et al. Extracellular vesicles as modulators of the cancer microenvironment. Semin Cell Dev Biol (2015), http://dx.doi.org/10.1016/j.semcdb.2015.01.013

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[14] Andre F, Schartz NE, Movassagh M, Flament C, Pautier P, Morice P, et al. Malignant effusions and immunogenic tumour-derived exosomes. Lancet 2002;360:295–305. [15] Lamparski H, Metha-Damani A, Yao J, Patel S, Hsu D, Ruegg C, et al. Production and characterization of clinical grade exosomes derived from dendritic cells. J Immunol Methods 2002;270:211–26. [16] Morse M, Garst J, Osada T, Khan S, Hobeika A, Clay T, et al. A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. J Transl Med 2005;3:9. [17] Escudier B, Dorval T, Chaput N, André F, Caby M, Novault S, et al. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of the first phase I clinical trial. J Transl Med 2005; 3:10. [18] Chaput N, Schartz NEC, Andre F, Taieb J, Novault S, Bonnaventure P, et al. Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection. J Immunol 2004;172:2137–46. [19] Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, Masurier C, et al. Tumorderived exosomes are a source of shared tumor rejection antigens for CTL crosspriming. Nat Med 2001;7:297–303. [20] Zeelenberg IS, Ostrowski M, Krumeich S, Bobrie A, Jancic C, Boissonnas A, et al. Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses. Cancer Res 2008;68:1228–35. [21] Weilin C, Jianli W, Chuansen S, Shuxun L, Yizhi Y, Qingqing W, et al. Efficient induction of antitumor T cell immunity by exosomes derived from heatshocked lymphoma cells. Eur J Immunol 2006;36:1598–607. [22] Dai S, Zhou X, Wang B, Wang Q, Fu Y, Chen T, et al. Enhanced induction of dendritic cell maturation and HLA-A*0201-restricted CEA-specific CD8+ CTL response by exosomes derived from IL-18 gene-modified CEA-positive tumor cells. J Mol Med 2006;84:1067–76. [23] Gehrmann U, Hiltbrunner S, Georgoudaki A-M, Karlsson MC, Näslund TI, Gabrielsson S. Synergistic induction of adaptive antitumor immunity by codelivery of antigen with ␣-galactosylceramide on exosomes. Cancer Res 2013;73:3865–76. [24] Andreola G, Rivoltini L, Castelli C, Huber V, Perego P, Deho P, et al. Induction of lymphocyte apoptosis by tumor cell secretion of FasL-bearing microvesicles. J Exp Med 2002;195:1303–16. [25] Huber V, Fais S, Iero M, Lugini L, Canese P, Squarcina P, et al. Human colorectal cancer cells induce T-cell death through release of proapoptotic microvesicles: role in immune escape. Gastroenterology 2005;128:1796–804. [26] Taylor DD, Gerc¸el-Taylor C¸, Lyons KS, Stanson J, Whiteside TL. T-cell apoptosis and suppression of T-cell receptor/CD3-␰ by Fas ligand-containing membrane vesicles shed from ovarian tumors. Clin Cancer Res 2003;9:5113–9. [27] Klibi J, Niki T, Riedel A, Pioche-Durieu C, Souquere S, Rubinstein E, et al. Blood diffusion and Th1-suppressive effects of galectin-9-containing exosomes released by Epstein-Barr virus-infected nasopharyngeal carcinoma cells. Blood 2009;113:1957–66. [28] Taylor D, Gerc¸el-Taylor C. Tumour-derived exosomes and their role in cancerassociated T-cell signalling defects. Br J Cancer 2005;92:305–11. [29] Sutherland CL, Rabinovich B, Chalupny NJ, Brawand P, Miller R, Cosman D. ULBPs, human ligands of the NKG2D receptor, stimulate tumor immunity with enhancement by IL-15. Blood 2006;108:1313–9. [30] Groh V, Wu J, Yee C, Spies T. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature 2002;419:734–8. [31] Clayton A, Tabi Z. Exosomes and the MICA-NKG2D system in cancer. Blood Cells Mol Dis 2005;34:206–13. [32] Clayton A, Mitchell JP, Court J, Linnane S, Mason MD, Tabi Z. Human tumor-derived exosomes down-modulate NKG2D expression. J Immunol 2008;180:7249–58. [33] Hedlund M, Stenqvist A-C, Nagaeva O, Kjellberg L, Wulff M, Baranov V, et al. Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function. J Immunol 2009;183:340–51. [34] Liu C, Yu S, Zinn K, Wang J, Zhang L, Jia Y, et al. Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function. J Immunol 2006;176:1375–85. [35] Reiners KS, Topolar D, Henke A, Simhadri VR, Kessler J, Sauer M, et al. Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity. Blood 2013;121:3658–65. [36] Clayton A, Mitchell JP, Court J, Mason MD, Tabi Z. Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2. Cancer Res 2007;67:7458–66. [37] Szajnik M, Czystowska M, Szczepanski MJ, Mandapathil M, Whiteside TL. tumor-derived microvesicles induce, expand and up-regulate biological activities of human regulatory T cells (Treg). PLoS ONE 2010;5:e11469. [38] Wieckowski EU, Visus C, Szajnik M, Szczepanski MJ, Storkus WJ, Whiteside TL. Tumor-derived microvesicles promote regulatory T cell expansion and induce apoptosis in tumor-reactive activated CD8+ T lymphocytes. J Immunol 2009;183:3720–30. [39] Wada J, Onishi H, Suzuki H, Yamasaki A, Nagai S, Morisaki T, et al. Surfacebound TGF-beta-1 on effusion-derived exosomes participates in maintenance of number and suppressive function of regulatory T-cells in malignant effusions. Anticancer Res 2010;30:3747–57. [40] Yu L, Yang F, Jiang L, Chen Y, Wang K, Xu F, et al. Exosomes with membraneassociated TGF-␤1 from gene-modified dendritic cells inhibit murine EAE independently of MHC restriction. Eur J Immunol 2013;43:2461–72.

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[41] Mokarizadeh A, Delirezh N, Morshedi A, Mosayebi G, Farshid A-A, Mardani K. Microvesicles derived from mesenchymal stem cells: potent organelles for induction of tolerogenic signaling. Immunol Lett 2012;147:47–54. [42] Yang C, Kim S-H, Bianco NR, Robbins PD. Tumor-derived exosomes confer antigen-specific immunosuppression in a murine delayed-type hypersensitivity model. PLoS ONE 2011;6:e22517. [43] Valenti R, Huber V, Filipazzi P, Pilla L, Sovena G, Villa A, et al. Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-{beta}-mediated suppressive activity on T lymphocytes. Cancer Res 2006;66:9290–8. [44] Yu S, Liu C, Su K, Wang J, Liu Y, Zhang L, et al. Tumor exosomes inhibit differentiation of bone marrow dendritic cells. J Immunol 2007;178:6867–75. [45] Xiang A, P C, L Y, L ZB, D JW, et al. Induction of myeloid-derived suppressor cells by tumor exosomes. Int J Cancer 2009;124:2621–33. [46] Liu Y, Xiang X, Zhuang X, Zhang S, Liu C, Cheng Z, et al. Contribution of MyD88 to the tumor exosome-mediated induction of myeloid derived suppressor cells. Am J Pathol 2010;176:2490–9. [47] Chalmin F, Ladoire S, Mignot G, Vincent J, Bruchard M, Remy-Martin JP, et al. Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloidderived suppressor cells. J Clin Invest 2010;120:457–71. [48] Battke C, Ruiss R, Welsch U, Wimberger P, Lang S, Jochum S, et al. Tumour exosomes inhibit binding of tumour-reactive antibodies to tumour cells and reduce ADCC. Cancer Immunol Immunother 2011;60:639–48. [49] Clayton A, Al-Taei S, Webber J, Mason MD, Tabi Z. Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production. J Immunol 2011;187:676–83. [50] Cao Y, Arbiser J, D’Amato RJ, D’Amore PA, Ingber DE, Kerbel R, et al. Forty-year journey of angiogenesis translational research. Sci Transl Med 2011;3:114rv3. [51] Welti J, Loges S, Dimmeler S, Carmeliet P. Recent molecular discoveries in angiogenesis and antiangiogenic therapies in cancer. J Clin Invest 2013;123:3190–200. [52] Abdollahi A, Folkman J. Evading tumor evasion: current concepts and perspectives of anti-angiogenic cancer therapy. Drug Resist Updat 2010;13:16–28. [53] Gesierich S, Berezovskiy I, Ryschich E, Zoller M. Systemic induction of the angiogenesis switch by the tetraspanin D6.1A/CO-029. Cancer Res 2006;66:7083–94. [54] Nazarenko I, Rana S, Baumann A, McAlear J, Hellwig A, Trendelenburg M, et al. Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosomeinduced endothelial cell activation. Cancer Res 2010;70:1668–78. [55] Phng LK, Gerhardt H. Angiogenesis: a team effort coordinated by notch. Dev Cell 2009;16:196–208. [56] Jubb AM, Turley H, Moeller HC, Steers G, Han C, Li JL, et al. Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer. Br J Cancer 2009;101:1749–57. [57] Sheldon H, Heikamp E, Turley H, Dragovic R, Thomas P, Oon CE, et al. New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes. Blood 2010;116:2385–94. [58] Al-Nedawi K, Meehan B, Kerbel RS, Allison AC, Rak J. Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR. PNAS 2009;106:3794–9. [59] Graner MW, Alzate O, Dechkovskaia AM, Keene JD, Sampson JH, Mitchell DA, et al. Proteomic and immunologic analyses of brain tumor exosomes. FASEB J 2009;23:1541–57. [60] Skog J, Wurdinger T, van Rijn S, Meijer DH, Gainche L, Curry WT, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 2008;10:1470–6. [61] Hong BS, Cho J-H, Kim H, Choi E-J, Rho S, Kim J, et al. Colorectal cancer cellderived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells. BMC Genomics 2009:10. [62] Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosomemediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654–9. [63] Umezu T, Ohyashiki K, Kuroda M, Ohyashiki JH. Leukemia cell to endothelial cell communication via exosomal miRNAs. Oncogene 2013;32:2747–55. [64] Grange C, Tapparo M, Collino F, Vitillo L, Damasco C, Deregibus MC, et al. Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche. Cancer Res 2011;71:5346–56. [65] Vaupel P, Mayer A. Hypoxia in cancer: significance and impact on clinical outcome. Cancer Metast Rev 2007;26:225–39. [66] Wysoczynski M, Ratajczak MZ. Lung cancer secreted microvesicles: underappreciated modulators of microenvironment in expanding tumors. Int J Cancer 2009;125:1595–603. [67] Orriss IR, Knight GE, Utting JC, Taylor SE, Burnstock G, Arnett TR. Hypoxia stimulates vesicular ATP release from rat osteoblasts. J Cell Physiol 2009;220:155–62. [68] Wang T, Gilkes DM, Takano N, Xiang L, Luo W, Bishop CJ, et al. Hypoxia-inducible factors and RAB22A mediate formation of microvesicles that stimulate breast cancer invasion and metastasis. Proc Natl Acad Sci USA 2014. [69] King H, Michael M, Gleadle J. Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer 2012;12:421. [70] Tadokoro H, Umezu T, Ohyashiki K, Hirano T, Ohyashiki JH. Exosomes derived from hypoxic leukemia cells enhance tube formation in endothelial cells. J Biol Chem 2013;288:34343–51. [71] Kucharzewska P, Christianson HC, Welch JE, Svensson KJ, Fredlund E, Ringnér M, et al. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxiadependent activation of vascular cells during tumor development. Proc Natl Acad Sci USA 2013;110:7312–7.

Please cite this article in press as: Webber J, et al. Extracellular vesicles as modulators of the cancer microenvironment. Semin Cell Dev Biol (2015), http://dx.doi.org/10.1016/j.semcdb.2015.01.013

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ARTICLE IN PRESS J. Webber et al. / Seminars in Cell & Developmental Biology xxx (2015) xxx–xxx

[72] Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer 2006;6:392–401. [73] Tuxhorn JA, Ayala GE, Smith MJ, Smith VC, Dang TD, Rowley DR. Reactive stroma in human prostate cancer. Clin Cancer Res 2002;8:2912–23. [74] Özdemir Berna C, Pentcheva-Hoang T, Carstens Julienne L, Zheng X, Wu C-C, Simpson Tyler R, et al. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 2014;25:719–34. [75] Rhim Andrew D, Oberstein Paul E, Thomas Dafydd H, Mirek Emily T, Palermo Carmine F, Sastra Stephen A, et al. Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma. Cancer Cell 2014;25:735–47. [76] Webber J, Steadman R, Mason MD, Tabi Z, Clayton A. Cancer exosomes trigger fibroblast to myofibroblast differentiation. Cancer Res 2010;70:9621–30. [77] Webber JP, Spary LK, Sanders AJ, Chowdhury R, Jiang WG, Steadman R, et al. Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes. Oncogene 2014;34:290–302. [78] Ostrowski M, Carmo NB, Krumeich S, Fanget I, Raposo G, Savina A, et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat Cell Biol 2010;12:19–30. [79] Polanska UM, Orimo A. Carcinoma-associated fibroblasts: non-neoplastic tumour-promoting mesenchymal cells. J Cell Physiol 2013;228:1651–7. [80] Cho JA, Park H, Lim EH, Lee KW. Exosomes from breast cancer cells can convert adipose tissue-derived mesenchymal stem cells into myofibroblast-like cells. Int J Oncol 2012;40:130–8. [81] Gu J, Qian H, Shen L, Zhang X, Zhu W, Huang L, et al. Gastric cancer exosomes trigger differentiation of umbilical cord derived mesenchymal stem cells to carcinoma-associated fibroblasts through TGF-␤/Smad pathway. PLoS ONE 2012;7:e52465. [82] Atay S, Banskota S, Crow J, Sethi G, Rink L, Godwin AK. Oncogenic KIT-containing exosomes increase gastrointestinal stromal tumor cell invasion. Proc Natl Acad Sci USA 2014;111:711–6. [83] Luga V, Zhang L, Viloria-Petit Alicia M, Ogunjimi Abiodun A, Inanlou Mohammad R, Chiu E, et al. Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 2012;151:1542–56.

[84] Ekström EJ, Bergenfelz C, von Bülow V, Serifler F, Carlemalm E, Jönsson G, et al. WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells. Mol Cancer 2014;13:88. [85] Zhu W, Huang L, Li Y, Zhang X, Gu J, Yan Y, et al. Exosomes derived from human bone marrow mesenchymal stem cells promote tumor growth in vivo. Cancer Lett 2012;315:28–37. [86] Roccaro AM, Sacco A, Maiso P, Azab AK, Tai Y-T, Reagan M, et al. BM mesenchymal stromal cell-derived exosomes facilitate multiple myeloma progression. J Clin Invest 2013;123:1542–55. [87] Bruno S, Collino F, Deregibus MC, Grange C, Tetta C, Camussi G. Microvesicles derived from human bone marrow mesenchymal stem cells inhibit tumor growth. Stem cells Dev 2013;22:758–71. [88] Hood JL, San RS, Wickline SA. Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis. Cancer Res 2011;71:3792–801. [89] Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med 2012;18:883–91. [90] Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev Cancer 2009;9:239–52. [91] Bobrie A, Krumeich S, Reyal F, Recchi C, Moita LF, Seabra MC, et al. Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression. Cancer Res 2012;72:4920–30. [92] Jung T, Castellana D, Klingbeil P, Hernández IC, Vitacolonna M, Orlicky DJ, et al. CD44v6 dependence of premetastatic niche preparation by exosomes. Neoplasia (New York, NY) 2009;11:1093–117. [93] Gong J, Jaiswal R, Mathys JM, Combes V, Grau GER, Bebawy M. Microparticles and their emerging role in cancer multidrug resistance. Cancer Treat Rev 2012;38:226–34. [94] Geddings JE, Mackman N. Tumor-derived tissue factor – positive microparticles and venous thrombosis in cancer patients. Blood 2013;122:1873–80. [95] Marleau A, Chen C-S, Joyce J, Tullis R. Exosome removal as a therapeutic adjuvant in cancer. J Transl Med 2012;10:134.

Please cite this article in press as: Webber J, et al. Extracellular vesicles as modulators of the cancer microenvironment. Semin Cell Dev Biol (2015), http://dx.doi.org/10.1016/j.semcdb.2015.01.013

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