Outsmart tumor exosomes to steal the cancer initiating cell its niche

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Review

Outsmart tumor exosomes to steal the cancer initiating cell its niche Florian Thuma, Margot Zöller ∗ Department of Tumor Cell Biology, University Hospital of Surgery and German Cancer Research Center, Heidelberg, Germany

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Keywords: Exosomes Metastasis Angiogenesis Cancer initiating cells Extracellular matrix

a b s t r a c t Exosomes are small vesicles that derive from endosomes and are delivered by many cells, including tumor cells that are a particular rich source of exosomes. Exosomes are suggested to be the most potent intercellular communicators. Being recovered in all body fluids, they can communicate with neighboring as well as distant cells. The latter was first described for dendritic cell exosomes that can initiate T cell activation. However, tumor exosomes (TEX) may impede this crosstalk. Besides with hematopoietic cells, TEX communicate with the tumor cell itself, but also with host stroma cells and endothelial cells. This crosstalk received much attention as there is strong evidence that TEX account for angiogenesis and premetastatic niche formation, which may proceed directly via binding and uptake of TEX by cells in the premetastatic organ or indirectly via TEX being taken up by hematopoietic progenitors in the bone marrow (BM), which mature toward lineages with immunosuppressive features or are forced toward premature release from the BM and homing into premetastatic organs. Knowing these deleterious activities of TEX, it becomes demanding to search for modes of therapeutic interference. I here introduce our hypothesis that metastasis formation may be hampered by tailored exosomes that outsmart TEX. The essential prerequisites are an in depth knowledge on TEX binding, uptake, binding-initiated signal transduction and uptake-promoted target cell reprogramming. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction 1.1. Metastasis and cancer initiating cells Metastasis frequently are the corner stone for curative therapy despite considerable progress in surgery, radiation and cytotoxic drug therapy [1,2]. Metastasis formation is the result of a cascade of events that primary tumor cells pass through by changing their phenotype and by their cross-talk with the host environment. In epithelial tumors the metastatic cascade is initiated through a pro-

Abbreviations: ADAM, A disintegrin and metalloproteinase; AML, actute myeloid leukemia; ASC, adult stem cells; BM, bone marrow; BMC, BM cell; CAF, cancerassociated fibroblast; CIC, cancer initiating cells; CML, chronic myeloid leukemia; CTL, cytotoxic T cells; DC, dendritic cell; ECM, extracellular matrix; EMT, epithelial mesenchymal transition; ESC, embryonic stem cell; ESCRT, endosomal sorting complex required for transport; HCV, hepatitis C virus; HSP, heat shock protein; LIC, leukemia-initiating cell; MDSC, myeloid-derived suppressor cells; MMP, matrix metalloproteinase; MSC, mesenchymal stem cells; M␾, macrophage; TEX, tumor exosomes; Th, helper T cells; Treg, regulatory T cells. ∗ Corresponding author at: Department of Tumor Cell Biology, Im Neuenheimer Feld 365, D 69120 Heidelberg, Germany. Tel.: +49 6221 565146; fax: +49 6221 565199. E-mail addresses: [email protected], [email protected] (M. Zöller).

cess called epithelial to mesenchymal transition (EMT), followed by individual tumor cells separating from the primary tumor mass to intravasate, extravasate and finally to settle and grow in distant organs [3,4]. The propensity to metastasize most likely relies on so called cancer initiating cells (CIC), a small subpopulation suggested accounting for primary tumor growth as well as metastatic spread [5]. CIC are long lived, slowly progressing through the cell cycle, radiation and drug resistant and use similar signaling pathways that guide the fate of embryonic and adult stem cells (ESC, ASC) [6], sharing with ESC over-expression of Oct4, Nanog, and c-Myc [7] and the signaling pathways Notch, Wnt and Hedgehog, important in shaping structure, cell fate and identity [8]. There is also compelling evidence for joint altered epigenetic regulation in ASC and CIC. Thus, polycomb genes, which play a role in transcriptional repression through histone modifications, associate with the promoter and regulatory regions of target genes in ASC as well as CIC [9]. Furthermore, the miRNA profile of tumor cells differs significantly from that of non-transformed cells with upregulation of several miRNA that support tumor growth, collectively called oncoMirs [10], which may act by targeting tumor suppressors [11]. Instead, tumor suppressor miRNA, e.g. let-7, miR-15a and others that suppress MET and Bcl2 are downregulated [12,13]. We recently experienced that a non-metastatic variant of a highly metastatic rat tumor line

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expressed the tumor suppressors let-7b, let-7d, let-7e and miR-101 at a high level, but oncoMir miR-494 and Pten-regulating miR21, known for promoting metastasis [14] and apoptosis-regulating miR-24-1 [15] were highly expressed in the metastasizing line [16]. 1.2. Metastasis and the niche The fate of ESC and ASC is determined by their position and is minutely regulated by few adjacent cells creating a defined environment, the niche [17]. CIC, too, appear to require a niche, possibly during oncogenesis and during settlement and growth in distant organs [18]. The latter is called the pre-metastatic niche, because it is initiated by the primary tumor before metastasizing cell arrival [19]. SC niches, composed of epithelial and mesenchymal cells and extracellular substrates, are important for maintenance of stemness [20] and function as an extrinsic regulatory system, which maintains and governs the location, adhesiveness, retention, homing, mobilization, quiescence/activation, symmetric/asymmetric division and differentiation [18,20]. The main contributors, Wnt, Hedgehog, Notch, TGF␤, several tyrosine kinase receptors all use similar intracellular signaling pathways like the Ras-Raf-MAPK and PI3K-Akt pathway [21]. Whether CIC require a niche has not been unequivocally answered. First, the niche for ASC provides a regulatory system, which may well function to prevent tumorigenesis by controlling ASC, but could also promote CIC proliferation [18]. Beyond this, CIC may be reprogrammed by exposing them to an embryonic microenvironment, which concept has received experimental support for malignant melanoma [22]. A preformed niche also can support CIC survival and homing [23], demonstrated for neural and colorectal CIC [24,25]. Important contributors to the CIC niche are cancer-associated fibroblasts (CAF), contributing to extracellular matrix (ECM) remodeling by provision of HGF, IL6, PDGF␤, prostaglandins and proteases [24,26] as well as by miRNA, where miR-31, which targets the chromatin remodeling homeobox gene SATB2 is strikingly downregulated [27]. In addition, exchange of miRNA between CIC and their niche plays an important role. Several miRNA implicated in cell proliferation were shown to become transferred from mammary cancer stroma cell into tumor cells [28]. Other important players are mesenchymal stem cells (MSC) [29] where nestin1+ MSC together with CAF and M␾ are particularly important for leukemia-IC [30]. Furthermore, tumor cell-derived IL1 induces PGE2 secretion by MSC, which operates in an autocrine manner to promote cytokine secretion that induce ␤-catenin signaling and CIC formation in adjacent tumor cells [31]. Further evidences for CIC requiring a niche is the finding that EMT is promoted by the tumor stroma. Myofibroblasts secret TGF␤ and by back-signaling force CIC into EMT [32]. Additional inflammatory mediators delivered by the stroma, like TNF␣ and IL6, sustain TGF␤ production, where IL6 attracts MSC to produce CIC supportive CXCL7 [33]. Thus, CIC shape their own environment by recruiting and activating specific cell types that support their maintenance. Additionally, CIC may even differentiate into niche cells, demonstrated for glioblastoma CIC [34]. Of particular interest with respect to the CIC niche is the study of Kaplan et al. [19] describing the formation of a pre-metastatic niche in organs, where tumor cells are likely to settle and grow, in advance to tumor cell arrival. It was suggested that resident fibroblasts become stimulated by tumor-derived growth factors to secrete fibronectin that promotes attachment of hematopoietic progenitors expressing VEGFR1 and VLA-4. In addition, stromal fibroblasts frequently express high amounts of CXCL12 that attract CXCR-4 expressing hematopoietic progenitors and CIC [35]. CAF also secrete CXCL12, attracting tumor cells as well as endothelial cell progenitors [36], demonstrated in several malignancies [37]. Via HGF expressing MSC, c-Met also becomes involved in

angiogenesis [38] and ␤-catenin, one of the key players in metastasis [38], translocates to the nucleus upon activation, e.g. via Wnt signaling, and interacts with the Tcf/Lef complex turning on cell cycle related genes like cyclin D1 and c-Myc [39]. ␤-catenin localization in the nucleus is further supported by integrin-linked kinase activation via matrix-bound ␤1 integrins and costimulatory signals like HGF, EGF, TGF␤ from the environment [40]. Taken together, CIC/metastasizing tumor cells depend on short ranged and long distance intercellular cross-talks, suggested to be the prime activity of exosomes [41–43]. Notably, many of the described contributors are widespread and it is difficult to imagine, particularly for long-distance communications, how selectivity is achieved. In view of these unexplained features and based on our finding that exosomes from a metastasizing rat pancreatic tumor line are essentially required to allow a non-metastasizing variant to settle in lymph nodes and lung [44], we proposed exosomes as the central actor. After a brief introduction on tumor exosomes (TEX), I will discuss how they communicate with surrounding and distant tissues and finally comment on exosomes as potential therapeutics. 2. Exosomes Exosomes are small 30–100 nm vesicles, which derive from the fusion of the intraluminal vesicles of multivesicular bodies (MVB) with the plasma membrane [41]. The molecular composition of exosomes reflects their origin from intraluminal vesicles. Besides a common set of membrane and cytosolic molecules, which includes tetraspanins, exosomes harbor subsets of proteins, such as adhesion molecules, molecules associated with vesicle transport, cytoskeletal proteins, signal transduction molecules, enzymes and others that are linked to cell type-specific functions. They also contain selected mRNA and miRNA [45,46]. 2.1. Exosome generation and composition It is well known that the relative abundance of proteins, mRNA and miRNAs differs between exosomes and donor cells. This implies active sorting into MVB. Protein sorting depends on monoubiquitinylation and the endosomal sorting complex required for transport (ESCRT). Besides, protein recruitment into tetraspanin networks and other internalization prone detergent resistant membrane domains can also be decisive, where raft microdomains enriched in sphingolipids, which form ceramide, play an important role [47–49]. Notably, recruitment via tetraspanin-enriched or raft microdomains is accompanied by exosomal recovery of protein complexes rather than singular molecules, that may have an impact on exosome targeting and the crosstalk with target structures [50]. mRNA recruitment may be guided by a zip code in the 3 -UTR [51]. miRNA recruitment is facilitated by physical and functional coupling of RISCs (RNA-induced silencing complexes) to components of the sorting complex. GW182 containing GW bodies, sorted into MVB, promote continuous assembly/disassembly of membrane-associated miRNA-loaded RISC [52,53]. The release of miRNA being actively controlled through a ceramide-dependent machinery associated with exosome secretion [54] points toward a contribution of tetraspanins known to interact with gangliosides [55]. According to their origin from endosomes/MVB, the exosomal protein profile is rich in molecules located in membrane domains prone for internalization as well as in molecules engaged in fission, scission and vesicular transport [46,56,57], where tetraspanins are frequently used to differentiate exosomes from other extracellular vesicles [46,58]. Notably, protein complexes as formed during internalization are maintained and recovered in exosomes. Thus, during stress-induced internalization the tetraspanin

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Tspan8 moves toward clathrin-coated pits, where it associates with ␣4␤1 or ␣6␤4, whereas in resting cells it mostly associates with ␣3␤1 [59]. This has striking consequences on exosome target cell selection as well as exosome activity [59,60]. Other abundantly recovered molecules are heat shock proteins that are of major importance for the interaction with immune cells [61,62], proteases, which besides others contribute to matrix modulation [63], signal transduction molecules, MHC molecules and cytoskeletal proteins [64]. Exosomal mRNA is less abundant than exosomal miRNA. Exosomal mRNA differs significantly from that of the originating cells, many exosomal mRNA being involved in cell cycle progression, angiogenesis, migration, or histone modification [16,60,65,66]. TEX also contain selected miRNA, which show specific profiles for defined tumor entities [64]. For exosome secretion the intracellular Ca++ level [67], the intracellular and extracellular pH [68] as well as Rab GTPases play a key role, where Rab25 regulates MVB docking or tethering [69] and rab27b exosome release [70]. In brief (Fig. 1A), the protein, mRNA and miRNA profiles of exosomes differ from that in the originating cells. While the selective recruitment of mRNA and miRNA awaits further elaboration, the protein profile is mostly defined by protein complexes in internalization prone membrane domains. As exosomes are detected in all body fluids, the selective enrichment of “marker” proteins as well as of miRNA makes exosomes a very attractive means for noninvasive diagnosis [64,71]. Importantly proteins, mRNA and miRNA are function-competent.

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support that the internalization process in the exosome donor cell and the exosome uptake by target cells use similar fusion/fission machineries, where maintenance of internalization complexes and re-use of these complexes for exosome uptake appear to be a common theme [82,83]. Furthermore, CD91 is a common receptor for several heat shock proteins (HSP). Accordingly, anti-CD91 interferes with exosome activity [84]. Of note, this also account for several viruses. Thus, hepatitis C virus binds to the large extracellular domain of CD81, which promotes uptake and recruitment into MVB and exosomes, facilitating the transfer of HCV [85]. Also, exosomes of Mycobacterium avium transfected M␾ contain pathogen-associated molecular patterns, which act as TLR ligands and lead to activation of uninfected M␾ with TNF␣ and Rantes secretion [86]. Considering binding, it is of note that exosomes bind with high avidity several matrix proteins [63]. Matrix protein binding is selective and requires defined tetraspanin – adhesion molecule complexes, particularly integrins and CD44 (Mu W et al., unpublished). Thus, exosomes display exquisite target cell selectivity in vitro and in vivo, which is based, at least in part, on target cell ligand interactions with exosomal tetraspanin-associated receptors. Maintenance of internalization complexes and re-use of these complexes for exosome uptake appear to be a common theme [46,72] (Fig. 1B). Importantly, the engagement of protein complexes in internalization prone membrane domains provides an explanation for the target cell selectivity that is difficult to imagine relying exclusively on single adhesion molecules, which frequently are expressed on many cells.

2.2. Exosome binding and uptake 2.3. Target modulation by exosomes There is consensus that exosome interact with selected target cells [59,60,72], yet the mode of selection requires further clarification, where it should be stressed that an unequivocal answer to this aspect is a conditio sine qua non for most discussed therapeutic applications of exosomes. Several mutually not exclusive mechanisms of exosome interaction with recipient cells are discussed: receptor-ligand interactions, attachment, fusion with the target cell membrane, or internalization [72–74]. Furthermore, there is evidence that exosome fusion is facilitated or requires an acid pH [68]. Exosomes are characterized by phosphatidylserine exposure on the outer membrane of exosomes, where phosphatidylserine is involved in exosome budding in the late endosomes and can trigger exosome uptake by binding to scavenger receptors, integrins and complement receptors and PS receptors (TIM), particularly TIM-4 [75,76]. In line with this, TEX bind very rapidly to macrophages (M␾), binding and uptake being efficiently blocked by anti-CD11b [77]. However, TEX-uptake in vivo was not dictated by scavenger receptors and the selectivity of TEX-binding argue for phosphatidylserine facilitating binding that may not be followed by uptake [59,77,78]. Already in 2004 it was described that allogeneic exosomes are taken up by dendritic cells (DC), Kupffer cells and some M␾, exosome targeting being mediated by milk fat globulin-E8, CD11a, CD54, phosphatidylserine, CD9 and CD81 on exosomes and requiring ␣v␤3, CD11a and CD54 as ligands on DC [79] suggesting that exosome binding and uptake by target cells likely varies depending on the pathway of endocytosis and the protein pattern on exosomes and target cells [80] as well as toward a contribution of tetraspanins. In fact, exosome bind their targets via adhesion molecules engaged in tetraspanin complexes [59,81] The most surprising finding considering the target cell ligands was the large overlap with exosomal binding structures, which included annexins, chaperons, and molecules involved in vesicular transport, tetraspanins and tetraspanin-associated molecules [59]. These findings strongly

First to note, exosomal proteins, mRNA and miRNA are function competent [46,54]. Accordingly, there are several modes, whereby exosomes can modulate their targets, where we will differentiate between binding-induced changes and target modulation by exosome uptake. The first mostly relies on activation of exosome ligands and protein cleavage by exosomal proteases. Exosome uptake-initiated changes can be brought about by transferred proteins, mRNA and miRNA. Though far from being comprehensively answered, there are reports confirming these distinct activities of exosomes, the review focusing on TEX with emphasis on their engagement in metastasis. 2.3.1. Exosome-binding induced target modulation 2.3.1.1. Matrix modulation by exosomes. As mentioned, exosomes are rich in proteases, which are active and modulate the exosomes protein profile, but as well the ECM and target cells. So far MMP2, 7, 9, 14, ADAM10, 15,17 and ADAMTS1,13 have been detected in TEX [63]. A tumor creates its own matrix, but also influences the host matrix to generate a surroundings promoting tumor cell migration and survival. The phenomenon is well known, but poorly understood and the impact of TEX is largely unexplored. First to note, exosome proteases can modulate the exosome protein profile. This has been described for L1 and CD44 shedding by ADAM10, for EpCAM, CD46, TNFR1 by unknown metalloproteinases, where proteolytic activity may be regulated through the protease association with tetraspanins, particularly described for ADAM10 and ADAM17 [87–89]. Yet exosomal proteases also modulate the ECM, where exosomal tetraspanins due to their association with proteases come into play. Tetraspanin associate with peptidases, ADAMs (A disintegrin and metalloproteinase), particularly ADAM10 and ADAM17, matrix metalloproteinases (MMP) and uPAR. Taking into account the association between tetraspanins and integrins,

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major ligands for ECM proteins, particularly the association of tetraspanins with MMP14 plays an important role in pericellular lysis [88,90–93]. The collagenolytic and laminin-degrading activity of exosomes facilitates angiogenesis and metastasis which includes modulation of the matrix in the premetastatic organ [22,28,78,94–96]. Degradation of aggrecan, due to ADAMTS1, 4 and 5 in glioma TEX, increases invasiveness of glioma cells [97,98]. For exosomal MMP2, MMP9 and MMP14 a positive correlation between exosomes and invasiveness has been described [94,99]. Cathepsin B on TEX also contributes to matrix remodeling [100]. Focalizing exosomal matrix degrading enzymes also allows for paving the path of metastasizing CIC toward the premetastatic niche, confirmed for a rat metastasizing pancreatic adenocarcinoma, where CD151 and Tspan8 complexes with ␣6␤4, MMP14 or TACE are particularly important in collagen IV and laminin degradation ([101], W Mu et al., unpublished). The ECM is not only a structural element, but a storage of bioactive compounds and an essential component in tissue repair

as well as in the crosstalk between tumor cells and the stroma [102]. Taking this into account, modulation of the ECM by exosomal proteases [103] could in addition to creation of space, account for cytokine/chemokine and protease liberation and generation of cleavage products that promote motility, angiogenesis and stroma cell activation [63]. Taken together, the modulation of the ECM by exosomal proteases creates a path for migrating cells, favors a tumor growth promoting microenvironment, angiogenesis and premetastatic niche establishment. 2.3.2. Exosome-initiated signal transduction Exosome-initiated signal transduction can be promoted by exosome binding and exosome uptake, which is difficult to differentiate as in most instances exosome binding is followed by exosome uptake. Keeping this in mind, I will first focus on target cell modulation that might preferentially be due to exosome binding and proceed with target cell modulation by uptaken exosomes.

Fig. 1. Tumor exosome composition and mode of action, a prerequisite for therapeutic interference: (A) TEX generation and composition: Exosomes derive from the fusion of intraluminal vesicles of multivesicular bodies with the plasma membrane. The composition of exosomes is shaped by proteins in internalization-prone membrane domains, monoubiquitinylation and ESCRT; mRNA and miRNA are recruited during inward budding. Rab proteins are of central importance in MVB docking, tethering and exosome release. (B) Exosomes can bind unspecifically, via PS receptors or antigen-specific receptors. Exosome uptake frequently is followed by exosome uptake via fusion with the target cell membrane or via internalization. Exosomes also bind to components of the ECM. Notably, ECM binding and target cell binding as well as uptake are facilitated by exosomal membrane complexes that bind to protein complexes in internalization prone membrane domains. (C) TEX-binding to the ECM via adhesion molecules initiates matrix degradation and liberation of growth factors via exosomal proteases; Mostly via clustering of TEX-target cell membrane receptors signal transduction is initiated that may promote activation/proliferation or cell death. (D) TEX-uptake can lead to transient or persisting target cell reprogramming, where the transfer of TEX receptors and, particularly, the transfer of exosomal miRNA are important. Notably, mRNA silencing via transferred miRNA may be accompanied by release from repression by the exosome miRNA primary target. (E) Undue effects of TEX can be prevented by hampering TEX release, blocking of exosome binding or blocking of receptors at the target cell or by competing via an excess of tailored exosomes with TEX. TEX-plasmapheresis offers an additional therapeutic option. Besides of interfering therapeutically with TEX, exosomes can be tailored to protect TEX-targets or to attack the TEX donor cells. Besides that exosomes have to be equipped with the fitting binding complexes, they can be loaded with and transfer cytotoxic drugs and, most promising, tumor suppressor miRNA or oncomir blocking reagents.

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Fig. 1. (Continued.)

2.3.2.1. Exosome signaling and immune cells. DC-exosomes are one of the best explored examples of exosome binding-initiated target cell activation. DC-exosomes can replace DC in immune response induction and exosome-based therapy was first explored in the context of DC-exosomes as a cancer vaccine [104]. DC-derived exosomes are particularly rich in CD9 and CD81, MHC I and II, where MHC molecules co-localize and associate with tetraspanins during vesicle formation [105]. DC also take up exosomes secreted by other cells, including tumor cells, which they internalize and process for presentation, CD9 and CD81 also being important for uptake by DC [79]. Thus, DC use exosomes as a source of antigen and produce exosomes that suffice for T cell activation, both features expanding the operational range of DC [106]. However, hope that DC-exosomes are a promising means for immunotherapy [107], was dampened by TEX interfering with immune response induction [108]. TEX can inhibit lymphocyte, predominantly CD4+ T cell proliferation in response to IL2, which is accompanied by impaired CD25 up-regulation and stronger suppressive activity of regulatory T cells (Treg), possibly due to exosome-associated TGF␤1 [103]. Tumor-exosomes also affect T cells by inducing FAS-mediated apoptosis [109] and by enzymatic activity, which leads to extracellular adenosine production negatively modulating tumor infiltrating leukocytes [110]. NK activity also becomes impaired, which relies on TEX inhibiting activation of Stat5, Jak3, cyclinD3 expression and perforin release [111] or on blocking NK cells via NKG2D binding as far as exosomes express the relevant receptors. Exosomal MICA*008 also provokes a NKG2D-independent reduction in NK cytotoxicity [112]. TEX also

suppress antigen-specific responses by inducing TGF␤1 and IL4 secretion and inhibiting DC maturation in draining lymph nodes [113]. Finally, tumor-exosomes can act as a decoy factor capturing tumor-directed drugs demonstrated for anti-CD20 in hematological malignancies [114]. Via stimulating TGF␤1 secretion by M␾, TEX suppress anti-tumor immune responses allowing for tumor growth and metastasis formation in allogeneic mice [115]. Another pathway of TEX-induced immunosuppression proceeds via exosomal TGF␤ and PGE2, which promote induction of myeloid-derived suppressor cells (MDSC) (CD14+, HLA-Drlow , TGF␤ secreting) [116]. TEX also support transition from CD4+CD25− to Treg via Smad2/3 and Stat3 phosphorylation [103,117,118]. By high ICAM1 expression, TEX block the interaction between T cells and EC, thereby decreasing T cell recruitment [119]. On the other hand, TEX can also support immune response induction. Exosomes express HSP, which function as an endogenous danger signal promoting NK activation and tumor cell lysis through granzyme B release [120,121]. Exosomes recovered from heat-stressed tumor cells are superior in inducing a tumor-antigenspecific cytototoxic T cell (CTL) response [122]. Radiation-induced exosomal release of HSP72 increases CTL and NK activity and induces costimulatory molecule expression in DC [123]. In line with this, vaccination with staphyloccocus enterotoxin A expressing TEX significantly inhibits tumor growth and prolongs the survival time by increasing IL2 and IFN␥ secretion and promoting T helper (Th), CTL and NK activation [124]. Increased immunogenicity of exosomes from heat-stressed tumor cells is further strengthened by exosomal chemokines that attract and activate DC and T cells, such

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that intratumoral injection efficiently inhibits tumor growth [125]. Finally, TEX can be a strong immunogen. Tumor antigens, which are non-immunogenic when presented by tumor cells, induce a potent Th, CTL and B cell response and led to a decrease in Treg, when presented by TEX [77,126, Gu et al., unpublished]. Taken together, though TEX can exert immunosuppressive features, they also can strengthen tumor-specific immune response induction such that TEX in combination with DC-exosomes could well contribute to cancer immunotherapy. 2.3.2.2. Exosome signaling and angiogenesis. Angiogenesis induction being one of the hallmarks of cancer, intense efforts have been taken to elaborate the contribution of TEX. TEX containing TNF␣, IL1␤, TGF␤ and TNFR1 recruit EC progenitors, promote angiogenesis [51] and stimulate EC by paracrine signaling [127]. Delta-like4 bearing TEX confer a tip cell phenotype to EC with filipodia formation, enhanced vessel density and branching [128]. However, not only TEX promote angiogenesis and the pathways, whereby exosomes affect EC appear to differ significantly depending on the exosome source [129]. One of the leading pathways proceeding via activation of PPAR␣ and NFkB activation [130]. Platelets interact via lipid components on EC proliferation, the effect being abolished by a stripper of bioactive lipids [131]. Shh expressing T cell-exosomes display multiple effects on EC attributed to Shh binding its receptor thereby inducing activation of NO synthase and the endogenous Shh pathway with increased FGF and VEGF expression, suggesting the Shh+ exosomes inducing reparative vascularization in ischemic tissue [132]. M␾-derived exosomes also can contribute to angiogenesis induction, which was suggested to proceed via CD40L expression on exosomes with CD40 on EC [133]. In a feedback, prostata TEX lead to activation of fibroblasts, which then shed exosomes that increase tumor cell migration via CX3CCX3CR1 [134]. Furthermore, EC-derived exosomes harbor MMP that favor invasion and capillary tube formation [135]. 2.3.2.3. Exosome signaling and tumor growth. Activated T cellexosomes promote tumor invasion via FAS signaling, where FasL+ exosomes initiate activation of the ERK and NF␬B pathway in melanoma cells with subsequent upregulation of MMP expression [136]. Another elegant examples of exosome-mediated signal transduction describes overexpression of CD9 or CD82 promotes formation and secretion of exosomes that contain ␤-catenin, thereby reducing its cellular content and impairing Wnt signaling. The reduction in ␤-catenin is ESCRT-independent, but occurs via tetraspanin-associated E-cadherin [137]. Besides indicating that the cargo of exosomes differs depending on ESCRT- or tetraspanininitiated internalization, this study demonstrates that by depletion of inhibitors or stimulators exosomes can opposingly affect signal transduction [138]. Also, TEX-promoted tumor growth may vary for individual tumors. Thus, a deficit in Rab27a leading to reduced exosome production affected growth of a tumor line that required recruitment of neutrophils, but not of another neutrophilindependent line [139]. Briefly, binding of TEX to hematopoietic cells, EC, stroma cells can severely affect the target cell, which may become activated or suppressed. Additionally, the export of proteins into TEX affects the tumor cell itself. Last not least, host exosome initiated signals contribute to tumor progression. It is important to note that the strength of exosomes relies on their accessibility throughout the body, which can be best exemplified with an organ such as the brain, where cell motility is very limited. Neurons express CD13 on the presynaptic membrane, where CD13 cleaves encephalin, which can no longer bind the opioid receptor. Yet, microglia exosomes expressing CD13 are required for controlling the catabolism of neuropeptides at distant sites [90]. It also has to be kept in mind that TEX-initiated signaling will vary with the origin and composition

of TEX, where most of the exosome-binding initiated signals rely on molecules associated with tetraspanins, which forces the importance of this constitutive exosome components not only in exosome binding, but also binding-induced signal transduction (Fig. 1C). 2.4. Exosome uptake promoted target cell modulation Early reports on the information transfer via exosomes showed that ES-exosomes transfer messages into hematopoietic progenitor cells that promoted survival and expression of early pluripotency markers [140] and that adult tissue exosomes, too, had the capacity to alter the phenotype of their target such that upon coculture BM cells (BMC) express markers found on the exosome donor cell [141], where uptake of exosome proteins, mRNA and miRNA are active contributors. 2.4.1. The transfer of exosomal proteins, mRNA and miRNA Exosomes can mediate disease spread via receptoroncoprotein- or miRNA-transfer [140,142], which frequently cannot be clearly deciphered. One of the first evidences to support TEX-uptake plays a critical role in autocrine stimulation of tumor growth revealed that the intercellular transfer of the oncogenic receptor EGFRvIII via TEX to glioma cells, lacking this receptor, causes transformation of indolent glioma cells [143] and reprograms growth factor pathways in EC [65]. Other oncogenes, like Ras, Myc, SV40T also induce signaling and gene expression [144–146], where e.g. exosomal amphiregulin, an EGFR ligand, increased tumor invasiveness 5-fold compared to the recombinant protein [147]. 2.4.1.1. TEX-uptake by tumor cells. TEX uptake-induced changes in recipient non-tumor cells can be transient, but also suffice to drive tumor growth as described for tissue transglutaminase and fibronectin [148] or high level c-Met uptake by BMC, which leads to their re-education to support premetastatic niche formation for melanoma cells, where in melanoma patients, too, circulating BM-derived cells express Met [149]. TEX also transport apoptosis inhibitory proteins [150] and present TGF␤ which drives differentiation of fibroblast toward tumor growth, angiogenesis and metastasis supporting myofibroblast [151]. Breast cancer TEX convert adipose-tissue derived MSC into myofibroblasts with increased expression of ␣-SMA, SDF1, VEGF, CCL5 and TGF␤, TGF␤RI and II, which is accompanied by SMAD pathway activation [152]. Lung cancer TEX uptake stimulates the environment to secret IL8, VEGF, LIF, oncostatin and MMP to support tumor growth [153]. Instead, uptake of tumor suppressor genes from non-transformed cells can mitigate the aggressiveness of cancer cells [154,155]. 2.4.1.2. TEX-uptake and metastasis. An involvement of exosomes in metastasis was first described for platelet-derived exosomes. These exosomes transferred the ␣IIb integrin chain to lung cancer cells, stimulated the MAPK pathway and increased expression of MT1-MMP, cyclin D2 and angiogenic factors as well as enhancing adhesion to fibrinogen and human umbilical vein endothelial cells [156]. We explored that TEX together with a soluble tumor matrix supported recruitment of hematopoietic progenitors from the BM as well as activation of stroma cells and leukocytes in premetastatic lymph nodes such that a non-metastatic tumor line settled and formed metastases [44]. Similar findings have been reported by Hood et al. [127], who explored premetastatic niche preparation by melanoma-TEX. The recruitment of tumor cells may also become facilitated by exosomal HSP90. A complex of exosomal HSP90 with MMP2 and tissue plasminogen activator promotes together with exosomal annexin II plasmin activation and thereby tumor cell motility [157] and, as already mentioned, the transfer of c-Met

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contributes to premetastatic niche formation mostly via bone marrow cell modulation [149]. Thus, pretreatment with TEX enhances migration and homing of tumor cells in sentinel lymph nodes due to stroma and hematopoietic cell as well as matrix modulation allowing for recruitment and growth of tumor cells [44,124,149,158]. Finally, uptake of exosomes from non-transformed cells in the tumor surrounding can affect tumor cells. This was described for fibroblast-exosomes that promote breast cancer motility via Wnt planar polarity signaling that includes as central elements Frizzled, Van-Gogh-like, Prickle and Disheveled [159]. 2.4.1.3. TEX-uptake and angiogenesis. TEX uptake also accounts for EC modulation. Colorectal cancer TEX are enriched in cell cycle-related mRNA, which promote EC proliferation [66]. Glioblastoma-TEX-induced angiogenesis relies on the transfer of exosomal proteins and mRNA in EC [65]. Also, EGFR-positive TEX are taken up by EC and elicit EGFR-dependent responses including activation of the MAPK and Akt pathway and VEGFR2 expression [160]. Angiogenesis was also reported to be induced by the transfer of Notch-ligand-delta-like-4, which inhibits Notch signaling and increases angiogenesis [125]. TEX expressing a complex of Tspan8 with CD49d preferentially are taken up by EC. Exosome uptake initiates progenitor EC maturation as well as EC activation including VEGFR transcription [60]. Chronic myeloid leukemia (CML)-exosomes induce angiogenic activity in EC, where a Src inhibitor affects exosome production as well as vascular differentiation [161]. 2.4.1.4. TEX mRNA and miRNA uptake. As mentioned TEX-uptake induced target cell modulation may frequently represent the combined result of protein transfer-initiated signal transduction, transferred mRNA translation and mRNA silencing by miRNA. Thus, a separation between these activities appears somewhat artificial. Nonetheless, a few reports describe preferential activities of mRNA and miRNA. Conversion of the BM niche by acute-ML (AML) cells is mediated by exosomes which transfer besides other IGF-IR, the mRNA being enriched in AML-exosomes, which promotes stroma cell proliferation. By the transfer of miR-150 in AML-exosomes to hematopoietic progenitors expression of CXCR4 becomes significantly reduced and HSC migration is impaired [162]. Also, CD105+ renal cell CIC-derived TEX carry proangiogenic mRNA and miRNA thereby triggering the angiogenic switch [28]. The group of Lyden describing exosomes from a metastatic melanoma line to educate BM progenitors through MET shifting them toward a pro-vasculogenic phenotype with c-Kit, Tie2 and MET expression also speculate on an important contribution of exosomal miRNA [146]. TEX from a metastasizing line contains a restricted repertoire of mRNA and miRNA, particularly the latter differing significantly from that of the donor line. TEX mRNA and miRNA are recovered in lymph node stroma and lung fibroblasts, and transferred miRNA significantly affects mRNA translation, which was exemplified for abundant TEX miR-494 and miR-542-3p, which target cadherin17. Concomitantly, MMP transcription, accompanying cadherin17 down-regulation, was up-regulated in LnStr transfected with miR-494 or miR-542-3p or co-cultured with TEX. Thus, TEX miRNA uptake affected premetastatic organ stroma cells toward supporting tumor cell hosting [16]. Exosomes from virus transfected cells also transfer viral miRNA [163]. EBV transfected nasopharyngeal carcinoma that transferred viral BART miRNA [164]. Leukemia cell contain miR-92a that is transferred into EC and downregulates CD49e, which selectively increases migration and tube formation [165]. In lung cancer TEX miR-21 and miR-29a act as a ligand of mouse TLR7 or human TLR8, functioning as agonist and leading to NF␬B activation and IL6 and TNF␣ secretion, which promotes metastasis [166]. Hepatocellular carcinoma-TEX contain

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a set of highly enriched miRNA that are not detected in the donor cell, including miR-584, miR-517c and others, one of the potential targets being identified as TGF␤ activated kinase 1 (TAK1), which activates JNK and MAPK pathway and NF␬B. Exosomal miRNA was transferred in coculture and promoted anchorage-independent growth and apoptosis resistance [167]. Last not least, stroma cells also release exosomes, whose miRNA influence tumor cells. BM stroma cell exosomes inhibit the growth of multiple myeloma, but those derived from patients with multiple myeloma force multiple myeloma progression, the latter exosomes showing a lower content of tumor suppressor miR-15a, but high levels of oncogenic proteins, cytokines and adhesion molecules [168]. Also tumor-associated M␾ secret exosomes with high miR223, that binds Mef2c, causing nuclear accumulation of ␤-catenin [169]. Monocyte exosomal miR-150, when transferred to EC promotes migration [170]. Taken together, though results on target cell modulation by TEXuptake are still sporadic, it is without question that transferred miRNA can reprogram target cells, the linkage between exosomal miRNA and the targeted mRNA remaining to be elaborated in many instances (Fig. 1D). Also in concern of the described impact of transferred proteins and mRNA, the question on long-lasting in vivo efficacy mostly awaits clarification. Exosomes being the most powerful means of intercellular communication that function even across long distance, it is utmost important to answer these open questions. 3. Exosomes as therapeutics Exosomes are easy to manipulate and efficiently transfer proteins and genes. This potentially offers a means to interfere with pathological angiogenesis and metastasis, two major targets in cancer therapy [58,171]. In addition, exosomes are discussed as cancer vaccine [108]. Nonetheless, in advance of discussing the possibilities to interfere with tumor growth and progression via exosomes, it should be mentioned that the indispensability of exosome transfer in human cancer remains questionable. In A431 PS blocking inhibits uptake of TEX by EC, but the antiangiogenic effect was only transient [143]. Also a blockade of cellular vesiculation (TSAP6, acidic sphingomyelinase) does not prevent tumorigenesis [172,173]. Furthermore, blocking of Rab27a involved in exosome biogenesis exerts distinct effects on primary versus metastatic tumor growth and also differs between tumors [135,149]. These findings should not be taken to discourage attempts to translate experimental studies on the power of exosomes into therapeutic settings, but should foster the point that clinical translation in many instances awaits progress in elaborating the mode of exosome activities, which are already used as a vaccine [107,174]. 3.1. Exosomes to substitute or support dendritic cells Exosome research became highly stimulated, when it was noted that antigen presenting cells release exosomes derived from MVB of the MHC class II compartment, which function similar to antigen presenting cells and stimulate T cells in vitro and in vivo [175]. Several studies used peptide-loaded DC-exosomes, where in some studies cyclophosphamide was given after chemotherapy to inhibit Treg. In all instances DC-exosomes were well tolerated, induced an antigen-specific response and or NK recovery and the diseasefree survival time was mostly prolonged. Notably, too, exosomes can be stored at −80 ◦ C and recovery of DC-exosomes exceeds that of loaded DC by 2-fold. Limitation was mostly restricted to the requirement of large amounts of DC-exosomes [176–178]. Though TEX can be immunosuppressive, there are several reports that exosomes delivered from DC after coculture with TEX

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might be superior to exosomes derived from peptide-pulsed DC, where TEX could be particularly helpful as antigen source, when immunogenic entities are unknown. Thus, DC pulsed with exosomes of an AML line provoked a strong anti-leukemia response [179]. In line with this, directing tumor-associated, non-mutated antigens like CEA and HER2 to exosomes by coupling to lactadherin can increase their immunogenicity [180]. Targeting prostatespecific antigen or prostatic acid phosphatase via lactadherin to exosomes was also shown to induce a superior immune response [181]. Furthermore, anticancer drug may force the release of HSP-bearing exosomes, which efficiently activate NK cells [182]. However, one also should be aware that exosomes may compete for anti-cancer drugs as demonstrated for HER-2-positive TEX that capture Trastuzumab [183] or CD20+ TEX that capture anti-CD20, where a blockade of the ATP-binding cassette transporterA3 that promotes exosome release mitigated the effect of exosomes [110]. 3.2. Outsmarting TEX Even taking into account that TEX must not be essentially required by individual tumors for survival and progression, it is without doubt that they are not beneficial for the host. Thus, great efforts are taken to hamper or redirect TEX, which are based on competition and/or tailoring. 3.2.1. Blocking TEX Blocking of exosome uptake could be performed at the exosome or the target cell level [159], where PS blocking of TEX only transiently inhibited angiogenesis [143]. Instead, in a rat model, where exosomes expressing the tetraspanin Tspan8 induced a lethal systemic consumption coagulopathy due to overshooting angiogenesis, blocking exosomes by a Tspan8-specific antibody completely prevented undue angiogenesis, although primary tumor growth was not impaired [184,185]. Based on this observation and ongoing studies that exosomes bind via tetraspanin-complexes to ligands also located in internalization prone membrane domains [59], we expect a scrutinized analysis of an individual tumors’ TEX-binding complex to be most promising for hampering undue TEX-initiated angiogenesis and premetastatic niche formation, where exosomes from non-transformed cells modulated to express the TEX-binding complex may be most promising [59]. As an alternative approach, TEX may be removed by affinity plasmapheresis known as Aethlon ADAPTTM [186]. Blocking of TEX also can affect drug and radiation resistance due to enhanced release of export transporter MRP2, ATP7A and ATP7B or Annexin A3 [187,188].

efficacy than purified AAV vectors [201]. Based on the observation that exosomes from non-tumor cells contain tumor-suppressive miRNA, it was suggested to use exosomes loaded with those miRNA, which was exemplified for miR-143, as a therapeutic strategy in cancer [162]. Also, systemic administration of miR26a, which induces cell cycle arrest via targeting cyclins D2 and E2, exerted a dramatic protective effect without toxicity in a mouse hepatoma model [202]. Additional approaches like miRNA inhibitors (miRNA sponges), antagomirs, locked-nucleicacid-modified oligonucleotides are reviewed in [203]. Of central importance will be the elimination of CIC themselves. This again requires a most scrutiny elaboration of exosome targeting structures of CIC, which should be facilitated by the definition of CIC markers, many of them being located in internalization prone membrane domains and thus facilitate exosome uptake [204]. Though at the present state of knowledge miRNA based therapies have to be considered as double-edged sword as most miRNA have a multitude of targets, loading exosomes with miRNA to reprogram CIC toward differentiation and/or apoptosis susceptibility can be expected to become feasible with further characterization of miRNA targets (Fig. 1E). As far as the above mentioned hurdles are solved, rapid progress in clinical translation can be expected [205]. 4. Conclusion The discovery of exosomes as intercellular communicators throughout the body has revolutionized many aspects of biological sciences and will bring a major breakthrough in the field of therapy. The power of exosomes is due to their ubiquitous presence, their particular protein profile and their equipment with mRNA and miRNA as well as their most efficient transfer in target cells. Together with the ease of transfecting exosomes, there should be hardly any limits in the use of exosomes as therapeutics. The therapeutic use of exosomes from non-transformed cells to compete TEX or to actively induce an immune response or passively to silence immunosuppression should not become a danger for the patient’s organism. Instead, for therapeutic approaches based on tailored TEX the following questions need to be answered most thoroughly in advance: (i) defining TEX targeting receptors and their ligands, which most likely will include modes to further restrict the panel of potential targets of natural TEX; (ii) a precise knowledge on miRNA targets and consequences on release from repression. Answering these questions will take time, but is not an insurmountable hurdle. Conflict of interest Florian Thuma and Margot Zöller declare no conflict of interest.

3.2.2. Tailored exosomes for drug delivery Hope in exosome-based therapy received a major boost with the discovery of horizontal transfer of mRNA and miRNA [189,190], which can be translated or mediate RNA silencing [140,191,192]. As exosomes are natural non-synthetic and non-viral products, are small and flexible, which allows them to cross biological membranes and to protect their cargo from degradation by a lipid bilayer [74], they are discussed as ideal and possibly the most potent gene delivery system [74,190,193–195]. Notably, exosome electroporation very efficiently transfers siRNA into exosomes [160]. Furthermore, special devices were developed, e.g. to cross the blood–brain barrier to deliver BACE1 siRNA, where mast cellexosomes were equipped with a brain penetrating peptide fused to the vesicular membrane protein Lamp2 [196,197]. To give a few additional examples. Curcumin or Stat3 inhibitor delivery confirmed exosomes to be well suited for drug delivery [198,199], where chemotherapeutic drug efficacy can be increased by lowering the pH of exosomes [68,200]. Adenoviral vectors associated with exosomes displayed higher transduction

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Please cite this article in press as: Thuma F, Zöller M. Outsmart tumor exosomes to steal the cancer initiating cell its niche. Semin Cancer Biol (2014), http://dx.doi.org/10.1016/j.semcancer.2014.02.011