Exosome Mediated Communication within the Tumor Microenvironment

Lara Milane, Amit Singh, George Mattheolabakis, Megha Suresh, Man-soor M. AmijiPII: S0168-3659(15)30001-8DOI: doi: 10.1016/j.jconrel.2015.06.029Reference: COREL 7734

To appear in: Journal of Controlled Release

Received date: 28 May 2015Accepted date: 19 June 2015

Please cite this article as: Lara Milane, Amit Singh, George Mattheolabakis, MeghaSuresh, Mansoor M. Amiji, Exosome Mediated Communication within the Tumor Mi-croenvironment, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.06.029

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Exosome Mediated Communication within the Tumor Microenvironment

Lara Milane, Amit Singh, George Mattheolabakis, Megha Suresh, and Mansoor M. Amiji*

Department of Pharmaceutical Sciences, School of Pharmacy, Bouve College

of Health Sciences, Northeastern University, Boston, MA 02115 (*Corresponding author: Tel. 617-373-3137, Fax: 617-373-8886, Email: m.amiji@neu.edu)

Running title: Exosome Mediated Communication

Abstract It is clear that exosomes (endosome derived vesicles) serve important roles in cellular communication both locally and distally and that the exosomal process is abnormal in cancer. Cancer cells are not malicious cells; they are cells that represent survival of the fittest at its finest. All of the mutations, abnormalities, and phenomenal adaptations to a hostile microenvironment, such as hypoxia and nutrient depletion, represent the astute ability of cancer cells to adapt to their environment and to intracellular changes to achieve a single goal survival. The aberrant exosomal process in cancer represents yet another adaptation that promotes survival of cancer. Cancer cells can secrete more exosomes than healthy cells, but more importantly, the content of cancer cells is distinct. An illustrative distinction is that exosomes derived from cancer cells contain more microRNA than healthy cells and unlike exosomes released from healthy cells, this microRNA can be associated with the RNA-induced silencing complex (RISC) which is required for processing mature and biologically active microRNA. Cancer derived exosomes have the ability to transfer metastatic potential to a recipient cell and cancer exosomes function in the physical process of invasion. In this review we conceptualize the aberrant exosomal process (formation, content selection, loading, trafficking, and release) in cancer as being partially attributed to cancer specific differences in the endocytotic process of receptor recycling/degradation and plasma membrane remodeling and the function of the endosome as a signaling entity. We discuss this concept and, to advance comprehension of exosomal function in cancer as mediators of communication, we detail and discuss; exosome biology, formation, and communication in health and cancer; exosomal content in cancer; exosomal biomarkers in cancer; exosome mediated communication in cancer metastasis, drug resistance, and interfacing with the immune system; and discuss the therapeutic manipulation of exosomal content for cancer treatment including current clinical trials of exosomal therapeutics. Often referred to as cellular nanoparticles, understanding exosomes, and how cancer cells use these cellular nanoparticles in communication is at the cutting edge frontier of advancing cancer biology. Keywords: exosomes, cancer, microRNA, signaling endosome, metastasis 1. Introduction One of the most groundbreaking discoveries in cell biology in the past thirty years was the discovery of exosomes; endosome derived vesicles (30-100 nm) shed by cells in processes of cellular housekeeping and communication. Exosomes were discovered in 1983 as transferrin associated 50 nm vesicles extruding from reticulocytes [1]. Although it has been thirty years since the discovery of exosomes, we are still in the infancy of understanding exosome biology

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and function. It is clear that exosomes are an important avenue for intracellular communication but some exciting and important questions remain to be answered. The most pivotal question that remains to be answered is; is exosomal release a form of deliberate cell communication? Considering how cancer cell exosomes can transform local healthy epithelial cells into cancerous cells, function in invasion of the extracellular matrix, and contribute to distal metastasis [2], it seems intuitive that this process would be deliberate; as if exosomes were the cells elevator pitch to their surroundings here is a quick message, this is what I want you to know. Our progressive understanding of the function of exosomes in health and disease certainly implies that the exosomal process (formation, content selection, loading, trafficking, and release) is an orchestrated and deliberate process. For example, a recent study using a chorioallantoic membrane of chick embryos as an in vivo tissue model, demonstrated that tumor cell xenografts within the membrane required exosome release for directional movement and correlated exosome release to the speed of cell movement through the membrane [3]. Inhibiting exosome release from the tumor cells disabled directional movement in the membrane [3]. Although we are gaining insight into both, how proteins are sorted into exosomes and selected for secretion, and the astounding functions of exosomes in cancer, the pivotal question still remains unanswered and naturally leads to a second question; is exosome release only deliberate in cancer cells? Exosomes are of endosomal origin so to understand the exosomal process and to explore these questions, we must turn to the endosomal process and the distinctions between cancer cell exosomes and exosomes released from healthy cells to investigate.

The difference between normal (healthy) cell exosomes and cancer cell exosomes has been well noted in numerous studies indicating that both the rate of exosomal release and the content (most notably microRNA) is increased and distinct. Various cancer cell lines have been shown to produce more exosomes relative to normal cells. For example, a study published in 2014 compared exosome release from a normal human mammary epithelial cell line and a breast cancer clone derived from the parent cell line. The study demonstrated that within a 24 hour period, exosome release from the normal (parent) cell line was (4.5 2.3) x 108 exosomes per 106 cells whereas exosome release for the cancer cell line was (53.2 1.6) x 108 exosomes per 106 cells [4]. Aligned with these, in vitro studies [4], in vivo studies [5] , and clinical analysis [2] have revealed an increase in cancer cell and tumor derived exosomes in cells and in patients. In addition to increased exosome release, the distinct content of exosomes has clinical significance. A seminal study by Melo, et al., demonstrated that, unlike exosomes released from normal cells, exosomes released from breast cancer cells secrete microRNAs that are associated with the RISC loading complex; this complex is essential for processing of pre-microRNA into mature microRNA which can functionally silence target genes [2]. The researchers demonstrated that exosomes from the sera of breast cancer patients as well as exosomes from breast cancer cells in culture, were capable of transforming normal epithelial cells into tumor forming cells and that this transformation was dependent on the RISC complex protein, Dicer [2]. The study went on to illustrate that CD43 (associated with metastatic breast cancer) is responsible for the increase of Dicer loading in breast cancer exosomes [2]. This study not only identifies the significance of Dicer in breast cancer derived exosomes, it demonstrates the potential of exosomes as mediators of tumorigenesis [2]. Until recently, the most commonly accepted theory of exosome sorting and loading was that exosome content is determined non-specifically under multivesicular formation and not through a deliberate sorting and packaging process [6]. Although completely logical, explaining the difference in cancer exosome content as a fundamental difference in cellular composition, this deduction fails to address why the rate of exosomal release can be increased in cancer, why exosomal content does not always parallel cellular content, and how exosomes seem to function in deliberate processes such as invasion. The theory of passive exosome loading is beginning to be

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disproved as we learn more about the lipids, receptors, and sorting machinery such as the endosomal sorting complex required for transcript (ESCRT) that coordinate to select and package exosomal content [7]. Although we are beginning to decipher the molecular mechanisms of exosomal sorting and release, and how the content of cancer exosomes differs from exosomes derived from healthy cells, still, little progress has been made to explain and understand how the exosomal process is distinct in cancer cells and why cancer cells can have an increased rate of exosome release and distinct exosomal content. A 2009 study demonstrated that exosome release and uptake is increased in an acidic pH, suggesting that the acidic microenvironment of tumors contributes to the increased rate of exosomal release and uptake by cancer cells [8]. We postulate that, in addition to factors such as microenvironmental pH, the aberrant exosomal process in cancer cells can be partially attributed to cancer specific differences in the endocytotic process of receptor recycling/degradation and plasma membrane remodeling and the function of the endosome as a signaling entity (Figure 1). Abnormalities in cancer cell surface receptors are well documented with notable over-expression of many different plasma membrane receptors such as growth factor receptors, glucose and nutrient importers, ABC transporters (dug efflux pumps), folate receptors, and various importers [9-12]. Cancer cells also have type-specific mutations or protein alterations that result in increased endocytosis [13]. Although endocytosis does not imply a direct release of exosomes, under conditions of increased endocytosis, paralleled exocytosis (and accompanying exosome release) is important for maintaining cell volume and membrane integrity [14, 15]. Receptor dysregulation in cancer can result in plasma membrane remodeling independent of ligand binding and receptor clustering (dimerization), for example, through receptor cross regulation; HER2 overexpression often characterizes types of lung, salivary, stomach, bladder, ovarian, and breast cancer and has been shown to increase recycling of the EGFR (Epidermal Growth Factor Receptor) [16]. Mutations in the p53 tumor suppressor gene (175H and 273H) in lung cancer cells have been shown to increase EGFR and integrin (51) recycling to the plasma membrane and studies have demonstrated that this increase was due to association of integrin and EGFR with RCP (Rab coupling protein; the Rab proteins function in intracellular trafficking) [17]. These studies also demonstrated that knockdown of RCP only effected integrin and EGFR trafficking in p53 mutant cells (not in wild type p53 cells) and this knockdown in the mutant cells impaired invasion and directional movement [17]. These results, combined with the results from the study of exosome function in directional movement of cancer cells (tumor cell migration through the chorioallantoic membrane of chick embryos) suggest that p53 mutations may enhance invasion by promoting receptor recycling and exocytosis with subsequent exosome release and directional movement. As well as cancer specific differences in endocytosis, receptor recycling, and plasma remodeling resulting in subsequent differences in the exosomal process, the function of the signaling endosome contributes to the formation of abnormal cancer exosomes. The signaling endosome is an effector that works in concert with cellular pathways, actively engaging with cytosolic components to load specific content through specific receptors or channels, such as the loading of microRNA through ceramide-rich lipid raft regions [18-22]. The function of the signaling endosome as a dynamic and responsive entity results in a cell specific endosomal process including exosome formation, content selection and loading, endosomal trafficking, and exosome release. As such, exosomes released from cells can function to achieve specific cellular directives such as digestion of the extracellular matrix for invasion and movement. As exosomes are derived from the endocytotic pathway, endocytosis can be considered the first step in the exosomal process. For a review of endocytosis in cancer, the reader is directed to Mosesson, Mills, and Yardens review; briefly, endocytosis can be clathrin mediated which is usually associated with clustered receptors, endocytosis can be cavaelae mediated (cavoelae are specialized lipid rafts), and endocytosis can be clathrin and cavoelae independent

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[13]. Endocytosis occurs as a signaling response to internalize extracellular molecules (non-specific), to internalize receptor ligands (receptor mediated), to recycle or degrade plasma membrane receptors (ligand bound and inactivated receptors) and to recycle or degrade membrane constituents [21, 23]. Cancer cells are associated with abnormal expression levels of plasma membrane receptors and components and these distinctions are often used to characterize cancer subtypes (biomarkers). For example, over expression of epidermal growth

The exosome process begins with endocytosis, follows the endosomal pathway, and ends with exocytosis. (1) Genotypic and phenotypic alterations in cancer result in cell-specific changes in the expression of plasma membrane receptors and plasma membrane constituents. (2 & 3) Cancer plasticity, altered plasma membrane receptor expression, characteristic changes in endocytosis (such as the overexpression of HER2 resulting in increased EGFR recycling), and increased plasma membrane remodeling results in endocytosis. (4) The endosome functions as a signaling entity; to coordinate with cellular pathways and interact with cellular components. The signaling endosome responds to the celluar environment and cellular signals. As such, endosomes can be recruited to achieve specific cellular tasks such as promoting invasion through exosome release of proteases. (5) For loading of specific content into endosomes, proteins, molecules, and microRNAs are trafficked towards the endosome. (6) Intraluminal vesicles (ILV) begin to form. (7) The endosome functions as a true signaling entity to select content for exosome packaging and release, and for degradation and recycling (not shown). Lipid rafts in the endosome load microRNA while proteins, receptor ligands, and transcription factors can be loaded via the ESCRT machinery (endosomal sorting complex required for transcript), importers, channels, or receptors. (8) The Rab GTPases function in trafficking of multivesicular bodies (MVB) to the site of the plasma membrane. (9) SNARE (soluble NSF attachment protein receptors) dependent or independent mechanisms (such as ceramide signaling) function to fuse MVBs with the plasma membrane. (10) Exosomes exhibit an effect in their local microenvironment (such as enhancing invasion) but they also enter the systemic circulation to influence distal regions of the body.

Figure 1. The Exosomal Pathway in Cancer.

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factor receptor (EGFR) is often associated with triple negative breast cancer that lacks estrogen receptors, progesterone receptors, and HER2 receptors [24]. Also of note, the expression of plasma membrane receptors and constituents is under a state of constant and dynamic remodeling as the phenotype of the cancer cell changes; tumors are heterogeneous cell populations but individual cancer cells can also be highly plastic cells capable of rapidly adapting to hostile microenvironmental pressures [12]. This plasticity often results in changes in the expression of plasma membrane receptors and subsequent plasma membrane remodeling [12]. As receptors are marked for recycling or degradation they enter the endocytotic pathway, transitioning from early endosomes to multivesicular bodies and can subsequently lead to exosomal release. In support of our concept, a recent study demonstrated that endocytosis of lipid rafts in mesenchymal stem cells directly correlates to increased secretion of exosomes [25]. Lipid rafts are a scaffold for clathrin-independent endocytosis and plasma membrane remodeling [21]. Cancer cell-specific characteristics such a lipid constituents of the plasma membrane, expression of receptors, mutations in oncogenes, reactions to hypoxia, and acquisition of drug resistance assuredly result in signaling endosome responses and subsequent exosome alterations. Additional factors influence exosome release such as microenvironmental interactions and feedback loops between cancer cells and the extracellular matrix, stromal cells, other tumor cells, and healthy cells. [4]. To propel the field of exosomal biology and therapeutics in cancer we have compiled a comprehensive review of cancer exosome biology and therapeutics. A key to understanding exosome biology is understanding the endosome as a signaling entity or a transient organelle (signaling endosome), not a passive vesicle in which exosomes are formed. This is perhaps the first tenant to understanding exosomal communication; understanding the endosome as a signaling entity that determines the contents of exosomes through its interaction with cytosolic components. In this review we discuss the concept of the endosome as a signaling entity that dictates exosomal fate and our concept of plasma membrane remodeling governing the exosomal process in cancer (Figure 1), detail exosome biogenesis and function (Section 2), discuss exosomal content in cancer (Section 3), detail the application of exosomes in cancer biomarker screening (Section 4), discuss exosomal communication in cancer (Section 5), and detail the application of exosomes as cancer therapeutics (Section 6). Exosomes are an important constituent of the tumor microenvironment; as such, comprehension of cancer derived and associated exosomes is a critical component of cancer biology. 2. Exosome Biology, Formation, and Communication in Health and Disease 2.1 Biogenesis

Cells release many different types of vesicles such as exosomes derived from endocytotic multivesicular bodies, ectosomes formed from outward budding and pinching of the plasma membrane, and secretory vesicles formed at the trans-Golgi network [26]. The two most distinguishing features of exosomes are their endocytotic origin and their small size (from 30 - 100 nm); ectosomes are much larger (100 350 nm) while secretory vesicles can vary greatly in size [26].

Exosomal release is one of three possible fates for multivesicular bodies (MVB). Multivesicular bodies are formed from early endosomes. When plasma membrane receptors are marked for recycling or degradation through ubiquitination and when invagination of the plasma membrane occurs, endocytosis initiates and early endosomes are formed through internalization of the plasma membrane. As internal vesicles form within the endosome, the endosome transitions to become multivesicular bodies [6]. Multivesicular bodies are also referred to as late endosomes or multivesicular endosomes. The internal vesicles are termed intraluminal vesicles (ILVs) and they are formed from inward budding of the MVBs, with membrane orientations the same as the extracellular facing side of the cellular plasma

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membrane [27]. The three fates for multivesicular bodies are; recycling through the trans-Golgi network, lysosomal degradation, or secretion through exocytosis (exosome release) [6]. The endosomal origin of exosomes is confirmed by proteomic analysis; exosomes contain proteins from endosomes, the plasma membrane and the cytosol [28]. Exosomes also have high levels of lipids and constituents associated with lipid rafts such as phosphatidylserine, sphingomyelin, ceramide, cholesterol [27]. 2.2. Sorting

Much investigation has focused on determining how exosome content is selected and if this is a deliberate process; exosome content is rich in enzymes, microRNA, transcription factors, heat shock proteins, MHCs, cytoskeleton components, signal transducers, and tetraspanins (transmembrane proteins). The function of ESCRT in sorting ubiquitinated proteins provides insight to the sorting processes that govern exosomal content [7].

The early endosome recruits the ESCRT machinery for the formation of ILVs; the ILVs are pre-exosomes [27, 29]. The ESCRT machinery consists of four ESCRT protein complexes and associated proteins; ESCRT-0 functions in a ubiquitin-dependent fashion to cluster cargo, ESCRT-I and ESCRT-II are involved in budding, while ESCRT-III functions in vesicle scission [27, 29]. Accessory proteins include ALIX and VPS4; ALIX is involved in vesicle budding and VPS4 assists with scission [27, 29].

There are also ESCRT independent mechanisms of MVB, ILV, and exosome formation; these mechanisms can involve lipids such as ceramide and cholesterol, phospholipase D2, or tetraspanins [27, 29]. Thinking of the early endosome as a signaling entity, it is easy to conceive how exosomal content can be determined by the membrane constituents of the early endosome. The early endosome has receptors that can facilitate the encapsulation of cytosolic components; for example, tetraspanins have been demonstrated to facilitate the incorporation of specific cargo into exosomes. Tetraspanin CD9 has been shown to mediate exosomal loading of the metalloproteinase CD10 while CD63 has been shown to control the loading of LMP1 Epstein-Barr virus protein into exosomes [30-32]. Likewise, the lipid composition of the early endosome and MVB as well as membrane dynamics have been shown to govern exosomal content. For example, the formation of lipid rafts by ceramide has been demonstrated to mediate microRNA loading into exosomes [18, 19].

Additional studies suggest a dominating role of ceramide and nSMase2 (neutral sphingomyelinase 2; the rate limiting enzyme of ceramide biosynthesis) in the exosomal process as ceramide orchestrates an ESCRT independent process of exosome formation, loading, and release [19, 33]. This could have powerful implications for cancer therapeutics; would disrupting the ceramide content of exosomal membranes disable the ability of cancer cells to form and secrete exosomes and subsequently reduce metastasis? Or perhaps only impair microRNA loading into exosomes? The contents of cancer cell exosomes are distinct from exosomes released from healthy cells; cancer exosomes deliver functional complexes including excessive microRNA that can exert biological activity (without further activation) upon uptake by recipient cells exosome content in cancer is the topic of Section 3 below. 2.3 Exocytosis and Secretion

Exosome secretion through exocytosis is mediated through Rab GTPases, molecular motors, cytoskeletal proteins, and SNAREs (soluble NSF attachment protein receptors); secretion can also be effected by factors such as intracellular Ca2+ levels (increased Ca2+ results in increased exosome secretion) and extracellular/intracellular pH gradients [8, 34]. The Rab GTPases are responsible for trafficking of late endosomes/MVBs to the site of the plasma membrane [27, 35]. Different Rab isoforms appear to dominate MVB trafficking to the plasma membrane in different cell types; for example in HeLa cells, silencing (RNAi) of the Rab27a isoform increased the size of MVBs while silencing of the Rab27b isoform diverted the

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MVBs to the perinuclear region of the cell instead of towards the plasma membrane [35]. Following plasma membrane docking, the SNARE protein complexes facilitate membrane fusion of the MVBs with the plasma membrane although SNAREs do not appear essential for exosome release [27]. 2.4 Exosome Uptake

Exosomes are internalized by recipient cells through receptor-mediated endocytosis, pinocytosis, phagocytosis, or through fusion with the cell membrane resulting in direct release of contents into the cytoplasm. As exosomes can really be thought of as cellular derived nanoparticles, it is logical to expect that that these cellular nanoparticles can target specific cells depending on their surface modification. This has been an area of intense research for drug delivery applications and therapeutic development, but this targeting ability has also been demonstrated by endogenous exosomes. For example, a 2010 study exploring the effects of the tetraspanin Tspan8 on angiogenesis in a rat adenocarcinoma model, demonstrated that Tspan8 in exosomes recruited specific proteins for loading that facilitated endothelial cell binding and uptake (CD106 and CD49d), Tspan8 exosomes targeted endothelial cells, and induced angiogenesis [36]. This study depicts how the endosome is a signaling entity that dictates exosome fate; Tspan8 incorporated in the endosome and subsequently into exosomes selected proteins to ensure target cell uptake (endothelial cells) to exert a biological effect (angiogenesis) in the target cell. Although the paracrine effects of exosomes are well documented, numerous studies are beginning to emerge documenting distal uptake of exosomes. A seminal exosomal study (discussed in the later metastasis section) by Lieberman et al details that distal sites of animal models (the lungs of treated mice) can be transformed upon systemic administration of exosomes [5]. Although it is unclear how far endogenous exosomes can travel in the body and how long they can maintain stability in systemic circulation, studies of exogenous therapeutic exosomes (detailed in Section 5) allude to the tremendous stability of exosomes and their ability to target most tissues. Systemically administered exosomes targeting the acetylcholine receptor have even been shown to deliver siRNA into the brains of treated mice, demonstrating the clear therapeutic potential of exosomes to target even the most difficult tissue to access while maintaining systemic stability [37].

Positive and negative feedback may also regulate exosome uptake and release in a cell specific manner. In cell culture studies an interesting feedback loop has been noted; exposure to self (same cell derived) exosomes promotes exosome release [4]. The study examined dose responses (exosome release) from cells (a normal mammary epithelial cell line and a cancer derived cell line) after exposure to self-derived exosomes and exosomes from the other cell line. The results of the study demonstrated that self exosomes promote further exosome release (positive feedback), whereas exposure of breast cancer cells to exosomes from normal mammary epithelial cells inhibited exosome release and, likewise, exosome release from the normal mammary epithelial cells was inhibited upon exposure to cancer cell derived exosomes [4]. This feedback control most likely does not translate to all cell types but does reveal that feedback processes can influence exosome secretion and in this scenario, implies a process of self propagation of exosome secretion and non-self inhibition of exosome secretion. 2.5 The Plasma Membrane Remodeling/Endosome/Exosome Axis in Cancer

The concept of the endosome as a signaling entity (Figure 1) is not a new concept; for a comprehensive review on endocytosis, endosomes, and signaling, the reader is directed to Sorkin and Zastrows 2009 Nature Review in Molecular Cell Biology [22] and for a focused review of multivesicular bodies in signaling, the reader is directed to Dobrowolski and De Robertiss 2012 Perspective [20]. The endosome functions in cellular signaling in many important ways; the most obvious function of endosomes is in signal housekeeping as endocytosis can function to recycle or degrade receptors, the kinetics of receptor return to the

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membrane can be important for signal propagation and transduction [20]. The endosome can function to generate signals, as with some receptor tyrosine kinase (RTK) signaling and SMAD signaling [20, 21]. Endosomes enriched in APPL (adaptor protein containing phosphotyrosine-interaction domain, pH domain, and leucine zipper motif) are involved in RTK signaling while endosomes enriched in SARA (SMAD anchor for receptor activation) are involved in TGF- (transforming growth factor-) signaling [38, 39]. For example, when TGF- receptor is endocytosed into endosomes with SARA, TGF- receptor binds to SARA and forms a complex that recruits SMAD2; SMAD2 is then phosphorylated and released into the cytoplasm where it joins with SMAD4 forming an active transcription factor complex that can translocate to the nucleus [38].

The endosome also behaves as a signaling entity as it interacts with cytosolic components to select content for exocytosis or lysosomal degradation [21]. Through signaling interactions, endosomal constituents can determine exosome content. For example, a study examining the interaction between the tetraspanin protein CD9 and the metalloprotease CD10 demonstrated that CD9 controls the loading of CD10 into exosomes [30]. The study used K562 erythromyeloid cells that have a low level of basal CD9 expression, transfected the cells with a plasmid conferring CD10 expression and neomycin resistance via a lentiviral vector and selected for CD10 expressing (neomycin resistant) cells [30]. These cells were further transfected with puromycin resistant vectors containing either an empty plasmid, wild-type CD9, or a mutant CD9 with an altered binding site for CD10 (substitution at residue 82 of the C-terminal tail) [30]. The expression of both wild-type CD9 and mutant CD9 did not alter the total cellular expression of CD10; the level of wild-type CD9 and mutant CD9 was similar in exosomes [30]. The wild-type CD9 associated with CD10 in the exosomal fraction and increased the level of CD10 recruitment into exosomes 5-fold relative to the K562 with basal (low) CD9 expression, and 2.5 fold relative to the mutant CD9 cells [30]. Additional experiments in Nalm-6 cells demonstrated that knockdown of CD9 with short hairpin RNA reduced the level of CD9 expression and CD10 recruitment in exosomes [30]. CD9 is associated with the plasma membrane, cytosol, endosomes, and exosomes and is often used as an exosome biomarker [40]. This study is a demonstration of how endosomal tetraspanins function to load specific content into exosomes. A study that provides further insight into the connection between endocytosis, exosome release, and cancer is a 2011 study of the receptor tyrosine kinase, Met [41]. The study used two previously validated oncogenic Met mutants in NIH3T3 cells and demonstrated that Met mutants had a higher intracellular concentration of Met (52%) compared to wild-type cells (18%) and this intracellular concentration co-localized with early endosomes and late endosomes, but not with lysosomes, resulting in receptor return to the plasma membrane and resistance of the receptor to degradation [41]. The Met mutants had defective actin fibers and required endocytosis to remodel the cytoskeleton (remodeling was inhibited upon treatment with endocytotic inhibitors); the Met mutants also had a > 3 fold higher rate of migration through a membrane compared to wild-type cells, migration of the Met mutants was inhibited by blocking endocytosis [41]. Inhibiting endocytosis inhibited the oncogenic activity of the Met mutants. This was demonstrated by first, validating the ongogenic activity of the mutants in vivo; grafting the Met mutant cells into nude mice resulted in rapid 500 mm3 tumors (within 12 days), whereas at this time point, wild-type grafts did not result in palpable growths [41]. After validating the oncogenic activity, in a new set of experiments, endocytotic inhibitors were administered when tumor volumes were 50 mm3; 5 days after treatment tumor size decreased 40-50% demonstrating the role of endocytosis in maintaining the oncogenic activity of the Met mutants [41]. The collective results of these studies suggest that endocytosis and increased receptor recycling is critical to maintaining the oncogenic activity of mutant tyrosine kinase receptors. The release of exosomes may promote this oncogenic activity through enhancing motility (as migration was inhibited when endocytosis was inhibited) and by establishing a suitable tumor

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microenvironment that promotes growth (in vivo tumor growth was inhibited when endocytosis was inhibited).

In support of the concept that alterations in the expression of plasma membrane receptors and plasma membrane remodeling in cancer contributes to the aberrant exosomal process demonstrated by cancer cells; a recent study demonstrated that hypoxia increased the release of exosomes from cancer cells [42]. Hypoxia results in translocation of Hypoxia Inducible Factors (HIFs) from the cytoplasm to the nucleus of the cell where this alpha HIF isoform (HIF-1, HIF-2, or HIF-3) complexes with a HIF beta isoform and ancillary proteins to form an active transcription factor that binds to Hypoxia Responsive Elements (HREs) on target genes. Target genes include numerous plasma membrane receptors such as GLUT-1 glucose transporter, EGFR (Epidermal Growth Factor Receptor), and the drug efflux pumps; P-glycoprotein (P-gp, MDR1) and Multidrug Resistance Protein 1 (MRP1). There are over 100 genes with HREs; many of which are plasma membrane receptors including the transferrin receptor, which was the first marker associated with exosome detection. The hypoxic induction study examined three breast cancer cell lines (MCF7, SKBR, and MDB-MB-231 cells) and demonstrated that exosome secretion increased in a dose response manner to increasing levels of hypoxia [42]. Growth in 1% oxygen conditions resulted in an increase (1:1.5 relative to normoxic cells) in exosome secretion for each cell line; dropping the oxygen concentration to 0.1% hypoxia further increased the production of exosomes for each cell line, almost doubling the secretion of exosomes relative to normoxic cells [42]. Our own studies in a panel of cancer cell lines have shown that hypoxia increases the expression and nucleic translocation of HIF-1 and HIF-2, resulting in a subsequent increase in the protein levels of the plasma membrane receptors P-gp, MRP1, EGFR, and GLUT-1 [10]. As receptor expression changes (decreases or increases) this can increase plasma membrane remodeling while increasing receptor expression can also directly increase receptor activation and internalization or result in receptor clustering which promotes endocytosis [21]. Numerous alterations in cancer cells, including activation of the p53 oncogene, result in subsequent changes in plasma membrane proteins, as well as changes in endocytosis (discussed in introduction). The most consistent trait of cancer cells is their constant state of flux and ability to rapidly adapt to microenvironmental selection pressure; this phenomena is often referred to as plasticity and ensures membrane remodeling and associated exosome release [12, 43]. 3. Exosomal Content in Cancer Cancer derived exosomes have a vast array of content composed of microRNAs, mRNAs, transcription factors, proteins, and lipids (Figure 2 [2, 44-49]). The content is highly variable and dependent on cell origin, but regardless of origin, the contents of exosomes are highly functional and exert powerful effects in recipient cells. 3.1 MicroRNAs and mRNAs

MicroRNA (miRNA) are small, non-coding RNA that are usually 20-25 nucleotide long sequences, which upon entering the recipient cell bind to the target mRNA sequence and inhibit translation. It has been found that miRNA expression is dysregulated in most cancers. MiRNA signatures of exosomes of different cellular origin are distinct. Many miRNA are tumor-specific and therefore can be used as diagnostic and prognostic biomarkers [50]. The shuttling of miRNA molecules that may either be tumor-supportive (oncomiRs) or tumor-suppressive is especially important for cancer. While mRNA enters the recipient cell to be translated into proteins, miRNA exert silencing effects on genes through a process called RNA interference (RNAi). Exosomal RNA content has also been shown to differ from donor cell RNA as it contains high levels of small RNA and lacks 18s and 28s ribosomal RNA (rRNA). Recently, the presence of single and double stranded DNA molecules was also discovered in tumor-derived exosomes [50, 51].

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IL4 activated macrophages are known to secrete exosomes containing miR223, which promotes invasiveness of breast cancer cells [52]. The mir200 family present in breast cancer exosomes and miR105 present in breast, ovarian, gastric and prostate cancer exosomes cause metastasis, invasion, tumorigenesis and tumor progression [5, 53]. MiR494 found in small cell lung carcinoma and breast cancer exosomes promotes proliferation, survival and migration [54, 55] Mir34a, on the other hand, which is known to increase apoptosis and senescence is found in tumor derived exosomes (TEX) of the breast, prostate, brain and bladder [56-58]. Therefore, not all miRNA may be implicated in tumor supportive mechanisms. 3.2 Transcription Factors

Specific transcription factors are often upregulated in cancer to activate the expression of proteins that promote tumor progression, survival, and metastasis. HIF1 involved in upregulating metastatic potential, angiogenesis and proliferation in tumor cells. As discussed below, HIF1 released in nasopharyngeal carcinoma cell exosomes can increase metastasis in recipient cells [59]. Tumor derived exosomes that contain FasL and TNF induce T cell apoptosis, which in turn causes immune suppression, thereby supporting tumor progression [60-62].

Exosomes isolated from body fluids of cancer patients have also been shown to have up-regulated levels of vascular endothelial growth factor (VEGF), while TEX contain transcription factors that modify the tumor stroma to create a tumor friendly environment that

Schematic illustrating components of an exosome including mRNA, microRNA, DNA, heat shock proteins, enzymes, MHC receptors, tetraspanins, lipid rafts, cytoskeletal elements, and membrane transport proteins.

Figure 2. Exosome Content in Cancer.

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supports metastasis. These stroma modulating factors include TGF, matrix metalloprotease 2 (MMP2) and urokinase plasminogen activator [63-67]. TGF expressed on the surface of tumor derived exosomes is capable of differentiating fibroblasts into myofibroblasts, which increases vasculature, facilitates tumor progression and metastasis [68].

Exosomes that release c-Met can increase angiogenic and metastatic potential in recipient tumor cells [69]. EGFR has been detected in lung cancer cell exosomes, and co-culturing of these cells with dendritic cells and Th0 cells, results in an increase in regulatory T cells that are tumor antigen specific, which downregulate immune responses towards tumors [70]. 3.3 Proteins

Having originated from endosomes, exosomes express a number of proteins involved in membrane related activities including RabGTPase and Annexin, cellular adhesion proteins including integrins and tetraspanins, cytoskeletal proteins such as actin and myosin and heat shock proteins such as Hsp70 [71, 72]. In certain disorders of the central nervous system, numerous proteins including -amyloid protein, -synuclein and superoxidedismutase have also been found to be released from exosomes and are implicated in the pathogenesis of these diseases [73-77]. In patients with cancer, TEX represent a large portion of the circulating exosome population and elevated levels of tumor specific proteins are found in the blood of cancer patients [78].

Cell adhesion proteins and enzymes that degrade the extracellular matrix have a pivotal role in aiding metastasis. Cancer associated fibroblasts secrete CD81 rich exosomes containing Wnt1 into the tumor stroma, which is then taken up by breast cancer cells bringing about metastasis and cell migration [40, 79, 80]. Numerous studies have pointed out that exosomes regulate stromal cells in the pre-metastatic organ, simultaneously recruiting hematopoietic stem cells from the bone marrow to help in creating a pre-metastatic niche. Exosomes expressing the CD9-M2, L2 and X2 complexes selectively target bone marrow cells and those that express Tspan8 and CD151-31 and 64 complex with lymph node stroma [81, 82]. In addition, tetraspanins can also associate with MMPs such as MMP14 and bring about a breakdown of the extracellular matrix facilitating metastasis [81, 83, 84].

By inhibiting NKG2D on natural killer (NK) cells and inhibiting their proliferation, TEX decrease the cytotoxic effects of NK cells on tumor cells. Tumor cell exosomes also carry p-glycoprotein causing drug resistance [85], MMPs to bring about matrix degradation [86], truncated EGFR that enhances oncogenic signaling [87], and delta like4 (Dll4) [88] and VEGF that causes angiogenesis [89, 90]. 3.4 Lipids

As previously mentioned, exosomes have elevated sphingomyelin (SM), cholesterol, glycerophospholipid and ceramide content [50, 73, 91-94]. Elevated levels of SM and cholesterol were uncovered in exosomes from PC-3 prostate cancer cells. Prostate cancer cells secrete exosomes with elevated levels of glycophospholipids including HexCer and LacCer, making these potential biomarkers [95, 96].

In a study conducted by Toda et. al, it was demonstrated that, in addition to surface protein ligands, lipids including SM and monosialodihexosylganglioside were also involved heavily in intracellular trafficking of exosomes [96, 97]. Docosahexaenoic acid, which can be found at millimolar concentrations in exosomes, aids in breast cancer suppression by acting on the anti-estrogen receptor site, inducing apoptosis by modifying metabolism of cholesterol, this occurrence could have powerful implications for designing exosome based therapeutics for breast cancer [94, 96, 97]. Although the contents of exosomes are highly variable, they have cell type specific trends correlating to their origin cells; as such, exosomes are proving to be useful tools in cancer biomarker screening.

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4. Exosomes in Cancer Biomarker Screening Poor prognosis with existing therapeutic approaches against cancer is largely attributed to late stage detection of the disease, which in part is due to lack of appropriate disease-specific biomarkers. Therefore, considerable focus has shifted to studying and identifying cancer-specific molecular signatures that can be utilized for detection of the disease at an early stage. To this end, much like circulating tumor cells, exosomes carry a wealth of information about the disease characteristics in the form of proteins, lipids, nucleic acids and metabolites that are shed from tumor cells into the exosomes [98]. Besides being secreted by different types of cells in vitro, studies have confirmed the presence of exosomes from various biological fluids including blood, urine, saliva, ascites, cerebrospinal fluid, synovial fluid, breast milk, and bronchoalveolar lavage [99]. Compositional analysis of these exosomes demonstrates disease and cell-specific unique molecular fingerprints alongside other conserved components that carry valuable genomic and proteomic information. Biomarker mining from exosomes becomes an even more attractive alternative because the components are well protected within the lipid bilayer and therefore can be preserved without any significant loss of information. Kalra et al stored the exosomes isolated from colorectal carcinoma cells LIM 1863 in plasma for up to 3 months at 4 C, 37 C, -20 C and -80 C followed by western blot analysis for the TSG101 marker to demonstrate the protein stability within the lumen of the vesicles [100]. Ge et al did a more rigorous analysis on the stability of miRNA in plasma and exosomes stored at 4 C, -20 C and -80 C for up to 5 years. Their results confirm that while in plasma, miRNA degrades at 4 C and -20 C, the total miRNA in exosomes do not show any significant difference in their total miRNA content or the individual miRNA concentration compared to a fresh sample. Further, subjecting the stored sample to a freeze/thaw cycle also did not lead to considerable loss in miRNA content of the exosomes [101]. Exosomes therefore are robust biological entities that offer tremendous promise in studying cancer-specific molecular signatures to identify biomarkers that could serve as potential diagnostic tools in monitoring disease onset, response to therapy or prognosis. Figure 3 outlines the chronological steps involved in identifying and validating exosome-derived proteins and nucleic acids as potential biomarkers for disease diagnosis, prognosis, and response to therapy. A systematic approach to plasma proteome and nucleic acid analysis has led to the identification of several proteins that could serve as biomarkers for identifying and staging cancer and developing personalized therapeutic approaches to achieve maximum treatment responses [99, 102]. The complexity of the plasma protein content poses a considerable hurdle in this developmental path since the useful biomarkers are often in a very low concentration and are dominated by other abundant proteins [98]. Exosomes on the other hand provide a much cleaner clinical sample devoid of plasma proteins and thus make the analysis and biomarker validation easier. Analysis of exosomes is an avenue for developing novel protein biomarkers for monitoring cancer as opposed to the existing standard-of-care biomarkers that have failed to make a mark in the clinical environment [103].

Prostate-specific antigen is the benchmark FDA-approved biomarker for prostate cancer but its specificity has been under scrutiny due to inconsistent results. Duijvesz et al compared the protein content of exosomes derived from non-cancerous prostate epithelial cells (PNT2C2 and RWPE-1) and compared it with prostate cancer cell (PC346C and VcaP) derived exosomes by using tryptic digestion followed by LC-MS/MS analysis [104]. Their results revealed at least four potential prostate cancer biomarkers proteins (PDCD6IP, FASN, XPO1 and ENO1) that were found in abundance in the exosomes from cancer cells among which, the latter two appear to be prostate-specific biomarkers since they have not been reported in other exosomes [104]. In a similar study, Hosseini-Beheshti et al critically evaluated the exosomal content of six different prostate cancer cell lines with a varying degree of expression of the androgen receptor (AR) [105]. They found 3 proteins (ENPL, GRP78 and AXA2L) that were exclusively present

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only in the AR- cells-derived exosomes while two proteins signatures were exclusive to AR+ cells-derived exosomes [105]. Nilsson et al identified mRNAs for PCA-3 and TMPRSS2:ERG in the exosomes isolated from urine samples collected from patients suffering from prostate cancer and their finding suggests that these biomarkers can be utilized for the assessment of tumor genotypic/phenotypic and metastatic potential as well as monitoring therapeutic responses to androgen deprivation therapy [106]. Recently, Bryant et al did a comprehensive analysis of the miRNA composition of exosomes isolated from the serum of patients suffering from non-metastatic and metastatic prostate cancer and compared it with those from healthy individuals [107]. Their findings reveal that 12 miRs were differentially expressed in exosomes of prostate cancer patients compared to healthy individuals and 16 miRs were differentially expressed in metastatic patients compared to non-metastatic patient with miR-141 and miR-375 showing significant association with metastasis [107]. These reports show an early positive indication of potential biomarkers for prostate cancer but have to be verified from patient samples in a larger cohort.

Schematic for biomarker validation in exosome screening. Endocytotic vesicles (EV) are isolated followed by nucleic acid and protein isolation for biomarker discovery and identification. The biomarkers are validated and submitted for FDA approval in diagnostics, prognosis, or therapeutics.

Figure 3. Validating Biomarker Screening of Cancer Exosomes.

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In glioblastoma, a highly malignant form of brain tumors, reports have established that the presence of EGFRvIII mutant splice variant is of tremendous significance and is useful in monitoring responses in therapeutic and vaccine clinical trials [108, 109]. A detailed proteomic profiling of exosomes from brain cancer cells further revealed the presence of EGFR along with TGF-, a cytokine with immunosuppressive function. Surprisingly, the analysis of exosomes from the serum of patients with brain tumors also showed an increased level of TGF- along with EGFR and EGFRvIII, suggesting the potential role of these biomarkers in the diagnosis and prognosis of disease [61]. Skog et al used a nested RT-PCR method and detected EGFRvIII from microvesicles isolated from the serum of patients suffering from glioblastoma but not from any of the 30 control samples [64]. More recently, Shao et al developed a more ingenious method for isolating exosomes [110]. The exosomes isolated from clinical blood samples were tagged with antibodies, which was further tagged with magnetic nanoparticles to be captured on a microfluidic device by microfiltration. The advantage of using such nanotechnology-based biosensors lies primarily in the requirement of a very low sample volume for analysis with minimum pre-processing. Importantly, the captured exosomes could either be analyzed in situ by -NMR with far superior sensitivity compared to common techniques such as ELISA or flow cytometry; or could be isolated from the device for subsequent molecular profiling by alternate methods. Based on the analysis of exosomes from GBM patients and healthy individuals, authors could establish an increased presence of EGFR, EGFRvIII, PDPN and IDH1 R132H in the exosomes of the patients and they were able to distinguish between the sources of the exosomes with a 90% confidence. Most importantly, analyses of the exosomes from patients undergoing a therapeutic regimen showed a distinct predictive capability of drug efficacy with the studied biomarkers [110]. As an alternative, miR-21 has been reported as one of the biomarkers that is secreted in high concentration in exosomes shed by gliobalstoma cells as well as those isolated from patients cerebrospinal fluid (CSF). Among a cohort of 13 patients suffering with the disease, the miR-21 level in the exosomes was found to be at least 10-fold higher than in exosomes from healthy individuals. In a critical analysis in a cohort of 29 patients, miR-21 level analysis from the exosomes could distinguish disease-carrying individuals from healthy individuals with a 87% sensitivity and 93 % specificity [111]. Two independent reports confirmed an overexpression of miR-21 in the serum of patients suffering from large B-cell lymphoma [112] and in exosomes isolated from the serum of patients with esophageal squamous cell carcinoma [113], which questions the validity of miR-21 as a cancer-specific marker and warrants more in-depth assessment.

Taylor et al analyzed the exosomes shed into the sera by patients, to stage ovarian cancer and reported 0.320 0.056, 0.640 0.053, 0.995 0.084 and 1.420 0.228 mg/ml of EpCAM protein of total exosomal protein in patients with stage I, II, III and IV ovarian cancer compared to benign tumor bearing patients with protein levels of 0.149 0.065 mg/ml and healthy individuals with 0.039 0.030 mg/ml EpCAM levels. They further detected 218 miRs in exosomes with 31 showing elevated levels including miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205, and miR-214 [114]. Im et al developed a nano-plasmonic exosome (nPLEX) assay for surface plasmon resonance-based, label-free detection of exosome proteins. They determined as many as 71 ovarian cancer signature proteins markers from cancer cells; further established that EpCAM and CD24 levels are highly elevated in the exosomes derived from ascites of cancer patients and could be used as biomarkers to study the patients response to chemotherapy [115]. Several other such studies have been undertaken in past 5 years to assess various biomarkers in different cancer patients and have been summarized in Table 1. One hallmark feature with the majority of these reports is that actual clinical samples have been increasingly incorporated into the studies to develop a direct correlation between the biomarker level and disease status. This change in the paradigm promises a rigorous and robust approach to biomarker identification, analysis, and subsequent validation in larger populations of patients to eliminate previous pitfalls and ensure an improved

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clinical translation [103].

Biomarker Type Source Analysis Disease PDCD6IP, FASN, XPO1 and ENO1 Protein Cells LC-MS/MS Prostate [104] PCA-3, TMPRSS2:ERG mRNA Urine RT-PCR Prostate [106] MiR-141, MiR-375 mRNA Serum RT-PCR Prostate [107]

EGFR, EGFRvIII, TGF- Protein Serum SDS-PAGE Glioblastoma [61]

EGFRvIII mRNA Serum RT-PCR Glioblastoma [64]

EGFR, EGFRvIII, PDPN, IDH1 R132H Protein Blood -NMR, ELISA Glioblastoma [110]

MiR-21 micro RNA CSF RT-PCR Glioblastoma [111]

MiR-21 microRNA Blood RT-PCR ESCC [113]

MiR-155, MiR-210, MiR-21 microRNA Serum RT-PCR Lymphoma [112]

MiR-21, MiR-141, MiR-200a, MiR-200c, MiR-200b, MiR-203, MiR-205, MiR-214

microRNA Serum Microarray Ovarian [114]

EpCAM, CD24 Protein Ascites nPLEX Ovarian [115]

MiR-718 microRNA Serum Microarray HCC [116]

MiR-17-3p, MiR-21, MiR-106a, MiR-146, MiR-155, MiR-191, MiR-192, MiR- 203, MiR-205, MiR-210, MiR-212, MiR-214

microRNA Plasma Microarray Lung [117]

EpCAM Protein Plasma Magnetic cell sorting

Lung [117]

LRG1 Protein Urine LC-MS/MS Lung [118]

Apbb1ip, Daf2, Foxp1, Incenp, Aspn, BC031781, Gng2

mRNA Saliva Microarray Pancreatic [119]

NT5E/CD73 Protein ascites MS/MS Pancreatic [120]

NT5E/CD73 Protein Cells MS/MS CRC [121]

CD63, Caveolin-1 Protein Plasma Exotest (ELISA) Melanoma [122]

5. Exosome Mediated Communication 5.1 Tumor Metastasis

Exosomes promote metastasis through their direct role in invasion and through content specific effects that promote metastasis, transformation, and the establishment of the pre-metastatic niche. Exosomes have been demonstrated to have a fundamental role in the physical process of invasion, the first stage of metastasis. MVBs and exosomes are associated with invadopodia, the actin-rich protrusions of cancer cells that initiate invasion through degradation of the extracellular matrix [123]. A series of experiments have demonstrated that MVBs and exosomes recruit to the plasma membrane of invadopodia and that knockdown of Rab27a decreases exosome secretion and extracellular matrix digestion associated with maturing invadopodia [123]. Further experiments have demonstrated that exosomes extracted

Table 1: Exosome Biomarkers

Table 1: A summary of reports on protein or RNA biomarker mining in exosomes isolated from cells and clinical samples.

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from cells with maturing invadopodia have the ability to induce invadopodia formation in non-invading cells [123]. Exosomes appear critical for invasion, extracellular matrix digestion, and metastatic induction. Similar studies have investigated the interaction of TEX with the extracellular matrix and have identified critical roles of cancer exosomes in promoting invasion. For example, a study using exosomes derived from metastatic rat pancreatic adenocarcinoma cells demonstrated that these exosomes express high levels of CD49c and CD44 and by examining binding of these exosomes as well as exosomes derived from a CD44 knockout cell line, binding to hyaluronic acid in the extracellular matrix was demonstrated [124]. These CD44 exosomes were also loaded with a high level of proteases which functioned to degrade the extracellular matrix in an in vivo rat model of matrix remodeling [124].

Exosomal content also promotes metastasis and transfers metastatic potential. The metastatic process consists of a series of events starting with epithethial-mesenchymal transition (EMT; mobilizing cells) and then mesenchymal-to-epithelial transition (MET; establishing a secondary tumor site). Exosomes have been identified as contributing to the formation of the pre-metastatic niche; a primed site distant from the primary tumor where eventual metastasis occurs. Circulating exosomes from the primary tumor develop and prime sites for metastasis and may even attract cancer cells to the niche, functioning as the first mediators of metastatic node formation [125]. Cancer exosomes have been shown to deliver functional complexes capable of promoting both EMT (such as HIF-1) and MET (such as miR-200).

A seminal exosomal study performed an eloquent investigation of the metastatic capabilities of exosomes in vitro and in vivo. Judy Lieberman and her group at Boston Childrens Hospital demonstrated that exosomes and ectosomes released from metastatic cancer cells can transfer metastatic capability to non-metastatic cells and this transformation is governed by the microRNA-200 family, known mediators of MET [5]. The study used cells with distinct metastatic capabilities (metastatic 4T1E mouse cells and metastatic human cells CA1a and BPLER cells and poorly metastatic 4T07 mouse cells and poorly metastatic human mesenchymal MB-231 cells) to study in vivo metastatic induction in mouse and human xenograft models. Exosomes from the highly metastatic cells transferred miR200 to xenografted human breast cancer cells and promoted metastasis of the xenografts [5]. This study is a pivotal demonstration that exosomes can not only promote metastasis, they can also transform distal cells into metastasis cells. Many studies have identified other specific microRNAs that promote metastasis. For example, a 2015 study examined exosomal miR-122, a biomarker clinically associated with metastatic breast cancer [126]. The results confirmed a high expression of miR-122 in all of the exosomes derived from a panel of breast cancer cell lines whereas most parental cell lines had low intracellular levels of miR-122, suggesting that miR-122 is targeted for exosomal secretion by breast cancer cells [126]. miR-122 downregulates pyruvate kinase and subsequently decreases glucose metabolism and GLUT-1 expression; mice treated with metastatic derived exosomes with high miR-122 content resulted in rapid brain and lung metastasis [126]. Numerous studies portray the role of specific microRNAs from cancer derived exosomes that are capable of conferring metastasis.

In a very interesting illustration of exosomal activity in establishment of the pre-metastatic niche, Hood et al. demonstrated that melanoma exosomes rapidly stimulate the production of endothelial spheroids and regulated inflammatory cytokines leading to promotion of endothelial angiogenic responses that can contribute to the metastatic potential [127]. Similarly, Peinado et al. demonstrated that exosomes from highly metastatic melanomas augmented the metastatic potential of primary tumors by receptor-tyrosine kinase MET dependent alterations of bone marrow progenitors [128]. Furthermore, it has been reported that melanoma excreted exosomes exhibit a preference for sentinel lymph nodes, preparing the nodes for melanoma cell recruitment and facilitating lymphatic metastasis [129]. Exosomes have apparent functions in physical invasion and through content specific effects, mediate metastasis.

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5.2 Drug Resistance Innate, acquired and de novo drug resistance remain a hallmark of cancer and drug resistance is the major obstacle in devising therapies to achieve successful outcomes against the disease. Innate multidrug resistance (MDR) occurs when cancer cells have drug resistant phenotypes prior to any drug or treatment exposure. Innate drug resistance is often associated with the over expression of ABC transporters; drug efflux pumps that include P-glyoprotein (MDR1) and multidrug resistance protein 1 (MRP1). Acquired MDR is a gradual process where cancer cells exposed to drug, radiation or targeted therapy educate themselves and alter their genetic makeup to acquire drug resistance cancer phenotypes. De novo drug resistance on the other hand is a transient strategy acquired by the cells to attain an environment-mediated drug resistance, which involves signaling events in the tumor microenvironment that can lead to soluble factor-mediated drug resistance (SFM-DR) or cell adhesion-mediated drug resistance (CAM-DR) [130]. Cancer cells employ a combination of mechanisms to achieve multidrug resistance such as decreased drug influx, increased drug metabolism and detoxification, increased DNA repair mechanisms, increased drug efflux, and decreased apoptosis. Cancer cells further mitigate radiation and chemotherapy via multiple different mechanisms such as epigenetic modifications, poor drug penetration and through the presence of cancer stem cells [131]. Given the central role of exosomes in cellular communication, it is undoubtable that exosomes also contribute to MDR. One intuitive mechanism involving exosomes would be the sequestration of cytotoxic drugs in the intracellular vesicles and subsequent expulsion, to negate drug effect within the cells [132]. Luciani et al supported this by showing that melanoma, adenocarcinoma and lymphoma cells confiscate drugs such as cisplatin, 5-flurouracil and vinblastin in their endosomal compartment [133]. This was further confirmed when melanosomes secreted from melanoma cells that were exposed to chemotherapy, were laden with cytotoxic drugs [134]. The platinum drugs internalized by cancer cells are often segregated into endosomal compartments such as lysosomes or vesicles that are destined for secretion and this is often mediated by lysosome-associated protein (LAMP) and copper export proteins ATP7A and ATP7B [135]. Safaei et al showed that exosomes secreted by cisplatin resistant ovarian cancer cells showed 2.6-fold higher drug concentration along with higher content of LAMP and other cisplatin transporters such as MRP2, ATP7A and ATP7B [136]. Cancer cells exposed to radiation therapy also show an altered composition and rate of exosome secretion, which suggests their role in resistance to radiotherapy [67, 137]. The major contribution of exosomes in imparting MDR to cancer cells cements from their ability to transport molecular information such as proteins, mRNAs and miRNAs from one cell to another. Exosomes facilitate cell-cell crosstalk within the tumor environment, which plays a crucial role in augmenting MDR pathways. Boelens et al studied the exosome-mediated interaction of stromal and breast cancer cells through paracrine and juxtacrine signaling and demonstrated that such crosstalk helps cancer cells to abate the chemo- and radio- therapeutic insults [138]. The noncoding RNA content from the stromal exosomes could activate a STAT1-dependent response and subsequent NOTCH3 signaling pathway in the breast cancer cells, which leads to decreased cell apoptosis and an increase in the tumor growth. A mechanistic analysis reveals that the cancer cells induce secretion of exosomes rich in RNA polymerase III transcripts, which contains 5-triphosphate, from the stromal cells to engage RIG-I receptors on cancer cells to activate the STAT1 pathway [139, 140]. Similarly, transfer of drug efflux pump proteins such as P-gp from docetaxel-resistant cells via exosomes renders drug-sensitive cells resistant to the drug [139, 140]. Another report shows exosome-mediated transfer of miRNA from adriamycin and docetaxel resistant MCF-7 cells to drug sensitive cells, leading to a drug resistant phenotype [141]. Microarray and qPCR analysis confirmed that miR-100, miR-17, miR-222, miR-342p and miR-miR-451 were considerably elevated in the exosomes derived from both drug resistance cell lines and incubation of these exosomes with drug-sensitive cells led to

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an increase in these miRNA levels in the acceptor cells as well [141]. A detailed analysis of the miR-222 target gene PTEN in drug resistant and sensitive cells showed that while drug resistant cells show a reduced expression, the gene was highly expressed in the sensitive cells and its activity was severely hampered upon treatment with a miR-222 mimic or exosomes from docetaxel resistant cells. Xiao et al described a similar observation with their study on non-small cell lung cancer A549 cells, where treatment of resistant cells with cisplatin led to an increase in miR-21 content of the exosomes, which conferred resistance when exposed to drug sensitive cells [142]. All these studies suggest that exosome mediate drug resistance through both the direct shuttling of drugs out of the cells and through the horizontal transfer of molecules/molecular signals that bestow drug resistance to the otherwise sensitive cells. 5.3 Immune System Modulation The work of Raposo et al demonstrating that the presence of major histocompatibility complex (MHC) Class II-peptide complexes in exosomes secreted by both human and murine B lymphoblastoid cells, which could activate antigen-specific T cell responses, was truly seminal work; the report cemented an immunomodulatory role of exosomes in vivo [143]. Exosomes, along with other tumor derived extracellular vesicles (EVs), regulate the immune system through four distinct pathways involving antigen presentation to prime CD4+ or CD8+ T cells, activation or suppression of the immune system, and modulation of the complement system (Figure 4). Exosomes involve in antigen presentation with or without the engagement of antigen presenting cells (APCs) such as dendritic cells (DCs). The direct presentation (Figure 4A, Pathway a) involves interaction of the MHC-peptide complex, costimulatory and adhesion molecules on the exosome surface with the corresponding T cells receptor, resulting in priming/activation of the T cells [143]. The direct T cell stimulation capability of exosomes however is poor compared to the APCs, which could be attributed to small size and limited sites of interaction [144]. Indeed, increasing the concentration of the MHC-peptide complex in exosomes or increasing the total particle size by immobilizing them on latex beads leads to significant improvement in their ability to stimulate T cells [145-147]. Indirect presentation on the other hand ensues with the interaction of exosomes with the APCs (Figure 4A, Pathway b and c) through surface receptors such as integrins or intercellular adhesion molecule 1 (ICAM1; CD54) [145, 148]. The exosome bound to the APCs could either decorate the surface and subsequently interact with the T cell (Figure 4A, Pathway b; also known as cross-dressing) or could be internalized by the APCs, where the antigen is further processed for presentation (Figure 4A, Pathway c) [148]. The internalized exosome is degraded within the APCs and the antigen peptide is processed for presentation on the surface of the APCs (Figure 4A, Pathway d) or could be secreted as APC-derived exosomes where the antigen is presented on the MHC from the parent APC (Figure 4A, Pathway e). The fate of exosomes interacting with DCs depends on the status of the parent cell; immature DC show efficient exosome internalization capability following processing of peptides for presentation on their surface or release as DC-derived exosomes whereas mature DCs mostly present the bound exosomes on their surface [149]. The cross dressing of exosomes was confirmed in a study where DCs lacking MHC class II molecule IAb were incubated with exosomes containing an IAb-peptide complex, following incubation DCs could stimulate CD4+ T cells, suggesting that exosomes served as a source of IAb while DCs provide the costimulatory molecule [150].

There is mounting evidence that besides presenting antigens, exosomes also serve as a source of native antigen including not only proteins/peptides but also functional mRNA and miRNA to regulate the immune system and therefore have promising therapeutic potential [145]. Wolfers et al studied exosomes derived from different syngeneic and allogeneic tumor models to show a strong T cell-mediated antitumor response and cross protection, suggesting that exosomes could be a source of antigens to mount an immune response. Using tumor-derived exosomes as a source of native antigen for antitumor protective therapy, they demonstrated a

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strong CD8+ T cell response leading to rejection of tumors not only by the autologous exosomes but by allogeneic exosomes as well, confirming that the antitumor response is not mediated by the MHC class I-peptide complex [151]. Similarly, microarray analysis of exosomes from murine (MC/9) and human mast cells (HMC-1) as well as primary murine mast cells contain an enormous

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Figure 4. Exosome Regulation of the Immune System in Cancer.

Schematic of the immune/exosome (tumor-derived extracellular vesicles; EVs) interface. As depicted in each panel, there are 4 different scenarios for exosome/immune interfacing involving antigen presentation to prime CD4+ or CD8+ T cells (A), activation (B) or suppression (C) of the immune system, and modulation of the complement system (D). Panel A: Exosomes involve in antigen presentation with or without engagement of antigen presenting cells (APCs) such as dendritic cells (DCs). In the direct presentation pathway (a), the MHC-peptide complex on the exosome surface interacts with the corresponding T cells receptor, resulting in priming/activation of the T cells. Indirect presentation (b and c) involves interaction of exosomes with the APCs. The exosome bound to the APCs could either decorate the surface and subsequently interact with the T cell (b; also known as cross-dressing) or could be internalized by the APCs, where the antigen is further processed for presentation (c). The exosome is degraded within the APCs and the antigen peptide is processed for presentation on the surface of the APCs (d) or could be secreted as APC-derived exosomes where the antigen is presented on the MHC from the APC (e). Panel B: Exosomes can directly activate macrophages, neutrophils, natural killer (NK) cells, and APCs. APCs subsequently activate CD4+ or CD8+ T cells. Panel C: Exosomes also function to suppress an immune response, protecting cancer cells from immune cell recognition and destruction. Tumor derived exosomes can trigger apoptosis in T cells, reduce activity of NK cells and macrophages, and increase the population of myeloid-derived suppressor cells (MSDCs). Panel D: Exosomes avoid immune recognition through the expression of surface molecules that prevent activation of the complement system, resulting in a subsequent escape from opsonization.

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pool of functional mRNAs that are absent in the cytoplasm of parent cells, could be translated in vitro to corresponding proteins and importantly, could be shuttled between transferring capability of exosomes have been related to immune response against pathogens mast cells (therefore termed exosomal shuttle RNA as previously mentioned) [62]. Exosomes can directly activate immune effector cells including macrophages, neutrophils, natural killer cells, and APCs (Figure 4B). Proteomic analysis of exosomes derived from cancer cells demonstrate a repertoire of cancer-specific antigens such as heat shock proteins (HSPs), melan A, mesothelin and carcinoembryonic antigen (CEA), that can activate APCs to mount an anti-cancer immune response, a phenomenon often referred to as immune surveillance [145, 151, 152]. These immunogenic abilities of tumor-derived exosomes have galvanized the efforts to use them as anti-cancer vaccines.

Exosomes interestingly perform an equally contrasting role of immunosuppression in cancer, which helps cancer cells to escape from immune recognition and therefore promotes tumorigenesis [153]. In this regard, exosomes play a multifaceted role in affecting the immune system at different levels to obliterate the innate and adaptive immune response against cancer cells. TEX induce T cell apoptosis, reduce NK cells activity, inhibit IFN- dependent class II expression of macrophages and alter monocyte differentiation to increase the myeloid-derived suppressor cell (MSDCs) population, which leads to a collective failure of the immune system in containment of cancer growth (Figure 4C) [153-158].

The ability of tumor and APCs-derived exosomes to evade immune recognition (Figure 4D) is another intriguing aspect that has been a focus of research, especially to improve the therapeutic use of exosomes in vaccines and drug delivery. Cells express surface molecules such as CD46 (membrane cofactor protein), CD55 (decay accelerating factor) and CD59 that prevent activation of the complement system and thus they escape opsonization [159]. Clayton et al demonstrated that APC-derived exosomes express CD55 and CD59 but not CD46 on their surface, which inhibits the formation of complement membrane attacking complex and helps them avoid clearance. They further showed that blocking CD55 with an antibody increased the surface binding of C3b upon incubation in serum and resulted in exosomal lysis. CD59 blocking with an antibody gave a similar result and the exosomal lysis increased significantly when both CD55 and CD59 were blocked, suggesting their preventive role [160]. More importantly, this indicates that exosomes are not shed by cells as a means of a secretory pathway to remove redundant molecules but are functional cellular nanocarriers. Collectively, these studies illustrate the mechanism behind the stability of exosomes in the blood by avoiding opsonins, complement systems and coagulation factors. Exosomes appear capable of delivering almost any biological message, and in cancer, exosomal communication contributes to metastasis, drug resistance, and cancer immunology. The question now is how to manipulate exosomes for cancer therapy? 6. Therapeutic Manipulation of Exosomal Content

Due to their eloquent function as natural nanoparticles, the therapeutic potential of exosomes is immense. As a starting point, studying exosomes as a model of an ideal drug delivery system is aiding the development of more effective therapeutics. Beyond using exosomes, natures nanoparticles, as a model for drug delivery, there is great interest in applying exosomes as therapeutics and in manipulating exosomal content for therapeutic outcomes [161]. Although exosomal content can significantly vary depending the cell of origin [162], exosomes can be manipulated for drug delivery applications. We discuss the therapeutic application of exosomes to control metastasis and for cancer immunotherapy (with illustrative examples in Table 2) and discuss the current status of exosome based clinical trials. 6.1 Therapeutic Applications of Exosomes in Preventing Metastasis

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The main approaches for inhibiting exosome-mediated metastatic potential has been focused on blocking the production of exosomes by tumor cells and blocking the communication of tumor cells with the immune system and other cells. It has been shown that exosomes from 4T1 cancer cells are released in a Rab27a-dependent manner, and it has been suggested that inhibiting Rab27a results in reduction of exosomal release and a decrease of primary tumor growth and metastatic potential [163]. A study performed on HeLa cells by Ostrowski et al. showed that knocking down Rab27a and b inhibited the production of exosomes. This was achieved by silencing two Rab27 effectors, Slp4 and Slac2b by using shRNAs [164]. Alternatively, exosome production can be inhibited by blocking H+/Na+ and Na+/Ca2+ channels [165, 166]. Chalmin et al. used the molecule dimethyl amiloride, which can block Ca2+ channels, in combination with cyclophosphamide treatment, an alkylating agent capable of eliminating regulatory T-cells, to examine the effects on CT26 tumor bearing mice and the potential inhibition of exosome secretion [167]. Dimethyl amiloride treatment resulted in reduction of exosomes in vitro and in vivo, while also inhibiting Stat-3 activation and demonstrated a synergistic effect of the combination treatment [167]. The combination treatment was ineffective in nude mice, demonstrating a critical role of T cells and the regulatory effect of exosomes [167]. Another therapeutic approach that can be used, although it is not specific for only inhibiting metastasis, is selective loading of exosomes and the use of exosomes as therapeutic carriers; surface modification and active targeting of tumor or immune cells can also be used with this approach [168, 169]. 6.2 Therapeutic Applications of Exosomes for Immunotherapy

Cancer immunotherapy is the exploitation of the immune system to recognize and attack tumor cells; it is based on the principle that the immune system can be boosted, guided or even activated in order to attack tumor cells, resulting in inhibiting tumor growth, metastasis or further more in tumor regression [170]. In a number of publications, it has been reported that exosomes originating from dendritic cells can strongly stimulate the immune system [171-173]. This can be achieved even without the presence of mature dendritic cells, through a CD8+T cell priming pathway [173]. The presence of MHC proteins, class I and II, in exosomes originating from dendritic cells allow exosomes to exhibit strong antigen presenting properties [169]. Segura et al. [174] demonstrated that the ability of exosomes to stimulate strong immune responses correlated with the maturity of the dendritic cells they originated from. Although the exosomes secreted from immature and mature dendritic cells share similar morphological characteristics and protein content, the exosomes originating from mature dendritic cells are enriched in MHC class II, B7.2 and ICAM-1 proteins while demonstrating a 50 to 100 fold stronger T cell stimulation [174].

In an interesting study, dendritic cells were cultured in vitro and were pulsed with L1210 lymphocytic leukemia cell antigen and lipopolysaccharide. It was demonstrated that the isolated and purified exosomes derived from the pulsed dendritic cells were able to induce spleen cell proliferation in vitro and activate them to kill L1210 cells. Furthermore, the dendritic cell derived exosomes, in combination with cyclophosphamide and polyinosinic-polycytidylic acid sodium salt, suppressed L1210 tumor growth in vivo and produced improved survival time in mice [175]. In another study, Zitvogel et al. used exosomes derived from dendritic cells which in turn were pulsed with acid-eluted peptides (AEP-P815 or AEP-TS/A) to immunize mice in vivo carrying P815 tumors. A week after a single intradermal administration of the exosomes, tumor growth was halted and by day 60, 40 to 60% of the animals were tumor free, illustrating the applicability of exosomes as a cell-free vaccine alternative to dendritic cell therapy for cancer immunotherapy [176].

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Disease Origin of Exosomes Conclusion

Melanoma TEX Exosomes promote metastatic niche at distant

sites [127]

Melanoma TEX Exosomes augmented metastatic potential of

primary tumors [128]

Melanoma TEX Exosomes accumulate at sentinel lymph nodes

for promoting metastatic niche [129]

Mouse Breast Cancer

TEX Inhibition of Rab27a inhibited TEX secretion, primary tumor growth and metastatic potential

[163]

Cervical Cancer TEX Inhibition of Rab27a and b, inhibited exosome

production [164]

Mouse colon cancer TEX

Inhibition of Na+/Ca2+ channels and exosome secretion using dimethyl amiloride. Improved

anticancer efficacy for cyclophosphamide treatment [167]

Prostate Cancer TEX Exosomes transferred drug resistance from

resistant cells to non-resistant cells [140]

Breast Cancer TEX Curcumin modified the ubiquintinated protein

content of the TEX, inhibiting their immunosuppressive properties [177]

Leukemia DEX

Exosomes pulsed with tumor specific antigen induced spleen cell proliferation and activated

them against tumor cells, inhibiting tumor growth [175]

Leukemia TEX Exosome immunization caused partial tumor

growth inhibition [178]

Mouse Mastocytoma DEX Exosomes pulsed with acid-eluted peptide

immunized mice and caused tumor regression [176]

Melanoma DEX Exosomes carried Mart-1 tumor antigen to

dendritic cells for specific lymphocyte stimulation [179]

Mouse Mastocytoma TEX Exosomes carried native tumor specific

antigens to dendritic cells for potent T-cell-dependent antitumor effect [180]

Glioma TEX

Exosomes carried antigen presenting molecules and tumor antigen, capable of

causing dendritic cells to activate T lymphocytes against glioma cells [181]

Lymphoma TEX

Exosomes by heat-shocked tumor cells contained higher number of HSP proteins and were able to induce dendritic cell maturation

and potent T cell responses [182] Mouse

plasmacytoma TEX

Exosomes carrying surface HSP caused more efficient dendritic cell maturation [183]

Table 2. Investigational Exosome Therapies

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6.3 Clinical Trials and the Future of Exosome Therapeutics The clinical translation of exosome therapeutics is being tremendously aided by improved methods for the isolation and purification of exosomes in a current good manufacturing practice (cGMP) like environment. Anosys SA, Inc. (France) pioneered the methodology for exosome purification and laid down the quality control criteria for their clinical application [184]. Exosomes prove to be clinically significant in a wide area of anticancer therapy since their role in cancer immune biology, angiogenesis, drug resistance, disease progression and metastasis is well studied, characterized and outlined. However, their first clinical implementation came as anticancer vaccines due to obvious advantages. As outlined earlier, exosomes derived from tumors cells and components from immune system are involved in antigen presentation, transport of native antigen and modulation of the immune response. Exosomes carry the necessary repertoire of surface molecules to avoid immune recognition and clearance from circulation, which make them an ideal candidate for therapeutic applications. Institut Gustave Roussy and Institut Curie (France) conducted a Phase I clinical trial to assess the safety and surrogate markers for efficacy of DC-derived exosomes (also known as Dex) in melanoma patients [185]. A total of 15 patients suffering from metastatic melanoma (Stage IIIB or IV) were recruited for the study and 1.5 ml of their blood was drawn for separation of CD14+ monocyte cells by leukapheresis. The monocytes were differentiated into immature DCs by treatment with GM-CSF and IL4; loaded with melanoma antigen gene (MAGE) peptide (10 g/mL) and subsequently the exosomes were isolated and purified for vaccination. No Grade 2 toxicity was observed in any of the patients and 5 out of 15 patients showed clinical efficacy to the vaccination regimen. Concomitantly, another Phase I clinical trial was undertaken at Duke University (Durham, USA) where 13 patients suffering from stage IIIb/IV NSCLC were recruited for vaccination with an aim to ascertain the safety profile of an exosome-based vaccine and its efficacy [186]. The patients were vaccinated four times subcutaneously or intra-dermally at a weekly interval and 9 out of the 13 patients completed the therapy. No acute exosome-related toxicity was observed in any patient while only grade 1-2 toxicity was observed in a subset of patients, which included reaction at site of injection, flu-like illness and peripheral arm pain. 52 665+ days post-vaccination survival was recorded in patients; MAGE-specific T cell response was observed in 1 out of 3 patients and lytic activity of NK cells was reinstated in 2 out of 4 patients. Another Phase I clinical trial in 40 patients with advanced colorectal cancer (stage III/IV) was initiated using ascites-derived exosome (Aex) with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) [187]. Both vaccination regimens were well-tolerated by patients with some counts of grade 1-2 adverse effects were noted including reaction at injection site, nausea, pain, fever and fatigue. Importantly, a significant carcinoembryonic antigen (CEA)-specific antitumor cytotoxic T lymphocytes (CTSs) response was observed in patients vaccinated with the Aex and GM-CSF combination, suggesting their clinical benefits. The promising safety profile from the initial clinical studies has led to initiation of several clinical trials based on exosome-mediated intervention (Table 3). The favorable outcome of past clinical trials and the future pipeline suggests that exosome-mediated intervention is aggressively moving towards clinical translation. It is not surprising since exosomes present several liposome-like desirable attributes such as the ability to load hydrophobic as well as hydrophilic drugs, improved residence time and minimal inherent toxicity. However, like any other delivery systems, exosome-mediated therapeutic approaches have several uphill challenges. One of the major obstacles remains the successful loading of the active pharmaceutical ingredient into the luminal space without disrupting their biological properties. Source of exosomes and scalability is another challenge that limits their applicability.

Table 2: Illustrative studies involving exosomes from different cell origin, their action, and possible therapeutic utilization *DEX: Dendritic cell derived exosomes; TEX: Tumor cell derived exosomes

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Exosomes carry the signature proteins from the host and therefore their application in a non-host patient could have immunogenic implications t