Exosomes: Emerging biomarkers and targets for ovarian cancer

ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■

Contents lists available at ScienceDirect

Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

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Mini-review

Exosomes: Emerging biomarkers and targets for ovarian cancer Q2 Maggie K.S. Tang, Alice S.T. Wong * Q3 School of Biological Sciences, University of Hong Kong, Pokfulam Road, Hong Kong

A R T I C L E

I N F O

Article history: Received 14 May 2015 Received in revised form 13 July 2015 Accepted 13 July 2015 Keywords: Exosomes Ovarian cancer Protein RNA

A B S T R A C T

The limitations of current chemotherapies have motivated research in developing new treatments. Growing evidence shows that interaction between tumors and their microenvironment, but not tumor cells per se, is the key factor in tumor progression and therefore of obvious scientific interest and therapeutic value. Exosomes are small (30–100 nm) extracellular vesicles which have emerged as key mediators and communicators between cancer cells and other major cell types in the tumor microenvironment such as stromal fibroblasts, endothelial cells, and infiltrating immune cells as well as noncellular extracellular matrices through paracrine mechanisms. This review is to highlight the emerging role of exosomes in particular types of cancer, such as ovarian cancer, owing to its unique route of metastasis, which is capable of rapidly translating exosome research for clinical applications in diagnosis, prognosis, and potential treatment. © 2015 Published by Elsevier Ireland Ltd.

31 Introduction

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Exosomes were first identified in 1983 from sheep reticulocytes. They were merely called ‘externalized vesicles’ [1], whereas the term ‘exosomes’ was later proposed by Johnstone et al. in 1987 [2]. Nowadays, exosomes specifically refer to those disk-shaped membranous vesicles with a diameter of 30–100 nm. Exosomes can be isolated from cultured supernatants of cell lines and various types of body fluids including urine, blood and ascites. In vitro studies suggest that exosomes are important mediators of intercellular communication. However, the exact biological function of exosomes is still anticipated. The biogenesis of exosomes begins with an inward budding of the plasma membrane, resulting in the incorporation of membrane proteins in the early endosomes. The limiting membrane of the endosomes further invaginates, and cytosolic proteins and RNAs are selectively targeted and enclosed within the internal vesicles to form multivesicular bodies (MVBs) within the cytoplasm. These MVBs then fuse with the plasma membrane and release the exosomes to the extracellular space [3]. The exact mechanism of how exosomes interact with target cells is still under debate. Based on in vitro studies, three models are proposed, (1) direct fusion, (2) endocytosis and (3) binding through exosomal surface protein [4–8] (Fig. 1). Although exosomes are secreted by most cell types, there are also data that suggest enhanced exosome release under pathological conditions, such as cancer. It is reasonable to speculate that these vesicles may play an important role in tumorigenesis since (1) they can

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Q1

* Corresponding author. E-mail address: [email protected] (A.S.T. Wong).

mediate distant intercellular communication, (2) tumor-derived exosomes usually carry tumor antigens, and (3) functional proteins and/or RNAs can be transferred to recipient cells via exosomes. More recently, exosomes have been identified in malignant ascites in patients of ovarian cancer, a highly aggressive tumor that is the leading cause of death of all gynecologic cancers worldwide [9–11]. Unlike most solid tumors, ovarian cancer rarely disseminates through the vasculature but has a high propensity to metastasize within the peritoneum. The formation of malignant ascites is a hallmark of advanced/metastatic ovarian cancer [12]. This unique metastatic mechanism also poses distinct therapeutic challenges, in which current treatments are not effective (5-year survival <25%). Therefore, a better understanding of the ascitic microenvironment is critically essential to our knowledge of ovarian cancer biology and may have important clinical applications. Exosomes are an active area in cancer research; it will not be surprising to realize how fast information could accumulate. This review summarizes the key characteristics of exosomes and provides new information on their implications in cancer development and progression with a particular focus on ovarian cancer. We will also discuss the role of exosomes as predictive biomarkers and potential therapeutic targets in ovarian cancer with the supporting preclinical and clinical data.

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Exosomal cargo and its potential significance Over the past decade, a growing body of research clearly indicates the importance of exosomes. However, the problem of inconsistent nomenclature and lack of standard methods to obtain highly pure exosomes remains. The nomenclature could be based on site and tissue type, such as dexosomes (from dendritic cells) [13,14] and oncosomes (from cancer cells) [15,16]. Exosomes are

http://dx.doi.org/10.1016/j.canlet.2015.07.014 0304-3835/© 2015 Published by Elsevier Ireland Ltd.

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Recipient cell Endocytosis Exosomes Surface protein

Binding of surface protein Exosomal cargo

Direct fusion

Inward budding of plasma membrane

Endosome

Membrane fusion Invagination of endosome and incorporation of protein and RNA

Donor cell 1 2 3 4

MVB

Fig. 1. The biogenesis of exosomes. The biogenesis of exosomes begins with an inward budding of the plasma membrane, leading to the formation of early endosomes. Further invagination of the endosomal membrane results in the incorporation of cytosolic protein/RNA within the exosomes. The resulting multivesicular bodies (MVBs) then fuse with the plasma membrane and release the exosomes to the extracellular space. Exosomes can interact with the recipient cells via endocytosis, direct fusion, or binding of surface proteins.

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sometimes muddled up with microvesicles, in which both are common extracellular vesicles (EVs). While exosomes are originated from the intracellular multivesicular bodies, microvesicles are shed directly from the plasma membrane [3]. Exosomes are typically 50–100 nm in size, whereas microvesicles can be up to 1000 nm [3]. Despite these differences, it remains a challenge to differentiate exosomes and microvesicles. In addition, there are EVs from various sources in the extracellular space, which could hardly be separated by one common method. Moreover, a single cell type can secrete EVs of different intracellular origins [17], which make purification of a single population of exosomes even more complex.

It is therefore absolutely essential to confirm the presence and purity of exosomes by employing various tests, for example, morphology (electron microscopy), density (sucrose gradient centrifugation), protein composition (Western blotting), and size (nanoparticle tracking). Ultracentrifugation is an original and still most widely used protocol for exosome purification. Yet its clinical use has been severely hampered by the tedious and time-consuming procedure. Moreover, as density gradient setup may vary among individuals and labs, there is also a need to ensure greater accuracy in size and concentration measurement. Recent advances in exosome proteome allow the rapid development of an immunological approach for the

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Table 1 Exosomal cargo of ovarian cancer-derived exosomes. Example of exosomal cargo Antigen presenting MHC class I and II molecules Surface molecules EpCAM L1CAM Integrin α2, α3, α5, αV, β4 Sialoglycoproteins N-glycans Proteases ADAM10 ADAM15 ADAM17 MMP1, 10, 14, 15 Tetraspanins Tetraspanin 4, 6, 9, 14, 15 CD9 CD63 Heat-shock proteins Hsp27, 60 Hsp22, 70

Origin

Possible biological significance

References

OVCAR-3, SKOV-3 and AD10 cells

Modulate immune response

[19]

Malignant ascites derivedexosomes OVMz and SKOV-3ip cells OVCAR-3 and IGROV1 cells SKOV-3 cell SKOV-3 cell

Interact with recipient cells

[20]

OVMz and SKOV-3ip cells MDAH2774 cell OVMz and SKOV-3ip cells OVCAR-3 and IGROV1 cells

Proteolytic cleavage and modulation of the extracellular matrix

[21,24] [25] [21] [22]

OVCAR-3 and IGROV1 cells Malignant ascites derivedexosomes OVCAR-3 and IGROV1 cells

Exosomal cargo sorting and target cell selection

[22] [26]

OVCAR-3 and IGROV1 cells Malignant ascites derivedexosomes Hsp90 Malignant ascites derivedexosomes MVB formation, membrane transport and fusion Tsg101 OVCAR-3 and IGROV1 cells Rab 7, 11, 13, 14, 35, 43 OVCAR-3 and IGROV1 cells Rap 1A, 1B, 2A, 2B, 2C OVCAR-3 and IGROV1 cells Annexin A1, A5 Malignant ascites derivedexosomes Annexin A2, A4, A6, A11 OVMz and SKOV-3ip cells miRNA miR-214, miR-140, miR-147, Serum from normal control, patients of benign disease and miR-135b, miR-205, miR-150, patients of ovarian cancer, as well miR-149, miR-370, miR-206, as primary ovarian tumor cell miR-197, miR-634, miR-485-5p, cultures miR-612, miR-608, miR-202, miR-373, miR-324-3p, miR-103, miR-593, miR-574, miR-483, miR-527, miR-603, miR-649, miR-18a, miR-595, miR-193b, miR-642, miR-557, miR-801, let-7e miR-21, miR141, miR200a, miR-200c, miR-200b, miR-203, miR-205, miR-214 let-7a-f and miR-200a-c SKOV-3 and OVCAR-3 cells

[21] [22] [23] [23]

[22] Intercellular communication and modulation of immunity

[22] [27] [28]

Intracellular trafficking and exosome biogenesis

[22] [22] [22] [26] [22]

Up-regulated in ovarian tumor derived-exosomes when compared to cells

[29]

Significantly elevated in ovarian cancer when compared to benign disease

Correlate with ovarian cancer invasiveness

[30]

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isolation of exosomes using specific surface markers. However, a robust platform for large-scale analysis still awaits further investigation. Large amounts of protein and/or RNA have been identified from the isolated exosomes. These exosomal cargos are of key interest to researchers because they could help to (1) understand the biogenesis of exosomes so to develop effective isolation and identification methods, (2) reflect the identity of the originated cell so to be used as biological markers, and (3) mediate the observed biological functions that could be target of functional blocking therapy. To determine if a certain exosomal protein or RNA is specific to a certain cell type or disease state, a large sample size is necessary. However, it may be difficult for a single research group to obtain the required materials. As a result, free web-based databases were set up to allow researchers to share and compare their Q4 findings; one example is ExoCarta (www.exocarta.org) [18]. To date, there are 13,333 protein entries, 2375 mRNA entries and 194 lipid entries gathered from 146 studies on the ExoCarta database. The function of exosomes seems to be dependent on its protein and/or RNA cargo. Some of the reported cargos found on ovarian cancer-derived exosomes and their potential biological significances

are listed in Table 1. By profiling the exosomal cargo, it is possible to predict the potential function(s) of the isolated exosome. However, several intriguing questions remain. For example, how can exosomes participate in many different cellular functions and respond to different stimuli? One possible explanation is that an exosome is capable of mediating multiple biological functions. Another possibility, and one that is supported by recent evidence, is that heterogeneous populations of exosomes are being produced such as apical and basolateral surfaces [17]. Yet, how these proteins and/ or RNAs are specifically targeted to different exosomes still awaits further investigation. Whether a similar function can be achieved by freshly isolated (primary) exosomes remains to be determined. Roles in ovarian tumor development and progression Exosomes appear to be a new and powerful signal mediator between cancer cells and their microenvironment [31]. Major cell types in the tumor microenvironment include stromal cells, endothelial cells, and infiltrating immune cells, all of which communicate with cancer cells. The major non-cellular component of the tumor microenvironment is the extracellular matrix. A wide range of

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Table 2 Exosomes in ovarian cancer. Origin

Extraction method

Identification method

Biological function

References

CP70 cell

Differential centrifugation

Not shown

[33]

Effusion supernatant of ovarian cancer patients SKOV-3 and OVCAR-3 cells

Commercial kit

Not shown

Differential centrifugation

Contribute to cisplatin resistance in a miR-21-3p dependent manner Induce larger tumor burden, infiltrative tumors and shorter survival in vivo Release of exosomes correlates with invasive potential Induce cytokine secretion

[35]

[34]

Ascites of ovarian cancer patients Plasma samples from ovarian cancer patients SKOV-3/Cis cell

Differential centrifugation

Electron microscopy, Western blot and nanoparticle tracking analysis Western blot and nanoparticle tracking analysis Electron microscopy

Differential centrifugation

Electron microscopy and Western blot

SKOV-3 and OVCAR-3 cells

Differential centrifugation

Not shown

Ascites of ovarian cancer patients Ascites of ovarian cancer patients OVMz, SKOV-3ip cells, ascites and serum of ovarian cancer patients GG, SKOV-3, M130 cells and ascites of ovarian cancer patients OVMz and SKOV-3ip cells

Differential centrifugation

Western blot

Distinguishable exosomal protein profile, could be potential biomarker Secrete annexin A3 that may be associated with platinum resistance Induce characteristics of tumor associated myofibroblasts in mesenchymal stem cells Prevent binding of antibodies to cancer cells

Differential centrifugation

Electron microscopy and Western blot

Impair the anti-tumor immunity

[11]

Differential centrifugation

Sucrose-density gradient centrifugation, Western blot and cytofluorographic analysis Sucrose-density gradient centrifugation and Western blot

Promote tumor grow in vivo

[26]

Express gelatinolytically active protein that may degrade extracellular matrix

[20]

Differential centrifugation

Sucrose-density gradient centrifugation and Western blot

[21]

Differential centrifugation

Sucrose-density gradient centrifugation and Western blot Not shown

Express proteolytically active ADAM10 and ADAM17 and mediate ectodomain cleavage of L1 and CD44 Ectodomain cleavage of L1 adhesion molecule Expel cisplatin

[39]

Western blot

Induce apoptosis of T cell

[40]

OVMz and ascites of ovarian cancer patients Ovarian carcinoma 2008 cells and the 2008/C13*5.25 subline Ascites of ovarian cancer patients

Differential centrifugation

Differential centrifugation

Differential centrifugation

Chromatography or differential centrifugation

[30] [9]

[36] [37] [10]

[38]

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biological functions includes, but is not limited to, angiogenesis, metastasis, chemoresistance, and immune responses may be involved, some of which are reviewed by Beach et al. [32]. Table 2 summarizes the functions of exosomes from both ascitic samples of ovarian cancer patients and cancer cell lines. Stromal cells Tumor metastasis is also facilitated through the formation of premetastatic niche. Melanoma-derived exosomes have been shown to play an important role in altering bone marrow progenitor cells to support the growth and metastasis of tumor in vivo [41]. In ovarian cancer, tumor-derived exosomes could activate adipose tissuederived mesenchymal stem cells to tumor-supporting myofibroblasts, contributing to tumor progression [37,42]. Thus exosomes could be an important mediator between tumor and their microenvironment in the establishment of pre-metastatic niche. Endothelial cells Tumor angiogenesis is one of the vital components of successful tumor development. It is a multistep process involving endothelial cell growth, migration, and differentiation [43]. Various studies demonstrated an interaction between cancer exosomes and endothelial cells [44,45]. Our recent data suggested a proangiogenic effect of exosomes from ovarian cancer cells, more importantly; such effect can also be observed in exosomes from patient samples (unpublished observations). Further exploring how these tumor-derived exosomes interact with endothelial cells could be particularly attractive as both the progressive growth of ovarian cancer and the formation of ascitic fluid are critically dependent on angiogenesis and the extent of which is a significant indicator of poor prognosis.

Immune cells Immune cells such as natural killer cells, dendritic cells, and T cells are important mediators of anti-tumor response. Exosomes in plasma of patients with ovarian cancer were found to express immunosuppressive factors such as IL-10 and TGFβ1 to promote T regulatory cell function and impair anti-tumor immunity [19]. More recently, malignant ascites-derived exosomes of ovarian cancer patients have been observed to induce apoptosis of peripheral blood lymphocytes and dendritic cells [11], suggesting another mechanism of impairing the anti-tumor immunity by these tumorderived exosomes. Extracellular matrix Another characteristic of cancer cells is their ability to metastasize. To achieve this, cancer cells dissociate from the primary tumor, in which reduction of specific cell surface adhesion molecules is observed. Indeed, both adhesion molecules and cell surface-anchored proteases have been reported on exosomes. For example, exosomes from ovarian cancer ascites have also been shown to serve as a platform for L1-CAM cleavage [38]. Proteases also appear to have an important role in regulating extracellular matrices. Likewise, EpCAM and CD24, known to be overexpressed in ovarian carcinomas, are secreted in exosomes of cultured cell lines and malignant ascites, which contain gelatinolytic enzymes of the matrix metalloproteinase family [20]. Exosomes as predictive biomarkers in ovarian cancer While serum CA125 is a widely used marker for ovarian cancer, not all ovarian cancer patients have increased CA125 levels [46].

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Moreover, it may also be elevated in other cancers, such as breast, colon, and endometrial, and benign conditions, including endometriosis, uterine fibroids, and pelvic inflammatory disease, as well as in as many as 1% of healthy women. In a recent randomized study of around 70,000 asymptomatic women, CA125 screening together with transvaginal ultrasound did not seem to reduce the mortality rate, but rather result in unnecessary surgeries due to false-positive test results, in which some experienced serious complications [47]. Thus, there is a need to identify new diagnostic biomarkers for early detection and exosomes, which are rich reservoirs of tumor-specific proteins, have been especially important in the discovery of biomarkers. Exosomes possess several unique Q5 advantages, including (1) being extremely stable (under various conditions of freezing, cold-storage, and thawing for up to years), abundant (108–13 exosomes/ml plasma), tumor-specific, and their content correlates with tumor staging and treatment outcome. The presence of exosomes in blood and other body fluids such as urine also suggests an important advantage over invasive biopsies. By comparing exosomes captured in ovarian cancer patients with those in healthy individuals, there was a significant difference in both number and protein content [35]. TGFβ1 and MAGE3/6 are significantly more prevalent in malignant ovarian tumors than in benign lesions [35]. For screening biomarkers, claudin 4-positive exosomes were present in the plasma samples from 32 out of 63 ovarian cancer patients but only 1 out of 50 healthy individuals, raising the possibility that Q6 it could be used as a highly sensitive and specific indicator [48]. Other markers such as CD24 and EpCAM being expressed by ovarian carcinomas are exploited for the potential for diagnostics [49]. Chemoresistance remains one of the major obstacles in cancer therapy. Exosomes isolated from a cisplatin-resistant human ovarian cancer cell line, 2008/C13*5.25 subline, have been shown to have a high level of cisplatin export transporters that are able to export cisplatin through an exosomal pathway [39]. More recently, exosome microRNAs (miRNAs) were found to be linked to cisplatin resistance in ovarian cancer cells [33]. Thus, their presence could be a useful prognostic marker to predict the therapeutic response to cisplatin treatment in patients with ovarian cancer. Besides proteins, exosomes also contain RNAs. Previously, the use of miRNA profile was shown to be highly informative for diagnosis of cancer [50]. Using miR021, miR141, miR200a, miR200c, miR200b, miR-205 and miR-214 as diagnostic miRNAs, the exosomal miRNA profiles highly reflect the tumor miRNA profiles. More importantly, the levels of these miRNAs in exosomes from plasma of ovarian cancer patients were significantly higher when compared to benign disease and could not be detected in normal controls, and differences in miR-200c and miR-214 expression with tumor stages [29], suggesting that the exosomal miRNA profile may not be limited to be used as a marker for early detection, but also in staging of ovarian carcinomas. The development of a simple and sensitive method has been challenging, in part mainly due to the time-consuming procedure in (1) isolating and purifying exosomes and (2) analyzing protein or RNA-based biomarkers on exosomes. Several commercial companies have already launched the development of an exosomebased diagnostic platform. Validation studies have shown promising results for successful differentiation of serum from ovarian cancer patients against non-cancer controls. The tested sera also include samples from glioblastoma, melanoma, colorectal cancer and pancreatic cancer, suggesting that the potential use of exosomes as a diagnostic biomarker is not limited to a single type of cancer, but is relevant to multiple tumor types (http://www.exosomesciences .com/newslist.aspx?newsid=7). There also seems to be a correlation between exosome content and clinical outcome. A recent study that examines ovarian cancer patients’ exosomal proteins before and after chemotherapy has shown that the exosome levels were relatively unchanged in patients who were irresponsive to

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chemotherapy, whereas significantly altered levels were observed in responders, suggesting that the protein content of exosomes may be useful in predicting treatment response [35]. Recently, exosomes were shown not only to serve as vehicles for the transport of miRNAs, but also to process precursor miRNAs into mature ones [51]. It has been reported that the RNase III gene family member Dicer, a key component of the miRNA processing machinery, is present in some tumor-derived exosomes and triggers the transformation of nontumorigenic cells into tumor forming ones through altering the transcriptome of recipient cells [51], opening new opportunities for the development of exosome-based diagnosis and therapies. The advancement in exosome proteomics enables the rapid development of immunological isolation of exosomes via specific exosomal proteins. Together with fluorescence activated cell sorting, highly pure and intact exosomes can be captured [52]. Alternatively, the successful isolation of exosomes with size-exclusion chromatography using syringe packed with Sepharose [53]. Such method is cheap and quick, but may need further optimization to increase vesicle recovery. More recently, microfluidic approaches allow a more rapid and cost effective way of isolating exosomes from a small amount of body fluids [54]. These microfluidic extraction methods include the use of immunological separation [55], physical sieving [56] or porous trapping [56]. There are also platforms to incorporate on-chip analysis capabilities. For example, the use of a PDMS chip that allows integrated exosome enrichment, protein processing and immunoassay detection via a specially designed cascading microfluidic circuit [57]. More recently, the development of a new surface-plasmon-resonance platform termed nano-plasmonic exosome (nPLEX) [49]. Both devices enable direct proteomic analysis of captured exosomes and can be used to differentiate exosomespecific protein signature between healthy donor and cancer patients, including ovarian cancer [49,57]. Another example is a microfiltration device that couples microfluidics and magnetic sensing technologies, in which extracellular vesicles were isolated by size, and label with magnetic nanoparticles. It allows efficient quantification of extracellular vesicles as well as average expression level of target markers, in which only small volumes of blood sample are needed [58]. Table 3 summarizes the features of these exosome capturing devices. Exosomes as potential therapeutic targets in ovarian cancer Current clinical applications of exosome research can be categorized into three directions, (1) exosome as a therapeutic target, (2) exosome-based immunotherapy, and (3) exosome-mediated delivery. Based on the US National Institute of Health’s clinical trials database (clinicalstrials.gov), there are several ongoing exosomebased trials that are anticipated to add important pieces of information in the near future. Exosome as a therapeutic target Several studies have evaluated the selective target elimination of circulating exosomes to treat cancer. For example, the use of a preexisting adaptive dialysis-like affinity platform technology device, in which exosomes bearing pathogenic antigens can be specifically bound and removed by their corresponding antibodies or affinity agents [59]. In vitro studies have successfully demonstrated removal of cancer-derived exosomes from patient blood using a Hemopurifier (http://www.aethlonmedical.com/products/hemopurifier/index.html). In collaboration with Sarcoma Oncology Center in California, an ex vivo study has been carried out to further elucidate its effectiveness in advanced stage patients for different types of cancer (http://www.aethlonmedical.com/products/hemopurifier/index.html). Another therapeutic avenue involves the targeting of exosome

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Table 3 Exosome capturing devices. Exosome devices

Capturing principles

Tested samples

Sepharose CL-2B column

Size-exclusion chromatography

Platelet-free supernatant

Features

PDMS chip

Immunoprecipitation with magnetic beads

Non-small-cell lung cancer and ovarian cancer plasma samples

• • • • •

Nano-plasmonic exosome (nPLEX) sensor

Surface plasmon resonance

Ascites of ovarian cancer patients

• •

Magnetic nanosensor

Membrane filters and magnetic sensing technologies

Packed red blood cell units

• • •

Hemopurifier

Specific antibodies or affinity agents

Colorectal, lymphoma, melanoma, ovarian and breast cancer samples

• •

Simple apparatus Low cost Quick isolation Diversified choice of immunocapturing antibodies Streamlined chemifluorescence-assisted sandwich immunoassay Highly sensitive, label-free, rapid and multiplexed protein analysis Sensor can be functionalized with different antibodies Quick Small input volume Quantify microvesicle concentration and average protein expression Commercially available Ongoing ex vivo evaluation on advanced-stage cancer patients

References [53]

[57]

[49]

[58]

[59]

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biogenesis. Rab family members are key players in the generation of exosomes. For example, Rab5 plays an important role in governing the biogenesis of MVBs and early endosome formation, and Rab27a regulates the fusion of MVBs with the plasma membrane, releasing exosomes into the extracellular space [60]. Targeted inhibition of Rab27a was found to lead to a dramatic disappearance of endosome-associated exosomes, whereas CD9- and Mfge8positive vesicles were unaffected, suggesting that exosome release is in part dependent on Rab27a, and that it could be a promising target in cancer therapy. Exosome-based immunotherapy Exosomes have also been shown to generate an anti-tumor immune response. With the evidence that ovarian cancer is immunogenic, exosome-based immunotherapy should be further pursued [61]. Based on the most studied functions of exosomes in adaptive immunity, which was first reported by Raposo et al. [62], active research was carried out to elucidate the possibility of utilizing exosomes as cell-free antigen presenting vectors in stimulating in vivo T-cell associated immunity to achieve both long-term and tumor-specific protection. The ability of dendritic cell-derived exosomes to present peptide-loaded MHC molecules has entered phase I clinical trials in France [63]. Treatment of dendritic cellderived exosomes preexposed to the melanoma-associated antigen 3 (MAGE3) in patients with metastatic melanoma has been shown to be feasible with no grade II toxicity, with one patient showing partial response [63]. In another phase I clinical trial conducted on non-small-cell lung cancer patients, dendritic cell-derived exosomes loaded with the same antigen were found to prolong disease stabilization [64]. In ovarian cancer, the 5-year survival rate of patients with tumor infiltrating T-cells was significantly higher than those without such T-cells [65]. While clinical trial is still pending, it is anticipated that, in combination with a Toll-like receptor 3 agonist, these ascites-derived exosomes could improve progression-free survival of advanced ovarian cancer patients undergoing chemotherapy [66]. Exosome-mediated delivery Due to the low stability and bioavailability of chemopreventive agents such as curcumin, a large amount has to be administered to achieve therapeutic effects. Plant-derived exosomes have been shown to be able to increase the stability and bioavailability of these chemopreventive agents and are taken up by human intestinal cells

and immune cells effectively, suggesting that these foreign exosomes may be used as delivery vectors in treating human diseases [67,68]. Indeed, a phase I clinical trial is currently ongoing to evaluate the feasibility and effectiveness of these plant-derived exosomes to deliver curcumin to colon tissues (NCT01294072). Curcumin, together with triptolide, has previously been demonstrated to induce apoptosis of ovarian cancer cells [69], suggesting that this approach can be useful, alone or in combination with other drugs, for the treatment of ovarian cancer.

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Conclusion and future perspective Intensive research and many breakthrough discoveries in the past three decades have been made on exosomes. However, we are far from a complete understanding of the biology of these vesicles and many questions remain to be addressed. The limitation to isolate large quantities of pure exosomes could impact our overall study of exosomes, so it will be necessary in future studies to develop a rapid and efficient method. Devices which enable cost-effective, larger-scale purification, and efficient analysis of rare exosomal markers, await further development. High-throughput expression analysis will prove useful to unravel the molecular functions of exosomes and how the different signaling networks are orchestrated. Further evaluation will also be needed to characterize and differentiate the different subpopulations of exosomes and to determine the function of exosomes in vivo. There is also a need for real-time intravital imaging of exosomes dynamic to further the understanding of exosomes and also an avenue toward the detection and prognosis prediction of cancer. Exosomes could also serve as promising therapeutic targets. However, these therapies warrant further investigation using additional animal studies and large cohort clinical trials.

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Acknowledgements Our work is supported by the Hong Kong Research Grant Council General Research Fund (HKU781013M), Collaborative Research Fund (CUHK8/CRF/11R), and Theme-based Research Fund (T12-401/13R). A.S.T.W. is Croucher Senior Research Fellow.

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Conflict of interest There is no conflict of interest.

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Q11 References [1] B.T. Pan, R.M. Johnstone, Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor, Cell 33 (1983) 967–978. [2] R.M. Johnstone, M. Adam, J.R. Hammond, L. Orr, C. Turbide, Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes), J. Biol. Chem. 262 (1987) 9412–9420. [3] C. Théry, Exosomes: secreted vesicles and intercellular communications, F1000 Biol. Rep. 3 (2011) 15. [4] C. Théry, M. Ostrowski, E. Segura, Membrane vesicles as conveyors of immune responses, Nat. Rev. Immunol. 9 (2009) 581–593. [5] I. Parolini, C. Federici, C. Raggi, L. Lugini, S. Palleschi, A. De Milito, et al., Microenvironmental pH is a key factor for exosome traffic in tumor cells, J. Biol. Chem. 284 (2009) 34211–34222. [6] A.E. Morelli, A.T. Larregina, W.J. Shufesky, M.L. Sullivan, D.B. Stolz, G.D. Papworth, et al., Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells, Blood 104 (2004) 3257–3266. [7] H.C. Christianson, K.J. Svensson, T.H. van Kuppevelt, J.P. Li, M. Belting, Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 17380–17385. [8] K.J. Svensson, H.C. Christianson, A. Wittrup, E. Bourseau-Guilmain, E. Lindqvist, L.M. Svensson, et al., Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1, J. Biol. Chem. 288 (2013) 17713–17724. [9] N.P. Bretz, J. Ridinger, A.K. Rupp, K. Rimbach, S. Keller, C. Rupp, et al., Body fluid exosomes promote secretion of inflammatory cytokines in monocytic cells via Toll-like receptor signaling, J. Biol. Chem. 288 (2013) 36691–36702. [10] C. Battke, R. Ruiss, U. Welsch, P. Wimberger, S. Lang, S. Jochum, et al., Tumour exosomes inhibit binding of tumour-reactive antibodies to tumour cells and reduce ADCC, Cancer Immunol. Immunother. 60 (2011) 639–648. [11] P. Peng, Y. Yan, S. Keng, Exosomes in the ascites of ovarian cancer patients: origin and effects on anti-tumor immunity, Oncol. Rep. 25 (2011) 749–762. [12] M.S. Stack, D.A. Fishman, Ovarian Cancer, vol. 149, Cancer Treatment and Research, Springer., US, 2010. [13] J.B. Le Pecq, Dexosomes as a therapeutic cancer vaccine: from bench to bedside, Blood Cells Mol. Dis. 35 (2005) 129–135. [14] S. Patel, A. Mehta-Damani, H. Shu, J.B. Le Pecq, An analysis of variability in the manufacturing of dexosomes: implications for development of an autologous therapy, Biotechnol. Bioeng. 92 (2005) 238–249. [15] M. Morello, V.R. Minciacchi, P. de Candia, J. Yang, E. Posadas, H. Kim, et al., Large oncosomes mediate intercellular transfer of functional microRNA, Cell Cycle 12 (2013) 3526–3536. [16] D. Di Vizio, J. Kim, M.H. Hager, M. Morello, W. Yang, C.J. Lafargue, et al., Oncosome formation in prostate cancer: association with a region of frequent chromosomal deletion in metastatic disease, Cancer Res. 69 (2009) 5601– 5609. [17] B.J. Tauro, D.W. Greening, R.A. Mathias, S. Mathivanan, H. Ji, R.J. Simpson, Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids, Mol. Cell. Proteomics 12 (2013) 587–598. [18] R.J. Simpson, H. Kalra, S. Mathivanan, ExoCarta as a resource for exosomal research, J. Extracell. Vesicles 1 (2012). [19] M. Szajnik, M. Czystowska, M.J. Szczepanski, M. Mandapathil, T.L. Whiteside, Tumor-derived microvesicles induce, expand and up-regulate biological activities of human regulatory T cells (Treg), PLoS ONE 5 (2010) e11469. [20] S. Runz, S. Keller, C. Rupp, A. Stoeck, Y. Issa, D. Koensgen, et al., Malignant ascites-derived exosomes of ovarian carcinoma patients contain CD24 and EpCAM, Gynecol. Oncol. 107 (2007) 563–571. [21] A. Stoeck, S. Keller, S. Riedle, M.P. Sanderson, S. Runz, F. Le Naour, et al., A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44, Biochem. J. 393 (2006) 609–618. [22] B. Liang, P. Peng, S. Chen, L. Li, M. Zhang, D. Cao, et al., Characterization and proteomic analysis of ovarian cancer-derived exosomes, J. Proteomics 80 (2013) 171–182. [23] C. Escrevente, N. Grammel, S. Kandzia, J. Zeiser, E.M. Tranfield, H.S. Conradt, et al., Sialoglycoproteins and N-glycans from secreted exosomes of ovarian carcinoma cells, PLoS ONE 8 (2013) e78631. [24] P. Gutwein, S. Mechtersheimer, S. Riedle, A. Stoeck, D. Gast, S. Joumaa, et al., ADAM10-mediated cleavage of L1 adhesion molecule at the cell surface and in released membrane vesicles, FASEB J. 17 (2003) 292–294. [25] H.D. Lee, Y.H. Kim, B.H. Koo, D.S. Kim, Ectodomain of ADAM15 is shed from secretory exosomes, BMB Rep. (2014) pii: 2901. [26] S. Keller, A.K. König, F. Marmé, S. Runz, S. Wolterink, D. Koensgen, et al., Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes, Cancer Lett. 278 (2009) 73–81. [27] F. Andre, N.E. Schartz, M. Movassagh, C. Flament, P. Pautier, P. Morice, et al., Malignant effusions and immunogenic tumour-derived exosomes, Lancet 360 (2002) 295–305. [28] M.P. Bard, J.P. Hegmans, A. Hemmes, T.M. Luider, R. Willemsen, L.A. Severijnen, et al., Proteomic analysis of exosomes isolated from human malignant pleural effusions, Am. J. Respir. Cell Mol. Biol. 31 (2004) 114–121. [29] D.D. Taylor, C. Gercel-Taylor, MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer, Gynecol. Oncol. 110 (2008) 13– 21.

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[30] M. Kobayashi, C. Salomon, J. Tapia, S.E. Illanes, M.D. Mitchell, G.E. Rice, Ovarian cancer cell invasiveness is associated with discordant exosomal sequestration of Let-7 miRNA and miR-200, J. Transl. Med. 12 (2014) 4. [31] R. Ge, E. Tan, S. Sharghi-Namini, H.H. Asada, Exosomes in cancer microenvironment and beyond: have we overlooked these extracellular messengers?, Cancer Microenviron. 5 (2012) 323–332. [32] A. Beach, H.G. Zhang, M.Z. Ratajczak, S.S. Kakar, Exosomes: an overview of biogenesis, composition and role in ovarian cancer, J. Ovarian Res. 7 (2014) 14. [33] R.C. Pink, P. Samuel, D. Massa, D.P. Caley, S.A. Brooks, D.R. Carter, The passenger strand, miR-21-3p, plays a role in mediating cisplatin resistance in ovarian cancer cells, Gynecol. Oncol. 137 (2015) 143–151. [34] O. Vaksman, C. Tropé, B. Davidson, R. Reich, Exosome-derived miRNAs and ovarian carcinoma progression, Carcinogenesis 35 (2014) 2113–2120. [35] M. Szajnik, M. Derbis, M. Lach, P. Patalas, M. Michalak, H. Drzewiecka, et al., Exosomes in plasma of patients with ovarian carcinoma: potential biomarkers of tumor progression and response to therapy, Gynecol. Obstet. (Sunnyvale) 3 (Suppl. 4) (2013). [36] J. Yin, X. Yan, X. Yao, Y. Zhang, Y. Shan, N. Mao, et al., Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients, J. Cell. Mol. Med. 16 (2012) 337–348. [37] J.A. Cho, H. Park, E.H. Lim, K.H. Kim, J.S. Choi, J.H. Lee, et al., Exosomes from ovarian cancer cells induce adipose tissue-derived mesenchymal stem cells to acquire the physical and functional characteristics of tumor-supporting myofibroblasts, Gynecol. Oncol. 123 (2011) 379–386. [38] P. Gutwein, A. Stoeck, S. Riedle, D. Gast, S. Runz, T.P. Condon, et al., Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells, Clin. Cancer Res. 11 (2005) 2492–2501. [39] R. Safaei, B.J. Larson, T.C. Cheng, M.A. Gibson, S. Otani, W. Naerdemann, Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drugresistant human ovarian carcinoma cells, Mol. Cancer Ther. 4 (2005) 1595–1604. [40] D.D. Taylor, C. Gercel-Taylor, Tumour-derived exosomes and their role in cancer-associated T-cell signalling defects, Br. J. Cancer 92 (2005) 305–311. [41] H. Peinado, M. Alecˇkovic´, S. Lavotshkin, I. Matei, B. Costa-Silva, G. Moreno-Bueno, et al., Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET, Nat. Med. 18 (2012) 883–891. [42] R. Lis, C. Touboul, C.M. Raynaud, J.A. Malek, K. Suhre, M. Mirshahi, et al., Mesenchymal cell interaction with ovarian cancer cells triggers pro-metastatic properties, PLoS ONE 7 (2012) e38340. [43] J. Folkman, K. Watson, D. Ingber, D. Hanahan, Induction of angiogenesis during the transition from hyperplasia to neoplasia, Nature 339 (1989) 58–61. [44] T. Umezu, K. Ohyashiki, M. Kuroda, J.H. Ohyashiki, Leukemia cell to endothelial cell communication via exosomal miRNAs, Oncogene 32 (2013) 2747–2755. [45] P. Kucharzewska, H.C. Christianson, J.E. Welch, K.J. Svensson, E. Fredlund, M. Ringnér, et al., Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 7312–7317. [46] B. Zhang, F.F. Cai, X.Y. Zhong, An overview of biomarkers for the ovarian cancer diagnosis, Eur. J. Obstet. Gynecol. Reprod. Biol. 158 (2011) 119–123. [47] E.E. Partridge, R.T. Greenlee, T.L. Riley, J. Commins, L. Ragard, J.L. Xu, et al., Assessing the risk of ovarian malignancy in asymptomatic women with abnormal CA 125 and transvaginal ultrasound scans in the prostate, lung, colorectal, and ovarian screening trial, Obstet. Gynecol. 121 (2013) 25–31. [48] J. Li, C.A. Sherman-Baust, M. Tsai-Turton, R.E. Bristow, R.B. Roden, P.J. Morin, Claudin-containing exosomes in the peripheral circulation of women with ovarian cancer, BMC Cancer 9 (2009) 244. [49] H. Im, H. Shao, Y.I. Park, V.M. Peterson, C.M. Castro, R. Weissleder, et al., Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor, Nat. Biotechnol. 32 (2014) 490–495. [50] J. Lu, G. Getz, E.A. Miska, E. Alvarez-Saavedra, J. Lamb, D. Peck, et al., MicroRNA expression profiles classify human cancers, Nature 435 (2005) 834–838. [51] S.A. Melo, H. Sugimoto, J.T. O’Connell, N. Kato, A. Villanueva, A. Vidal, et al., Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis, Cancer Cell 26 (2014) 707–721. [52] M.F. Peterson, N. Otoc, J.K. Sethi, A. Gupta, T.J. Antes, Integrated systems for exosome investigation, Methods (2015) pii: S1046-2023(15)00161-00169. [Epub ahead of print]. [53] A.N. Böing, E. van der Pol, A.E. Grootemaat, F.A. Coumans, A. Sturk, R. Nieuwland, Single-step isolation of extracellular vesicles by size-exclusion chromatography, J. Extracell. Vesicles 3 (2014). [54] A. Liga, A.D. Vliegenthart, W. Oosthuyzen, J.W. Dear, M. Kersaudy-Kerhoas, Exosome isolation: a microfluidic road-map, Lab Chip 15 (2015) 2388–2394. [55] C. Chen, J. Skog, C.H. Hsu, R.T. Lessard, L. Balaj, T. Wurdinger, et al., Microfluidic isolation and transcriptome analysis of serum microvesicles, Lab Chip 10 (2010) 505–511. [56] R.T. Davies, J. Kim, S.C. Jang, E.J. Choi, Y.S. Gho, J. Park, Microfluidic filtration system to isolate extracellular vesicles from blood, Lab Chip 12 (2012) 5202– 5210. [57] M. He, J. Crow, M. Roth, Y. Zeng, A.K. Godwin, Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology, Lab Chip 14 (2014) 3773–3780. [58] J. Rho, J. Chung, H. Im, M. Liong, H. Shao, C.M. Castro, et al., Magnetic nanosensor for detection and profiling of erythrocyte-derived microvesicles, ACS Nano 7 (2013) 11227–11233. [59] A.M. Marleau, C.S. Chen, J.A. Joyce, R.H. Tullis, Exosome removal as a therapeutic adjuvant in cancer, J. Transl. Med. 10 (2012) 134.

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[60] J.H. Hurley, G. Odorizzi, Get on the exosome bus with ALIX, Nat. Cell Biol. 14 (2012) 654–655. [61] L.E. Kandalaft, G.T. Motz, J. Duraiswamy, G. Coukos, Tumor immune surveillance and ovarian cancer: lessons on immune mediated tumor rejection or tolerance, Cancer Metastasis Rev. 30 (2011) 141–151. [62] G. Raposo, H.W. Nijman, W. Stoorvogel, R. Liejendekker, C.V. Harding, C.J. Melief, et al., B lymphocytes secrete antigen-presenting vesicles, J. Exp. Med. 183 (1996) 1161–1172. [63] B. Escudier, T. Dorval, N. Chaput, F. André, M.P. Caby, S. Novault, et al., Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derivedexosomes: results of the first phase I clinical trial, J. Transl. Med. 3 (2005) 10. [64] M.A. Morse, J. Garst, T. Osada, S. Khan, A. Hobeika, T.M. Clay, et al., A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer, J. Transl. Med. 3 (2005) 9.

[65] L. Zhang, J.R. Conejo-Garcia, D. Katsaros, P.A. Gimotty, M. Massobrio, G. Regnani, et al., Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer, N. Engl. J. Med. 348 (2003) 203–213. [66] H. Navabi, D. Croston, J. Hobot, A. Clayton, L. Zitvogel, B. Jasani, et al., Preparation of human ovarian cancer ascites-derived exosomes for a clinical trial, Blood Cells Mol. Dis. 35 (2005) 149–152. [67] D. Sun, X. Zhuang, X. Xiang, Y. Liu, S. Zhang, C. Liu, et al., A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes, Mol. Ther. 18 (2010) 1606–1614. [68] S. Ju, J. Mu, T. Dokland, X. Zhuang, Q. Wang, H. Jiang, et al., Grape exosome-like nanoparticles induce intestinal stem cells and protect mice from DSS-induced colitis, Mol. Ther. 21 (2013) 1345–1357. [69] Y.Y. Cai, W.P. Lin, A.P. Li, J.Y. Xu, Combined effects of curcumin and triptolide on an ovarian cancer cell line, Asian Pac. J. Cancer Prev. 14 (2013) 4267–4271.

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