Salivary exosomes as potential biomarkers in cancer

Oral Oncology 84 (2018) 31–40

Contents lists available at ScienceDirect

Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology

Review

Salivary exosomes as potential biomarkers in cancer a,b

Soumyalekshmi Nair

a,b,c

, Kai Dun Tang

, Liz Kenny

d,e,f

, Chamindie Punyadeera

a,b,c,⁎

T

a

The School of Biomedical Sciences, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, Australia Translational Research Institute, Brisbane, Australia The Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, Woolloongabba, Queensland, Australia d School of Medicine, University of Queensland, Queensland, Australia e Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia f Central Integrated Regional Cancer Service, Queensland Health, Queensland, Australia b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Extracellular vesicles Salivary exosomes Tumour progression Metastasis

Over the past decade, there has been emerging research in the field of extracellular vesicles, especially those originating from endosomes, referred to as ‘exosomes. Exosomes are membrane-bound nanovesicles secreted by most cell types upon fusion of multivesicular bodies (MVBs) to the cell plasma membrane. These vesicles are present in almost all body fluids such as blood, urine, saliva, breast milk, cerebrospinal and peritoneal fluids. Exosomes participate in intercellular communication by transferring the biologically active molecules like proteins, nucleic acids, and lipids to neighboring cells. Exosomes are enriched in the tumour microenvironment and growing evidence demonstrates that exosomes mediate cancer progression and metastasis. Given the important biological role played by these nanovesicles in cancer pathogenesis, these can be used as ideal noninvasive biomarkers in detecting and monitoring tumours as well as therapeutic targets. The scope of the current review is to provide an overview of exosomes with a special focus on salivary exosomes as potential biomarkers in head and neck cancers.

Introduction The intercellular communication between cancer cells and surrounding stromal cells is crucial for modulating tumorigenesis as well as for the development and progression of tumour metastasis. Understanding the molecular mechanisms involved in this cellular cross-talk provides new insights into the identification of valuable biomarkers as well as therapeutic targets that can stop tumour progression [1]. Cell to cell communication by extracellular vesicles is a burgeoning area of research and these are considered key players in mediating communication between tumour cells and healthy, normal cells [2]. Exosomes are included as part of “liquid biopsy” together with circulating tumour cells and circulating tumour DNA. Broadly, extracellular vesicles (EVs) are categorized into three sub-populations based on their cellular origin and morphology; namely, exosomes, microvesicles (MVs) and apoptotic bodies. Exosomes are nano-sized vesicles (30–100 nm in diameter), originating from the endosomal pathway and secreted into the surrounding extracellular space by exocytosis. Exosomes have been isolated from a wide number of body fluids such as

blood, urine, saliva, breast milk, cerebrospinal and peritoneal fluids [3,4]. MVs (100–1000 nm in diameter) are larger than exosomes and are formed by the direct outward budding of the plasma membrane. Like exosomes, MVs are covered by phospholipid bilayer and contain molecular cargo including proteins and nucleic acids. The apoptotic bodies (1–5 µm in diameter) are released as blebs (protrusions of the cell membrane) when cells undergo apoptosis [5]. The recent advancement in understanding EV- mediated cell signaling has significantly contributed in elucidating the molecular mechanisms of cancer progression and has expanded the frontiers of cancer biology. Among the different sub-population of EVs, exosomes have been implicated as the most important indicators of cancer cell microenvironment and novel mediators of oncogenic cell communication [6]. Growing evidence demonstrates that there exist specialized sorting mechanisms that package, specific signaling molecules into exosomes. Enrichment of tumour-specific molecules in exosomes promote tumour progression as well as selectively target these vesicles to specific celltypes mediating organotropic metastasis [7,8] This review summarizes exosome-mediated cell signaling as vital players in mediating cancer progression and metastasis, with special focus on the impact and utility

⁎ Corresponding author at: The School of Biomedical Sciences, Room 603D, Institute of Health and Biomedical Innovations, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, QLD 4059, Australia. E-mail address: [email protected] (C. Punyadeera).

https://doi.org/10.1016/j.oraloncology.2018.07.001 Received 10 November 2017; Received in revised form 21 May 2018; Accepted 9 July 2018 1368-8375/ © 2018 Published by Elsevier Ltd.

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processes like fertilization, embryo implantation and early pregnancy [26]. Recent studies have revealed that exosomes are important mediators of intercellular communication by transferring their molecular cargo between cells and potentially donating their own cytoplasm to the recipient cell [4,27]. The proteins and different types of RNAs including mRNAs and non-coding RNAs shuttle by the exosomes are functional and can influence the transcriptome of the recipient cell, thereby having an impact on the phenotype and behavior of donor/ recipient cells [27,28]. Current literature illustrates that the molecular cargo encapsulated in exosomes like proteins and RNAs which are specific to cancer cells can be identified and validated as biomarkers of tumours. The proteomic analysis of BRCA1 deficient breast cancer secretome identified non-invasive biomarkers of breast cancer [18]. Certain microRNAs (miRNAs) like miR-21 and miR-1246 are selectively enriched in breast cancer exosomes and can be used as indicators of breast cancer [29]. Also, proteomic analysis of urinary exosomes revealed that a significant number of proteins were differentially abundant in prostate cancer patients [30]. Logozzi et al. reported high levels of exosomes, expressing CD-63 and caveolin 1 in plasma of melanoma patients [31]. A cell surface glycoprotein, Glypican-1, specifically enriched in cancer exosomes has been reported as a specific marker of pancreatic cancer showing the potential for early detection of this cancer-type [32]. Furthermore, a tumour specific protein EGFvIII has been detected in the serum MVs of glioblastoma patients showing its importance as a biomarker of glioblastoma [33]. The potential exosome biomarkers that can be used in cancer is summarized in Table 1.

of salivary exosomes in the management of head and neck cancers. Exosome biogenesis and signaling Biogenesis of exosomes involves the inward budding of the late endosomal membrane giving rise to membrane-covered vesicles called MVBs. MVBs are 250–1000 nm in diameter and contain several intraluminal vesicles (ILVs) which are 30–100 nm in diameter. ILVs are formed from the endosomal membrane of MVBs, by segregation of cargo at the membrane followed by inward invagination, and release of a membrane vesicle containing a portion of cytosol into the intra-luminal space of MVBs. Some of these MVBs formed during the maturation of endosomes are directed to the lysosomal pathway for degradation, while some fuse with the plasma membrane leading to release of ILVs. The ILVs released into the extracellular milieu are called exosomes [9]. Several different molecules actively participate in different stages of exosome biogenesis. Endosomal Sorting Complex Required for Transport (ESCRT) is a highly conserved group of protein complexes involved in membrane modelling and formation of MVBs and ILVs. The common exosomal markers like ALIX (ALG-2-interacting protein X) and TSG101 (Tumour susceptibility gene 101 protein) are proteins present in the ESCRT machinery [10]. Also, ESCRT- independent mechanisms involving ceramide-induced aggregation of lipid microdomains leading to the formation of exosomes, has been implicated [11]. The tetraspanin proteins like CD-63 and CD-81 and the heat shock proteins like HSP-60, HSP-70 and HSP-90 are part of the ESCRT-independent mechanisms. The Rab family of GTPases are involved in the trafficking and docking of MVBs to the plasma membrane and fusion of the MVBs with the plasma membrane is mediated by soluble NSF-attachment protein receptor (SNARE) complex of proteins [12,13]. Depending on the mechanisms involved in their biogenesis and secretion, exosomes are heterogeneous in their morphology and its content represent the phenotype of the cell from which they originate. They are enclosed by the phospholipid bilayer from the cell and contain almost all the known molecules of the cell including proteins, nucleic acids and lipids. They can migrate a long distance from their cell of origin and can cross physiological barriers like blood-brain barrier [14,15]. Exosomes contain a vast array of biologically active molecules which typically reflects the content of the cell from which they originate. When released into the extracellular space, exosomes can act on the same cell giving autocrine action or proximally giving a paracrine action or can even act distally giving endocrine functions while transported by blood and lymph [16]. The biological signaling of the exosomes on their target cell is mediated by different mechanisms. These include (1) internalization of exosome by recipient cells by phagocytosis or endocytosis [17,18]; (2) attaching to target cell receptor and affecting downstream signaling by receptor-ligand interaction [19] or (3) fusing with the plasma membrane of the recipient cell and transferring their cargo into the cell [20]. All three modes of communication can affect the functions of the recipient cell and may change its behavior.

Exosome isolation Exosome isolation is an extensive and rapidly developing area of research. Different methods are available for the isolation and enrichment of exosomes from cell culture supernatant and different body fluids [34,35]. Usually, the body fluid or the cell culture supernatant will be differentially centrifuged at 2000g to remove the dead cells and then at 10,000g to remove the cellular debris and non-exosomal vesicles like MVs. Then, the supernatant containing exosomes is passed through a 200 nm filter which separates all larger particles of size above 200 nm (i.e. dead cells, apoptotic blebs and cellular debris from the smallvesicles). This is followed by any commonly used separation techniques such as ultracentrifugation at 100,000g; isopycnic centrifugation; immunoaffinity separation using antibodies to the surface markers of exosomes; size exclusion chromatography; ultrafiltration or polymerbased precipitation [36]. The ultracentrifugation separates exosomes on the basis of its sedimentation coefficient by spinning at high speed, greater than 100,000 g. It is the most widely used method and suitable for isolation of large volumes of the sample at low cost [37]. Density gradient centrifugation uses a gradient medium like sucrose or iodixanol to separate vesicles on the basis of their floatation densities. This is considered as ‘gold standard’ method for isolation of exosomes and ultracentrifugation combined with density gradient centrifugation is reported to give superior quality exosomal preparation for proteomic analysis [36]. Size exclusion chromatography is another method that gives high purity samples with minimal protein contamination but de-merited by less sample throughput and dilution of the final sample [38]. The easiest method of exosome isolation is the polymer-based precipitation in which exosomes are co-incubated with polymers like polyethylene glycol followed by low-speed centrifugation. This method gives a good yield of exosomes but possible co-isolation of non-exosomal particles affects the purity of preparation [35]. Immuno-affinity separation is the most specific method for the isolation of exosomes and capable of isolating even subpopulations of exosomes. A variety of platforms like immune beads, ELISA plates, modified chromatography columns and microfluidic devices have been developed to capture exosomes by targeting their surface markers [37]. Ultrafiltration is another method by

Functions of exosomes In the beginning, the exosomes were considered to be functioning as exporting or expelling unwanted cellular proteins from a cell [21]. Later, Raposo et al., reported that exosomes derived from antigenpresenting cells (APCs) have T cell stimulatory function [22]. Also, dendritic cell-derived exosomes were reported to elicit T cell-mediated anti-tumour immune response [23]. Since then exosomes have been classified to play extensive roles in antigen presentation as they express proteins involved in cell adhesion and co-stimulation like MHC class I and II molecules [24]. This immune stimulatory potential of exosomes has been explored in the development of exosomes based anti-tumour vaccines [25]. Additionally, exosomes play a significant role in modulating and attenuating immune response in normal physiological 32

Serum

Melanoma

33 miRNA Protein miRNA

Serum

Plasma Serum

Serum

Urine

Oesophageal squamous cell carcinoma Non-small cell lung cancer Nasopharyngeal carcinoma

Hepatocellular carcinoma

Bladder cancer

Protein

miRNA

miRNA

Serum

Colorectal cancer (CRC)

miRNA

Protein

Ascites fluid

Cervicovaginal lavages

Protein

Plasma

Cervical cancer

miRNA

Serum

Ovarian cancer

Protein

miRNA

Serum

Saliva

Pancreatobiliary tract cancer

EDIL-3 [147]

miR-718 [145]

NY-ESO-1, EGFR, PLAP, EpCAM and Alix [141] miR-24-3p [143]

miRNA-21 [139]

let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a [135]

miR-21, miR-146a[132]

L1CAM, CD24, ADAM10, and EMMPRIN[127]

Claudin-4[124]

miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR203, miR-205 and miR-214[120]

EGFRvIII[33]

miR-1246, miR-4644[118]

Survivin [111] miR-21, miR-1246[29] miR-373[113]

PCA-3 [108] TMPRSS2:ERG (fusion gene mRNA) [108]

Long non-coding RNA mRNA Protein miRNA

TM256, LAMTOR1, VATL, ADIRF [105]

MIA, S100 [103]

CD44v6, Tspan8, EpCAM, CD104 [99] Glypican I [32] miR-1246, miR-4644, miR-3976, miR-4306 [99]

Biomarker

Protein

Protein

miRNA

Protein

Type of exosome biomarker

Glioblastoma

Serum Plasma

Breast cancer

Urine

Serum

Pancreatic cancer

Prostate cancer

Biological fluid

Cancer

Table 1 Exosomal biomarkers in various cancer types.

Mutations in EGFR signaling pathway is associated with malignant transformation [142] miR-24- targets Jab1/CSN5 and mediates tumorigenesis and resistance to radiation [144] miR-718 targets the EGR-3 (Early Growth Response Protein 3) regulate proliferation, invasion and migration[146] Activate EGFR and mediated tumour progression[147]

miR-21 regulates CCL20 and regulate apoptosis [133] miR-146 regulate E5-E7 oncogenic proteins and cancer progression [134] Let-7 bind to KRAS and positive response to chemo drugs in metastatic CRC [136] Let-7 and miR-21 downregulate p53 miR-21 inactivates the TGF-β signaling [137] miR-23- regulate TGF-β signaling [138] miRNA-21- target PDCD4 and cause malignant transformation [140]

LAMTOR1- regulates mTOR signaling, endosomal assembly, cell proliferation [106] VATL- enhances invasion and metastatic capacity [107] PCA-3- regulates apoptosis and androgen receptor signaling [109] Promoters of TMPRSS2 drives the over-expression of ETS family transcription factor leading to oncogenic transformation [110] Survivin inhibits apoptosis and increases angiogenesis [112] miR-21 targets ANKRD46 in breast cancer cell lines [114] and associated with tumour progression, metastasis and poor patient survival [115] miR-1246 targets CCNG2 and promote cell proliferation, invasion and drug resistance [116] miR-373 targets several genes associated with invasion and metastasis and effect on breast cancer is controversial [117] miR-1246 targets CCNG2 and promote drug resistance and cancer stem cell-like properties [102] EGFRvIII mutation causes constitutive expression of EGFR, increase mitogenic factors including Akt, downregulate Bcl-2 and repress apoptosis [119] miR-21 targets Bcl-2, TPM1, PDCD4, maspin, PTEN and cause tumour progression [121] miR-200 – target ZEB1 and ZEB2 and regulate EMT [122] miR-205- is tumour suppressive and targets HER2 pathway [123] Claudin-4 enhances angiogenesis [125] and increase invasion and metastasis by increasing MMPs [126] EMMPRIN targets multiple pathways regulating proliferation, cell cycle, apoptosis, invasion and metastasis [128] ADAM10 cleaves the Nectin-4 protein leading to its release in circulation [129] L1CAM increases IL-1β and NF-kB levels and increase motility and invasion [130] CD24- phenotype is associated with cancer stem cell-like properties and enhanced chemo-resistance [131]

CD44v6 promotes tumour growth and metastasis by targeting MET and VEGFR-2 pathways [100] Tspan 8 promotes metastasis by associating with the integrins in ECM [101] miR-1246 target tumour suppressive gene CCNG2 and increase chemo-resistance and cancer stem-like properties [102] MIA interacts with the proteins in ECM and increases progression and metastasis of metastatic melanomas [104]

Reported biological function in the specific cancer type

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Table 2 The comparison of various exosome isolation methods. Isolation methods Ultracentrifugation

Technique and principle by high-speed centrifugation at or • Performed above 100,000g for 90–120 min to obtain the exosome pellet

particles on the basis of size, shape and • Separates density.

Advantages cost involved • Low for isolation from large • Suitable volumes • No chemical additives

Limitations

• Time-consuming labour-intensive • Highly sample throughput • Low exosome yield • Lesser contamination by microvesicles • Possible and other small non-vesicular particles varies with the physical • Efficiency properties like viscosity of the biological fluid

Isopycnic centrifugation

Chemical precipitation/ Polymer based precipitation

by high-speed centrifugation at or efficient in separating exosomes • Performed • Highly • Time-consuming above 100,000g in a cushion of specific gradient from particles of similar density like labour-intensive • Highly medium like sucrose/iodixanol. viral particles and protein aggregates Low sample throughput • particles on the basis of their size, exosome preparations of high exosome yield • Separates • Produce • Lesser shape and density in a specific gradient medium. purity contamination by the separating • Possible medium sample is incubated with polymers like laborious contamination by nonspecific • The • Less • Possible polyethylene glycol (PEG) and further particles and precipitating polymer No requirement of laborious equipment • precipitated by low-speed centrifugation or yield of exosomes of isolation varies with the • High • Efficiency filtration. manufacturer Scalable for larger sample volumes • water-excluding polymer interlinks/ • The intertwines the molecules of the solution thereby

• Ultrafiltration

• •

Size exclusion chromatography

Immuno-affinity capture

changing their solubility and expelling them out of the solution. Many exosome precipitation kits compatible with different body fluids are available commercially. The sample is passed through membrane filters with defined molecular weight or size exclusion limit Exosomes are separated based on their size

time consuming than • Less ultracentrifugation addition of chemicals • No • Do not need special equipment

sample is passed through a stationary phase pure exosomal preparations • The • Highly of porous beads. devoid of contaminating proteins particles like proteins pass through the chemical additive • Smaller • No pores and eluted late whereas exosomes do not Exosome structure and biological • enter the pores and eluted at an early stage. activity are not affected molecules on the basis of their size Highly reproducible • Sorts • based on receptor-ligand interaction Highly selective and can yield pure • Separation • by binding antibodies to exosome-specific populations of exosomes. antigens that are membrane-bound. Possible to isolate even sub• populations of exosomes platforms can be used for the immuno• Different isolation of exosomes like the affinity chromatography columns, immune beads, ELISA plates, microfluidic devices and immune chips

of exosomes to filter • Attachment membrane leads to lesser exosomal yield

• • • •

and possible damage to exosomal membrane and its biological activity Non-vesicular particles of the same size range are co-isolated leading to less pure exosomal preparations Clogging and entrapment of vesicles in the filter decreases the efficiency of isolation Low sample throughput Ideal for mostly small volumes of concentrated samples

contamination with binding • Possible antibodies functional activity of exosomes can be • The affected by hard elution buffers of loss of exosome on attachment • Chance with the binding antibody leading to lesser yield

reagent cost • High sample should be made cell-free before • The antibody binding exosome-specific marker, as well as the • The exosome subtype-specific marker should be well characterized and established

body fluids have shown both predictive and/or diagnostic potential when discriminating healthy individuals from cancer patients [41,42]. The biologically active molecules packed in exosomes, when horizontally transferred between different cell types in the proximity as well as distant locations, are functional in the recipient cells and are capable of changing the recipient cell’s biological properties by accelerating tumour growth, metastasis, angiogenesis and drug resistance [43]. The following sections illustrate the dynamic roles of exosomes in cancer progression and metastasis (Figure 1).

which exosomes in a sample are concentrated based on their size but exosomal attachment to the membrane may cause possible damage to the exosome membrane and consequently affect its bioactivity [39]. The basic principle, advantages, and disadvantages of each method appear in Table 2.

Exosomes and their roles in tumour pathogenesis Exosomes in large numbers are shed into the tumour microenvironment and emerging studies have identified exosomes as dynamic players in mediating the molecular cross-talk that drives tumour progression, invasion and metastasis [40]. The circulating levels of tumour exosomes significantly increase, paralleling the aggressive nature of the cancer [41]. Tumour-associated exosomes have been identified in cancer patients and the levels of exosomes released into

Exosomes mediate tumour cell development, progression, invasion and metastasis Exosomes derived from tumour cells transfer molecular cues to surrounding normal healthy cells in the tumour microenvironment. Al34

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T-CELL APOPTOSIS & TREG EXPANSION

TUMOUR MICROENVIRONMENT

ANGIOGENESIS

EXOSOMES INHIBITION OF NK CELL

PRE-METASTATIC NICHE MVB

EARLY ENDOSOME

LYSOSOME

GOLGI

INDUCTION OF MYELOIDDERIVED SUPPRESSOR CELLS

NUCLEUS

DRUG RESISTANCE

Fig. 1. An overview of the diverse roles of exosomes mediating tumour progression and metastasis.

adenocarcinoma cells (PDAC) derived exosomes express high levels of macrophage migration inhibitory factor (MIF) that inhibits the migration of macrophages to a pre-metastatic niche in the liver and increases the metastatic load in PDACs [50]. Tumour-associated stromal cells such as fibroblasts produce exosomes which shuttle miRNAs and proteins between cells and promote the tumour progression and metastasis. Gastrointestinal stromal tumour cells invade the interstitial stroma by releasing oncogenic protein tyrosine kinase- containing exosomes, which on uptake by the stromal cells activate the matrix metalloproteinases I (MMPI) for tumour progression [51]. Internalization of mesenchymal stromal cells (MSC) derived exosomes by breast and small cell ovarian cancer cells alter the cellular functionalities and acquisition of new tumour properties [52]. Exosomes from tumour stromal fibroblasts increase the cell protrusions, motility and invasiveness of breast cancer cells by mediating the autocrine Wnt-PCP (Plantar cell polarity) signaling [53]. Finally, exosomes released from chronic myelogenous leukemia cells stimulate stromal bone marrow cells to produce interleukin-8 (IL-8) which activates the C-X-C Motif Chemokine Ligand 1 & 2 (CXCL 1 & 2) receptors, affecting the downstream signaling and modulating the malignant phenotype[54].

Nedawi et al. reported a truncated and oncogenic form of epidermal growth factor receptor known as EGFRvIII expressed by glioblastoma cells, when transferred to cells lacking EGFRvIII expression caused oncogenic activity and anchorage-independent growth in recipient cells [44]. Exosomes isolated from mutant KRAS expressing colon cancer cells contain proteins like KRAS, EGFR, SRC family kinases and integrins, which are tumour promoting and enhance the invasiveness in wild-type-KRAS-expressing cells [45]. Important insights into the microenvironment-mediated tumour progression have been provided by the elucidation of molecular cross-talk between astrocyte-derived exosomes and primary tumour cells metastasizing to the brain. Astrocytederived exosomes transfer miRNA that downregulates an important tumour suppressor protein, PTEN, in recipient cells, imparting a PTENloss phenotype and hence an increase in tumour growth. [46]. Also, the cellular disposal of tumour suppressor miRNAs mediated by exosomes helps in the acquisition of metastatic properties. Cellular disposal of miRNA, miR23b is increased in bladder carcinoma cells for the attainment of metastatic properties and helping tumour progression [47]. Growing evidence shows the important role of exosomes in mediating pro-metastatic niche formation and accomplishing long distance metastasis to bone marrow and lymph nodes. Melanoma exosomes mediate lymphatic metastatic progression by homing into the sentinel lymph nodes and inducing expression factors responsible for cell recruitment, matrix remodeling and angiogenesis, thereby making the tumour microenvironment conducive to tumour cell adherence and growth [48]. Further, exosomes from highly metastatic melanoma cells can permanently educate bone marrow progenitor cells via signaling mediated by a receptor tyrosine-kinase oncoprotein, MET and augment cell mobilization and tumour progression [49]. Pancreatic ductal

Tumour exosomes promote angiogenesis Hypoxia is an important mediator in the tumour microenvironment which may induce the release of exosomes from tumour cells. The hypoxic exosomes packaged with angiogenic factors help neovascularization and tumour progression. King et al., [55] demonstrated that in the presence of hypoxia, hypoxia-inducible factor (HIF) mediates an 35

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Shedden et al., reported a correlation between exosome shedding and anti-cancer drug resistance in National Cancer Institute (NCI) 60 cell line [73]. In multidrug-resistant tumours, cytotoxic drugs are sequestered and sorted into the exosome pathway, increasing the exosomemediated expulsion of drugs, leading to chemo resistance [74,75].

enhanced release of exosomes from breast cancer cell lines [55]. Multiple myeloma cells release more exosomes under hypoxic conditions with the upregulation of miR-135b, which accelerates endothelial tube formation via HIF-FIH (Factor inhibiting HIF) signaling pathway [56]. Hypoxic glioblastoma cells release microvesicles loaded with TF/VIIa coagulation-initiation-protease complex which activates the PAR-2 (Protease-activated receptor 2) on endothelial cells leading to PAR-2/ ERK1/2–dependent induction of the proangiogenic growth factor HBEGF, causing increased angiogenesis and tumour growth [57]. Additionally, glioblastoma cell-derived hypoxic enxosomes activate cell proliferation and tumour vascularization by programing the endothelial cells to secrete several growth factors and cytokines and to activatePI3K/AKT signaling in brain vascular pericytes [58]. Tumour-derived exosomes mediate neovascularization by shuttling different molecules that can activate the angiogenic activities in the stromal cells and increase endothelial cell proliferation and migration. Exosomal delivery of TGFβ (Transforming Growth Factor β) activates cellular differentiation of fibroblasts to a myofibroblast phenotype which increases tumour angiogenesis [59]. Exosomes derived from chronic myelogenous leukemia modulates neovascularisation by causing reorganization of vascular endothelial cells into tubes [60]. Uptake of exosomes containing the Tspan8-CD49d complex by endothelial cells leads to endothelial cell proliferation, migration, sprouting and maturation of endothelial cell progenitor cells in rat adenocarcinoma model [61].

Exosomes as drug targets Identifying the definite functional role of exosomes and their precise targets can pave way for targeting these molecules or pathways as therapeutic targets in cancer. The molecular cargo carried by exosomes can be transferred to recipient cells and harnessing this physiological phenomenon, exosomes carrying tumour-suppressive molecules or anticancer drugs can be utilized as novel therapeutic modalities in cancer treatment and management. Exosomes are vital players in the regulation and harmonization of various aspects of the immune system and their extensive role in cancer immune modulation make them promising immunotherapeutic candidates [26,76]. The proinflammatory activity of exosomes, especially the antigen presentation ability and capability to activate dendritic cells in anti-tumour response has been exploited as anti tumour vaccine [77]. Since EVs are non-viable products, exosome-based immunotherapy offers many advantages over the cell-based immunotherapy, which includes (i) ease of storage, transport and transplantation and (ii) lesser deleterious effects associated with cell differentiation and persistence of biological potency [26]. However, the capacity of the exosome to enhance or suppress the immune system depends on diverse factors, hence detailed characterization of exosomes as well as thorough understanding of its biological role are essential for developing exosomebased immune therapy.

Exosomes mediate immune evasion in tumour cells and propagate metastasis Tumour cells secrete immunologically active exosomes capable of evoking anti-tumour inflammatory responses. Exosomes derived from heat-treated malignant ascites promote dendritic cell (DC) maturation and induce a tumour-specific cytotoxic T lymphocyte (CTL) response [62]. Tumour-derived exosomes can activate the natural killer (NK) cell granzyme B activity initiating apoptosis of tumour cells [63]. In contrast, there are various reports of exosomes carrying immunosuppressive molecular signals that help immune evasion by tumour cells thereby enhancing tumour progression and metastasis. Tumour cell-derived exosomes induce T cell apoptosis by FasL mediated response as observed in ovarian cancer cells [64] and galectin-9 mediated response in nasopharyngeal carcinoma cells [65]. Exosomes induce the expansion of regulatory T cells, promote their resistance to apoptosis and immune suppressive function by TGFβ or IL-10 mediated signaling [66]. It has been shown that the MHC class I-related chain A present in tumour-derived exosomes downregulate the NKG2D receptors on the surface of NK cells and cause a significant reduction in NK cell cytotoxic activity [67]. In mice mammary tumour, exosomes inhibit the differentiation of bone marrow myeloid precursor cells into dendritic cells by inducing the expression of IL-6 [68]. In contrast, exosomes promote the differentiation of myeloid-derived suppressor cells whereby increasing tumour metastasis [69].

Salivary exosomes Analysis of salivary exosomes for biological content can provide new insights into tumour development, progression and metastasis. Saliva collection is easy, simple, non-invasive; saliva contains fewer proteins and is less complex than serum [78,79]. Blood or serum has a large number of molecules which makes the detection of low abundant and relatively hidden biomarkers difficult. Unlike blood, biomarkers in saliva are diluted and readily accessible and can be detected using highly sensitive amplification technologies. These characteristics make saliva an ideal biological fluid for biomarker discovery [80,81]. Ogawa et al., reported for the first time the presence of exosome-like vesicles in human saliva containing dipeptidyl peptidase (DPP) IV, galectin-3 and immunoglobulin A and proposed their potential role in protein catabolism and promoting local immunity within the oral cavity [82]. They further fractionated the salivary exosomes into two types, namely, exosome I and exosome II, with different size and protein composition and speculated that the variation in size and compositions may be attributed from their origin from different salivary glands and/ or cell-types [83]. Exosome II has more or less similar shape and size compared to exosomes from other sources whereas exosome I is larger and more electron dense than exosome II. A proteomic analysis of the exosomes revealed that both exosomes contain common exosomal proteins markers such as CD-63, Hsp 70, Alix, TSG, other plasma membrane proteins and GW 182, a member of RNA induced silencing complex required for miRNA function. The proteins like DPP IV, carbonic anhydrase, cystatin family proteins, IgG Fc binding protein and galectin-3 binding protein are more enriched in exosome II indicating the potent role of exosome II in protein catabolism and immune response whereas some proteins like moesin, radixin and annexins were exclusively found in exosome I. Both the exosomes contain high levels of IgA reaffirming the antigen presentation function of exosomes and its significance in the local immune defense in the oral cavity. They further performed a transcriptomic analysis in the two types of salivary exosomes using next-generation sequencing and identified a large

Exosomes are involved in mediating drug resistance in cancer Exosomes derived from tumour cells transfer proteins and miRNAs which are capable of inducing resistance to drugs and increase cell proliferation and invasiveness in recipient cells. Exosomes derived from hormone-refractory prostate cancer cells induce resistance to docetaxel by transferring P-glycoprotein to recipient cells [70]. Exosomes in ER (Estrogen-receptor)-positive breast cancer cells transfer miR-221/222, which downregulates the expression of p27 and ERα (target genes of tamoxifen), thereby inducing tamoxifen resistance [71]. Hu et al. reported that the priming of cancer stem cells by exosomes derived from fibroblasts cells isolated from colorectal carcinoma increased its resistance to chemotherapeutic agents [72]. Exosome-mediated export and expulsion of drug from tumour cells is an important mechanism conferring drug resistance in tumour cells. 36

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profiles [96]. The authors further investigated the role of these salivary exosomes and described them as key players in the suppression of NK cell-mediated immune surveillance. The discriminatory salivary exosomes interact directly with the NK cells in the oral cavity affecting the NK cell cytotoxicity and immune surveillance in tumour development [97]. The mechanism of development of discriminatory salivary biomarkers for systemic tumours delineates the definitive functional role of exosomes in mediating tumour progression and opens up new avenues for using simple saliva test for clinical screening and detection of cancer. A better understanding of the exosome-mediated delivery of tumour products from distal tumours to salivary gland has expanded the utility of salivary exosomes as potential biomarkers and the term “saliva-exosomics” has been coined for next-generation salivomics, highlighting the importance of integrating salivomics with other biomedical areas [98].

repertoire of coding and non-coding RNAs. This comprised of miRNAs, piRNAs, snoRNAs, lncRNAs of processed pseudogenes and mRNAs of various proteins [84,85]. In addition, Gallo et al., reported that majority of the miRNAs in saliva are concentrated in exosomes and proposed the exosomal miRNA as the first step to biomarker discovery [86]. These studies provided interesting insights into the wide variety of biologically active molecules like RNAs and proteins selectively enriched in salivary exosomes and their potential role in mediating intercellular communication. Palanisamy et al., isolated exosomes from saliva by ultracentrifugation and analysed using electron microscopy (EM) and atomic force microscopy (AFM), revealing unique ‘cup-shaped’ vesicles of < 100 nm size [87]. They identified 509 mRNA transcripts in salivary exosomes by microarray analysis. These transcripts were reasonably stable and biologically active when taken up by keratinocytes, modulating the protein expression in recipient cells. A study by Sharma et al., identified the human salivary exosomes as trilobed structures [88]. They analysed the substructural organization of exosomes and identified the single surface molecule characteristics using ultrasensitive low force AFM, Field Emission Scanning Electron Microscopy (FASEM) and force spectroscopy with antibody-coated AFM tips. They reported the reversible elastic nanomechanical properties of salivary exosomes undergoing structural deformation under high forces and proposed the development of engineered exosomes from saliva as drug delivery vesicles. Human salivary exosome protein has been characterized by analyzing the proteome of parotid gland salivary exosomes using MudPIT (multidimensional protein identification technology) mass spectrometry [89]. This in-depth analysis of the salivary exosome proteome revealed a rich protein repertoire comprising common exosomal proteins like tetraspanins, heat shock proteins, cytoskeletal proteins, syntenin, annexins and Rab family members; proteins involved in the process of exosome formation; and proteins involved in cell-adhesion indicating cell to cell communication function of exosomes. Parotid gland-specific proteins such as aquaporins (facilitate in saliva secretion), cytokeratin and specific epithelial markers were also identified highlighting their value as diagnostic biomarkers in immunological discorders such as Sjogren’s syndrome. Sharma et al., performed a nanoscale characterization of salivary exosomes from oral cancer patients, at single vesicle level using highresolution Atomic Force Microscopy (AFM). [90]. They identified increased expression of CD-63 on the surface of oral cancer exosomes. In addition, an increased exosome concentration was observed in oral cancer patient’s saliva samples and these vesicles appeared to be larger with heterogeneous morphology with the more inter-vesicular aggregation on AFM. Further, the variations in morphology and surface markers of salivary exosomes in oral cancer were analyzed by Zlotogorski-Hurvitz et al., [91]. They reported a higher exosome concentration in saliva from oral cancer patients, which is in agreement with previous studies that different types of cancers secrete higher amounts of exosomes than normal healthy individuals. Also, exosomes from oral cancer patients were identified to be larger in size than exosomes from the healthy group and had elevated CD-63 expression mostly in the form of glycosylated CD-63 with a lesser expression of CD9 and CD-81. Exosomes may be the key mediators linking oral health to systemic events. A tumour developing distally from the oral cavity can redefine the salivary biomarker profile by inducing cancer specific discriminatory changes in saliva reflecting the presence of a tumour [92]. Cancer-specific discriminatory transcriptomic signatures in saliva were reported in pancreatic [93] and ovarian cancer [94] and proteomic signature changes in lung cancer [95]. Exosomes are found to play a pivotal role in the development of this tumour specific salivary biomarker profile. In pancreatic ductal adenocarcinoma, exosomes from a distal tumour are capable of changing the biology of exosomes derived from salivary gland and induce changes in the salivary biomarker

Conclusion Exosomes are nanovesicles of 30–100 nm diameter is size, derived from the endocytic compartment of the cells and released into the extracellular space. Exosomes carry molecular content such as DNA, RNA and protein, reflecting their cellular origin and horizontally transfer these bioactive molecules to recipient cells, playing important roles in intercellular communication and molecular signalling. Thus, exosomes are crucial mediators of the cell-to-cell communication and are enriched in almost all the body fluids, both under physiological and pathological conditions thereby serving as ideal diagnostic/prognostic biomarker candidates. Current literature describes exosomes as vital players and mediators of cancer progression, invasion and metastasis in a number of tumour types. These extensive and diverse roles played by exosomes in cell to cell communication and immunomodulation make them promising candidates for novel therapeutic modalities in cancer management. Saliva is an ideal diagnostic medium for biomarker detection owing to its non-invasiveness, ease of collection and cost-effectiveness. Blood or serum has a large number of biomolecules, which makes the detection of low abundant biomarkers difficult. Unlike blood, biomarkers in saliva are diluted and readily available. Additionally, the proximal location or the direct contact of the oral and oropharyngeal cancers with the saliva makes it a promising surrogate medium for detecting these cancer-types. However, salivary exosomes isolation protocols present a major hurdle to the development of saliva-based diagnostics assays. Paralleling data from other biological fluids, there are inconsistencies in the yield and content of exosomes isolated from saliva with different isolation methods and this can have an impact on the utility of this medium as a diagnostic platform for cancer. Additionally, more efforts are to be made in developing and optimizing methodologies, which can exclude the steps of exosome isolation and detect exosomes directly from biological fluids like saliva. Further studies regarding characteristics and biological functions of salivary exosomes are warranted for a better understanding of the immunomodulatory mechanisms mediated by biomolecules present in saliva of cancer patients. This can expand the borders of saliva research and open up new avenues of biomarker discovery and therapeutic interventions in cancer. Conflict of interest statement None declared. References [1] Wessler S, Aberger F, Hartmann TN. The sound of tumor cell-microenvironment communication – composed by the Cancer Cluster Salzburg research network. Cell Commun Signal: CCS 2017;15:20. [2] Katsuda T, Kosaka N, Ochiya T. The roles of extracellular vesicles in cancer biology: toward the development of novel cancer biomarkers. Proteomics 2014;14:412–25.

37

Oral Oncology 84 (2018) 31–40

S. Nair et al.

challenge of high purity vesicle isolation. Mol Biosyst 2016;12:1407–19. [37] Greening DW, Xu R, Ji H, Tauro BJ, Simpson RJ. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. Methods Mol Biol (Clifton, NJ) 2015;1295:179–209. [38] Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 2015;4:27031. [39] Li P, Kaslan M, Lee SH, Yao J, Gao Z. Progress in exosome isolation techniques. Theranostics 2017;7:789–804. [40] Yu S, Cao H, Shen B, Feng J. Tumor-derived exosomes in cancer progression and treatment failure. Oncotarget 2015;6:37151–68. [41] Silva J, Garcia V, Rodriguez M, Compte M, Cisneros E, Veguillas P, et al. Analysis of exosome release and its prognostic value in human colorectal cancer. Genes Chromosomes Cancer 2012;51:409–18. [42] Cappello F, Logozzi M, Campanella C, Bavisotto CC, Marcilla A, Properzi F, et al. Exosome levels in human body fluids: A tumor marker by themselves? Eur J Pharm Sci: Off J Eur Fed Pharm Sci 2016;96:93–8. [43] Guo L, Guo N. Exosomes: Potent regulators of tumor malignancy and potential biotools in clinical application. Crit Rev Oncol/Hematol 2015;95:346–58. [44] Al-Nedawi K, Meehan B, Micallef J, Lhotak V, May L, Guha A, et al. Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 2008;10:619–24. [45] Demory Beckler M, Higginbotham JN, Franklin JL, Ham AJ, Halvey PJ, Imasuen IE, et al. Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS. Mol Cell Proteomics: MCP 2013;12:343–55. [46] Zhang L, Zhang S, Yao J, Lowery FJ, Zhang Q, Huang WC, et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 2015;527:100–4. [47] Ostenfeld MS, Jeppesen DK, Laurberg JR, Boysen AT, Bramsen JB, PrimdalBengtson B, et al. Cellular disposal of miR23b by RAB27-dependent exosome release is linked to acquisition of metastatic properties. Cancer Res 2014;74:5758–71. [48] Hood JL, San RS, Wickline SA. Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis. Cancer Res 2011;71:3792–801. [49] Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, et al. Melanoma exosomes educate bone marrow progenitor cells toward a prometastatic phenotype through MET. Nature Med 2012;18:883–91. [50] Costa-Silva B, Aiello NM, Ocean AJ, Singh S, Zhang H, Thakur BK, et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol 2015;17:816–26. [51] Atay S, Banskota S, Crow J, Sethi G, Rink L, Godwin AK. Oncogenic KIT-containing exosomes increase gastrointestinal stromal tumor cell invasion. PNAS 2014;111:711–6. [52] Yang Y, Bucan V, Baehre H, von der Ohe J, Otte A, Hass R. Acquisition of new tumor cell properties by MSC-derived exosomes. Int J Oncol 2015;47:244–52. [53] Luga V, Zhang L, Viloria-Petit Alicia M, Ogunjimi Abiodun A, Inanlou Mohammad R, Chiu E, et al. Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration. Cell 2012;151:1542–56. [54] Corrado C, Raimondo S, Saieva L, Flugy AM, De Leo G, Alessandro R. Exosomemediated crosstalk between chronic myelogenous leukemia cells and human bone marrow stromal cells triggers an interleukin 8-dependent survival of leukemia cells. Cancer Lett 2014;348:71–6. [55] King HW, Michael MZ, Gleadle JM. Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer 2012;12:421. [56] Umezu T, Tadokoro H, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH. Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1. Blood 2014;124:3748–57. [57] Svensson KJ, Kucharzewska P, Christianson HC, Skold S, Lofstedt T, Johansson MC, et al. Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells. PNAS 2011;108:13147–52. [58] Kucharzewska P, Christianson HC, Welch JE, Svensson KJ, Fredlund E, Ringner M, et al. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxiadependent activation of vascular cells during tumor development. PNAS 2013;110:7312–7. [59] Webber J, Steadman R, Mason MD, Tabi Z, Clayton A. Cancer exosomes trigger fibroblast to myofibroblast differentiation. Cancer Res 2010;70:9621–30. [60] Taverna S, Flugy A, Saieva L, Kohn EC, Santoro A, Meraviglia S, et al. Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis. Int J Cancer 2012;130:2033–43. [61] Nazarenko I, Rana S, Baumann A, McAlear J, Hellwig A, Trendelenburg M, et al. Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosomeinduced endothelial cell activation. Cancer Res 2010;70:1668–78. [62] Zhong H, Yang Y, Ma S, Xiu F, Cai Z, Zhao H, et al. Induction of a tumour-specific CTL response by exosomes isolated from heat-treated malignant ascites of gastric cancer patients. Int J Hyperther: Off J Eur Soc Hyperther Oncol N Am Hyperther Group 2011;27:604–11. [63] Gastpar R, Gehrmann M, Bausero MA, Asea A, Gross C, Schroeder JA, et al. Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells. Cancer Res 2005;65:5238–47. [64] Taylor DD, Gercel-Taylor C, Lyons KS, Stanson J, Whiteside TL. T-cell apoptosis and suppression of T-cell receptor/CD3-zeta by Fas ligand-containing membrane vesicles shed from ovarian tumors. Clin Cancer Res: Off J Am Assoc Cancer Res 2003;9:5113–9.

[3] Ela S, Mager I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013;12:347–57. [4] Lässer C, Seyed Alikhani V, Ekström K, Eldh M, Torregrosa Paredes P, Bossios A, et al. Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med 2011;9:9. [5] Gyorgy B, Szabo TG, Pasztoi M, Pal Z, Misjak P, Aradi B, et al. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci: CMLS 2011;68:2667–88. [6] Zhang X, Yuan X, Shi H, Wu L, Qian H. Xu W. Exosomes in cancer: small particle, big player. J Hematol Oncol 2015;8. [7] Teng Y, Ren Y, Hu X, Mu J, Samykutty A, Zhuang X, et al. MVP-mediated exosomal sorting of miR-193a promotes colon cancer progression. Nat Commun 2017;8:14448. [8] Hoshino A, Costa-Silva B, Shen T-L, Rodrigues G, Hashimoto A, Tesic Mark M, et al. Tumour exosome integrins determine organotropic metastasis. Nature 2015;527:329–35. [9] Kowal J, Tkach M, Thery C. Biogenesis and secretion of exosomes. Curr Opin Cell Biol 2014;29:116–25. [10] Colombo M, Moita C, van Niel G, Kowal J, Vigneron J, Benaroch P, et al. Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. J Cell Sci 2013;126:5553–65. [11] Trajkovic K, Hsu C, Chiantia S, Rajendran L, Wenzel D, Wieland F, et al. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science (New York, NY) 2008;319:1244–7. [12] Savina A, Vidal M, Colombo MI. The exosome pathway in K562 cells is regulated by Rab11. J Cell Sci 2002;115:2505–15. [13] Fader CM, Sánchez DG, Mestre MB, Colombo MI. TI-VAMP/VAMP7 and VAMP3/ cellubrevin: two v-SNARE proteins involved in specific steps of the autophagy/ multivesicular body pathways. Biochimica et Biophysica Acta (BBA) – Molecular. Cell Res 2009;1793:1901–16. [14] Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJA. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotech 2011;29:341–5. [15] Aryani A, Denecke B. Exosomes as a nanodelivery system: a key to the future of neuromedicine? Mol Neurobiol 2016;53:818–34. [16] Keller S, Sanderson MP, Stoeck A, Altevogt P. Exosomes: from biogenesis and secretion to biological function. Immunol Lett 2006;107:102–8. [17] Feng D, Zhao W-L, Ye Y-Y, Bai X-C, Liu R-Q, Chang L-F, et al. Cellular internalization of exosomes occurs through phagocytosis. Traffic 2010;11:675–87. [18] Montecalvo A, Larregina AT, Shufesky WJ, Stolz DB, Sullivan ML, Karlsson JM, et al. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood 2012;119:756–66. [19] Thery C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 2009;9:581–93. [20] Parolini I, Federici C, Raggi C, Lugini L, Palleschi S, De Milito A, et al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem 2009;284:34211–22. [21] Pan BT, Teng K, Wu C, Adam M, Johnstone RM. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol 1985;101:942–8. [22] Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med 1996;183:1161–72. [23] Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, et al. Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 1998;4:594–600. [24] Thery C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol 2002;2:569–79. [25] Chaput N, Schartz NE, Andre F, Zitvogel L. Exosomes for immunotherapy of cancer. Adv Exp Med Biol 2003;532:215–21. [26] Zhang B, Yin Y, Lai RC, Lim SK. Immunotherapeutic potential of extracellular vesicles. Front Immunol 2014;5:518. [27] Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007;9:654–9. [28] Simons M, Raposo G. Exosomes–vesicular carriers for intercellular communication. Curr Opin Cell Biol 2009;21:575–81. [29] Hannafon BN, Trigoso YD, Calloway CL, Zhao YD, Lum DH, Welm AL, et al. Plasma exosome microRNAs are indicative of breast cancer. Breast Cancer Res 2016;18:90. [30] Lorena Urbanelli SB, Sagini Krizia, Ferrara Giuseppina, Lanni Marco, Emiliani C. Exosome-based strategies for diagnosis and therapy. Recent Pat CNS Drug Discov 2015;10:10–27. [31] Logozzi M, De Milito A, Lugini L, Borghi M, Calabro L, Spada M, et al. High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients. PloS one. 2009;4:e5219. [32] Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, et al. Glypican1 identifies cancer exosomes and detects early pancreatic cancer. Nature 2015;523:177–82. [33] Skog J, Wurdinger T, van Rijn S, Meijer DH, Gainche L, Sena-Esteves M, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 2008;10:1470–6. [34] Muller L, Hong CS, Stolz DB, Watkins SC, Whiteside TL. Isolation of biologicallyactive exosomes from human plasma. J Immunol Methods 2014;411:55–65. [35] Zlotogorski-Hurvitz A, Dayan D, Chaushu G, Korvala J, Salo T, Sormunen R, et al. Human saliva-derived exosomes: comparing methods of isolation. J Histochem Cytochem 2015;63:181–9. [36] Abramowicz A, Widlak P, Pietrowska M. Proteomic analysis of exosomal cargo: the

38

Oral Oncology 84 (2018) 31–40

S. Nair et al.

[65] Klibi J, Niki T, Riedel A, Pioche-Durieu C, Souquere S, Rubinstein E, et al. Blood diffusion and Th1-suppressive effects of galectin-9-containing exosomes released by Epstein-Barr virus-infected nasopharyngeal carcinoma cells. Blood 2009;113:1957–66. [66] Szajnik M, Czystowska M, Szczepanski MJ, Mandapathil M, Whiteside TL. Tumorderived microvesicles induce, expand and up-regulate biological activities of human regulatory T cells (Treg). PloS One 2010;5:e11469. [67] Ashiru O, Boutet P, Fernandez-Messina L, Aguera-Gonzalez S, Skepper JN, ValesGomez M, et al. Natural killer cell cytotoxicity is suppressed by exposure to the human NKG2D ligand MICA*008 that is shed by tumor cells in exosomes. Cancer Res 2010;70:481–9. [68] Yu S, Liu C, Su K, Wang J, Liu Y, Zhang L, et al. Tumor exosomes inhibit differentiation of bone marrow dendritic cells. J Immunol (Baltimore, Md : 1950) 2007;178:6867–75. [69] Liu Y, Xiang X, Zhuang X, Zhang S, Liu C, Cheng Z, et al. Contribution of MyD88 to the tumor exosome-mediated induction of myeloid derived suppressor cells. Am J Pathol 2010;176:2490–9. [70] Corcoran C, Rani S, O’Brien K, O’Neill A, Prencipe M, Sheikh R, et al. Docetaxelresistance in prostate cancer: evaluating associated phenotypic changes and potential for resistance transfer via exosomes. PloS One 2012;7:e50999. [71] Wei Y, Lai X, Yu S, Chen S, Ma Y, Zhang Y, et al. Exosomal miR-221/222 enhances tamoxifen resistance in recipient ER-positive breast cancer cells. Breast Cancer Res Treat 2014;147:423–31. [72] Hu Y, Yan C, Mu L, Huang K, Li X, Tao D, et al. Fibroblast-derived exosomes contribute to chemoresistance through priming cancer stem cells in colorectal cancer. PloS One 2015;10:e0125625. [73] Shedden K, Xie XT, Chandaroy P, Chang YT, Rosania GR. Expulsion of small molecules in vesicles shed by cancer cells: association with gene expression and chemosensitivity profiles. Cancer Res 2003;63:4331–7. [74] Chen KG, Valencia JC, Lai B, Zhang G, Paterson JK, Rouzaud F, et al. Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas. PNAS 2006;103:9903–7. [75] Safaei R, Larson BJ, Cheng TC, Gibson MA, Otani S, Naerdemann W, et al. Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells. Mol Cancer Therap 2005;4:1595–604. [76] Xiao-Bo Li Z-RZ, Schluesener Hermann J, Shun-Qing Xu. Role of exosomes in immune regulation. J Cell Mol Med 2006;10:364–75. [77] Mignot G, Roux S, Thery Clotilde, Ségura Elodie, Zitvogel L. Prospects of exosomes in immunotherapy of cancer. J Cell Mol Med 2006;10:376–88. [78] Schulz BL, Cooper-White J, Punyadeera CK. Saliva proteome research: current status and future outlook. Crit Rev Biotechnol 2013;33:246–59. [79] Topkas E, Keith P, Dimeski G, Cooper-White J, Punyadeera C. Evaluation of saliva collection devices for the analysis of proteins. Clinica chimica acta; Int J Clin Chem 2012;413:1066–70. [80] Chai RC, Lim Y, Frazer IH, Wan Y, Perry C, Jones L, et al. A pilot study to compare the detection of HPV-16 biomarkers in salivary oral rinses with tumour p16(INK4a) expression in head and neck squamous cell carcinoma patients. BMC Cancer 2016;16:178. [81] Salazar C, Calvopina D, Punyadeera C. miRNAs in human papilloma virus associated oral and oropharyngeal squamous cell carcinomas. Expert Rev Mol Diagn 2014;14:1033–40. [82] Ogawa Y, Kanai-Azuma M, Akimoto Y, Kawakami H, Yanoshita R. Exosome-like vesicles with dipeptidyl peptidase IV in human saliva. Biol Pharm Bull 2008;31:1059–62. [83] Ogawa Y, Miura Y, Harazono A, Kanai-Azuma M, Akimoto Y, Kawakami H, et al. Proteomic analysis of two types of exosomes in human whole saliva. Biol Pharm Bull 2011;34:13–23. [84] Ogawa Y, Taketomi Y, Murakami M, Tsujimoto M, Yanoshita R. Small RNA transcriptomes of two types of exosomes in human whole saliva determined by next generation sequencing. Biol Pharm Bull 2013;36:66–75. [85] Ogawa Y, Tsujimoto M, Yanoshita R. Next-generation sequencing of proteincoding and long non-protein-coding RNAs in two types of exosomes derived from human whole saliva. Biol Pharm Bull 2016;39:1496–507. [86] Gallo A, Tandon M, Alevizos I, Illei GG. The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PloS One 2012;7:e30679. [87] Palanisamy V, Sharma S, Deshpande A, Zhou H, Gimzewski J, Wong DT. Nanostructural and transcriptomic analyses of human saliva derived exosomes. PloS One 2010;5. [88] Sharma S, Rasool HI, Palanisamy V, Mathisen C, Schmidt M, Wong DT, et al. Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy. ACS Nano 2010;4:1921–6. [89] Gonzalez-Begne M, Lu B, Han X, Hagen FK, Hand AR, Melvin JE, et al. Proteomic analysis of human parotid gland exosomes by multidimensional protein identification technology (MudPIT). J Proteome Res 2009;8:1304–14. [90] Sharma S, Gillespie BM, Palanisamy V, Gimzewski JK. Quantitative nanostructural and single-molecule force spectroscopy biomolecular analysis of human-salivaderived exosomes. Langmuir ACS J Surfaces Colloids 2011;27:14394–400. [91] Zlotogorski-Hurvitz A, Dayan D, Chaushu G, Salo T, Vered M. Morphological and molecular features of oral fluid-derived exosomes: oral cancer patients versus healthy individuals. J Cancer Res Clin Oncol 2016;142:101–10. [92] Yang J, Wei F, Schafer C, Wong DT. Detection of tumor cell-specific mRNA and protein in exosome-like microvesicles from blood and saliva. PloS One 2014;9:e110641. [93] Zhang L, Farrell JJ, Zhou H, Elashoff D, Akin D, Park NH, et al. Salivary transcriptomic biomarkers for detection of resectable pancreatic cancer.

Gastroenterology 2010;138:949–57. e1–7. [94] Lee YH, Kim JH, Zhou H, Kim BW, Wong DT. Salivary transcriptomic biomarkers for detection of ovarian cancer: for serous papillary adenocarcinoma. J Mol Med (Berlin, Germany) 2012;90:427–34. [95] Xiao H, Zhang L, Zhou H, Lee JM, Garon EB, Wong DT. Proteomic analysis of human saliva from lung cancer patients using two-dimensional difference gel electrophoresis and mass spectrometry. Mol Cell Proteomics: MCP 2012;11(M111):012112. [96] Lau C, Kim Y, Chia D, Spielmann N, Eibl G, Elashoff D, et al. Role of pancreatic cancer-derived exosomes in salivary biomarker development. J Biol Chem 2013;288:26888–97. [97] Katsiougiannis S, Chia D, Kim Y, Singh RP, Wong DT. Saliva exosomes from pancreatic tumor-bearing mice modulate NK cell phenotype and antitumor cytotoxicity. FASEB journal: official publication of the Federation of American Societies for. Exp Biol 2016. [98] Nonaka T, Wong DTW. Chapter six – saliva-exosomics in cancer: molecular characterization of cancer-derived exosomes in saliva. In: Hu TY, Tamanoi F, editors. The Enzymes. Academic Press; 2017. p. 125–51. [99] Madhavan B, Yue S, Galli U, Rana S, Gross W, Muller M, et al. Combined evaluation of a panel of protein and miRNA serum-exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity. Int J Cancer 2015;136:2616–27. [100] Matzke-Ogi A, Jannasch K, Shatirishvili M, Fuchs B, Chiblak S, Morton J, et al. Inhibition of tumor growth and metastasis in pancreatic cancer models by interference with CD44v6 signaling. Gastroenterology 2016;150(513–25):e10. [101] Yue S, Mu W, Erb U, Zoller M. The tetraspanins CD151 and Tspan8 are essential exosome components for the crosstalk between cancer initiating cells and their surrounding. Oncotarget 2015;6:2366–84. [102] Hasegawa S, Eguchi H, Nagano H, Konno M, Tomimaru Y, Wada H, et al. MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer. Br J Cancer 2014;111:1572–80. [103] Alegre E, Zubiri L, Perez-Gracia JL, González-Cao M, Soria L, Martín-Algarra S, et al. Circulating melanoma exosomes as diagnostic and prognosis biomarkers. Clin Chim Acta 2016;454:28–32. [104] Bosserhoff AK, Buettner R. Expression, function and clinical relevance of MIA (melanoma inhibitory activity). Histol Histopathol 2002;17:289–300. [105] Overbye A, Skotland T, Koehler CJ, Thiede B, Seierstad T, Berge V, et al. Identification of prostate cancer biomarkers in urinary exosomes. Oncotarget 2015;6:30357–76. [106] Schiefermeier N, Scheffler JM, de Araujo ME, Stasyk T, Yordanov T, Ebner HL, et al. The late endosomal p14-MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration. J Cell Biol 2014;205:525–40. [107] Xu X, Liu B, Zou P, Zhang Y, You J, Pei F. Silencing of LASS2/TMSG1 enhances invasion and metastasis capacity of prostate cancer cell. J Cell Biochem 2014;115:731–43. [108] Nilsson J, Skog J, Nordstrand A, Baranov V, Mincheva-Nilsson L, Breakefield XO, et al. Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. Br J Cancer 2009;100:1603–7. [109] Ferreira LB, Palumbo A, de Mello KD, Sternberg C, Caetano MS, de Oliveira FL, et al. PCA3 noncoding RNA is involved in the control of prostate-cancer cell survival and modulates androgen receptor signaling. BMC Cancer 2012;12:507. [110] Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Mehra R, Sun XW, et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science (New York, NY) 2005;310:644–8. [111] Khan S, Bennit HF, Turay D, Perez M, Mirshahidi S, Yuan Y, et al. Early diagnostic value of survivin and its alternative splice variants in breast cancer. BMC Cancer 2014;14:176. [112] Lv Y-G, Yu F, Yao Q, Chen J-H, Wang L. The role of survivin in diagnosis, prognosis and treatment of breast cancer. J Thorac Dis 2010;2:100–10. [113] Eichelser C, Stuckrath I, Muller V, Milde-Langosch K, Wikman H, Pantel K, et al. Increased serum levels of circulating exosomal microRNA-373 in receptor-negative breast cancer patients. Oncotarget 2014;5:9650–63. [114] Yan LX, Wu QN, Zhang Y, Li YY, Liao DZ, Hou JH, et al. Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivo tumor growth. Breast Cancer Res: BCR 2011;13. R2-R. [115] Yan L-X, Huang X-F, Shao Q, Huang MAY, Deng L, Wu Q-L, et al. MicroRNA miR21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA 2008;14:2348–60. [116] Li XJ, Ren ZJ, Tang JH, Yu Q. Exosomal MicroRNA MiR-1246 promotes cell proliferation, invasion and drug resistance by targeting CCNG2 in breast cancer. Cell Physiol Biochem: Int J Exp Cell Physiol Biochem Pharmacol 2017;44:1741–8. [117] Wei F, Cao C, Xu X, Wang J. Diverse functions of miR-373 in cancer. J Transl Med 2015;13:162. [118] Machida T, Tomofuji T, Maruyama T, Yoneda T, Ekuni D, Azuma T, et al. miR1246 and miR4644 in salivary exosome as potential biomarkers for pancreatobiliary tract cancer. Oncol Rep 2016;36:2375–81. [119] Xu H, Zong H, Ma C, Ming X, Shang M, Li K, et al. Epidermal growth factor receptor in glioblastoma. Oncol Lett 2017;14:512–6. [120] Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 2008;110:13–21. [121] Le Quesne J, Caldas C. Micro-RNAs and breast cancer. Mol Oncol 2010;4:230–41. [122] Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, et al. The miR200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008;10:593. [123] Iorio MV, Casalini P, Piovan C, Di Leva G, Merlo A, Triulzi T, et al. microRNA-205 regulates HER3 in human breast cancer. Cancer Res 2009;69:2195–200.

39

Oral Oncology 84 (2018) 31–40

S. Nair et al.

[136] Agostini M, Pucciarelli S, Calore F, Bedin C, Enzo M, Nitti D. miRNAs in colon and rectal cancer: a consensus for their true clinical value. Clinica chimica acta; Int J Clin Chem 2010;411:1181–6. [137] Mohammadi A, Mansoori B, Baradaran B. The role of microRNAs in colorectal cancer. Biomed Pharmacother 2016;84:705–13. [138] Moustakas A, Heldin C-H. The regulation of TGFβ signal transduction. Development 2009;136:3699–714. [139] Tanaka Y, Kamohara H, Kinoshita K, Kurashige J, Ishimoto T, Iwatsuki M, et al. Clinical impact of serum exosomal microRNA-21 as a clinical biomarker in human esophageal squamous cell carcinoma. Cancer 2013;119:1159–67. [140] Hiyoshi Y, Kamohara H, Karashima R, Sato N, Imamura Y, Nagai Y, et al. MicroRNA-21 regulates the proliferation and invasion in esophageal squamous cell carcinoma. Clin Cancer Res: Off J Am Assoc Cancer Res. 2009;15:1915–22. [141] Sandfeld-Paulsen B, Aggerholm-Pedersen N, Baek R, Jakobsen KR, Meldgaard P, Folkersen BH, et al. Exosomal proteins as prognostic biomarkers in non-small cell lung cancer. Mol Oncol 2016;10:1595–602. [142] Brambilla E, Gazdar A. Pathogenesis of lung cancer signaling pathways: roadmap for therapies. Eur Resp J: Off J Eur Soc Clin Resp Physiol 2009;33:1485–97. [143] Ye SB, Zhang H, Cai TT, Liu YN, Ni JJ, He J, et al. Exosomal miR-24-3p impedes Tcell function by targeting FGF11 and serves as a potential prognostic biomarker for nasopharyngeal carcinoma. J Pathol 2016;240:329–40. [144] Wang S, Pan Y, Zhang R, Xu T, Wu W, Wang C, et al. Hsa-miR-24-3p increases nasopharyngeal carcinoma radiosensitivity by targeting both the 3'UTR and 5'UTR of Jab1/CSN5. Oncogene 2016;35:6096–108. [145] Sugimachi K, Matsumura T, Hirata H, Uchi R, Ueda M, Ueo H, et al. Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation. Br J Cancer 2015;112:532–8. [146] Wang Z-d Qu, F-y Chen Y-y, Z-s Ran, H-y Liu, H-d Zhang. Involvement of microRNA-718, a new regulator of EGR3, in regulation of malignant phenotype of HCC cells. J Zhejiang Univ Sci B 2017;18:27–36. [147] Beckham CJ, Olsen J, Yin P-N, Wu C-H, Ting H-J, Hagen FK, et al. Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression. J Urol 2014;192:583–92.

[124] Li J, Sherman-Baust CA, Tsai-Turton M, Bristow RE, Roden RB, Morin PJ. Claudincontaining exosomes in the peripheral circulation of women with ovarian cancer. BMC Cancer 2009;9:244. [125] Li J, Chigurupati S, Agarwal R, Mughal MR, Mattson MP, Becker KG, et al. Possible angiogenic roles for claudin-4 in ovarian cancer. Cancer Biol Ther 2009;8:1806–14. [126] Agarwal R, D'Souza T, Morin PJ. Claudin-3 and claudin-4 expression in ovarian epithelial cells enhances invasion and is associated with increased matrix metalloproteinase-2 activity. Cancer Res 2005;65:7378–85. [127] Keller S, König A-K, Marmé F, Runz S, Wolterink S, Koensgen D, et al. Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes. Cancer Lett 2009;278:73–81. [128] Zhao Y, Chen S, W-f Gou, Z-f Niu, Zhao S, L-j Xiao, et al. The role of EMMPRIN expression in ovarian epithelial carcinomas. Cell Cycle 2013;12:2899–913. [129] Buchanan PC, Boylan KLM, Walcheck B, Heinze R, Geller MA, Argenta PA, et al. Ectodomain shedding of the cell adhesion molecule Nectin-4 in ovarian cancer is mediated by ADAM10 and ADAM17. J Biol Chem 2017;292:6339–51. [130] Bondong S, Kiefel H, Hielscher T, Zeimet AG, Zeillinger R, Pils D, et al. Prognostic significance of L1CAM in ovarian cancer and its role in constitutive NF-kappaB activation. Ann Oncol: Off J Eur Soc Med Oncol 2012;23:1795–802. [131] Meng E, Long B, Sullivan P, McClellan S, Finan MA, Reed E, et al. CD44+/CD24ovarian cancer cells demonstrate cancer stem cell properties and correlate to survival. Clin Exp Metast 2012;29:939–48. [132] Liu J, Sun H, Wang X, Yu Q, Li S, Yu X, et al. Increased exosomal MicroRNA-21 and MicroRNA-146a levels in the cervicovaginal lavage specimens of patients with cervical cancer. Int J Mol Sci 2014;15:758–73. [133] Yao T, Lin Z. MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20. Biochimica et Biophysica Acta (BBA)-Mol Basis Dis 2012;1822:248–60. [134] Gocze K, Gombos K, Juhasz K, Kovacs K, Kajtar B, Benczik M, et al. Unique microRNA expression profiles in cervical cancer. Anticancer Res 2013;33:2561–7. [135] Ogata-Kawata H, Izumiya M, Kurioka D, Honma Y, Yamada Y, Furuta K, et al. Circulating exosomal microRNAs as biomarkers of colon cancer. PloS One 2014;9:e92921.

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