Accepted Manuscript

Exosomes, new biomarkers in early cancer detection

Seyed Hamid Jalalian, Mohammad Ramezani, Seyed Ali Jalalian, Khalil Abnous,
Seyed Mohammad Taghdisi

PII: S0003-2697(18)31059-5
DOI: https://doi.org/10.1016/j.ab.2019.02.013
Reference: YABIO 13247

To appear in: Analytical Biochemistry

Received Date: 13 October 2018

Revised Date: 26 January 2019
Accepted Date: 13 February 2019

Please cite this article as: S.H. Jalalian, M. Ramezani, S.A. Jalalian, K. Abnous, S.M. Taghdisi,
Exosomes, new biomarkers in early cancer detection, Analytical Biochemistry (2019), doi: https://
doi.org/10.1016/j.ab.2019.02.013.

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 ACCEPTED MANUSCRIPT

1 Exosomes, new biomarkers in early cancer detection

2 Seyed Hamid Jalaliana,b,c, Mohammad Ramezanid, Seyed Ali Jalalianb, Khalil Abnousa*, Seyed
3 Mohammad Taghdisie,f,*

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a
4 Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical
5 Sciences, Mashhad, Iran
b
6 Students Research Committee, Department of pharmaceutical nanotechnology, School of Pharmacy,

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7 Mashhad University of Medical Sciences, Mashhad, Iran
c
8 Academic Center for Education, Culture and Research (ACECR)-Mashhad Branch, Mashhad, Iran.

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d
9 Nanotechnology Research Center, Institute of Pharmaceutical Technology, Mashhad University of
10 Medical Sciences, Mashhad, Iran.
e
11 Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of
12 Medical Sciences, Mashhad, Iran.
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13 Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical
14 Sciences, Mashhad, Iran
15 * Corresponding authors:
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16 Prof. Khalil Abnous, Pharmaceutical Research Center, Institute of Pharmaceutical Technology, Mashhad
17 University of Medical Sciences, Mashhad, Iran, abnouskh@mums.ac.ir, Tel.: +98 513 1801535, Fax.:
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18 +98 513 882 3255

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Dr. Seyed Mohammad Taghdisi, Targeted Drug Delivery Research Center, Mashhad University of
20 Medical Sciences, Mashhad, Iran, taghdisihm@mums.ac.ir, Tel.: +98 513 1801203, Fax.: +98 513 882
21 3255
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23 Abstract

24 Exosomes are endosomal-derived vesicles, playing a major role in cell-to-cell communication.

25 Multiple cells secret these vesicles to induce and inhibit different cellular and molecular
26 pathways. Cancer-derived exosomes have been shown to affect development of cancer in

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27 different stages and contribute to the recruitment and reprogramming of both proximal and distal
28 tissues. The growing interest in defining the clinical relevance of these nano-sized particles in

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29 cancers, has led to the identification of either tissue- or disease-specific exosomal contents, such
30 as nucleic acids, proteins and lipids as a source of new biomarkers which propose the diagnostic

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31 potentials of exosomes in early detection of cancers. In this review, we have discussed some
32 aspects of exosomes including their contents, applications and isolation techniques in the field of
33 early cancer detection. Although, exosomes are considered as ideal biomarkers in cancer
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diagnosis, due to their unique characteristics, there is still a long way in the development of
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35 exosome-based assays.

36 Keywords:
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37 Exosomes; Biomarker; Cancer; Diagnosis; Early detection

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39 1. Introduction

40 Cancer is the most leading cause of death before age 70 years in 91 of 172 countries according to
41 the World Health Organization (WHO) in 2015 [1]. In fact, cancer incidence and mortality are
42 rapidly growing worldwide due to several reasons like aging and growth of the population.

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43 However, early diagnosis of cancer can increase the survival rate of patients. One of the latest
44 concepts regarding this field is the analysis of extracellular vesicles released by cells where the

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45 exosomes are at the frontier.

46 Cells secrete multiple types of cell-derived microvesicles including exosomes, plasma-

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47 membrane-budded microvesicles and apoptotic bodies with the ability to carry biologically
48 active cargo such as proteins and nucleic acids. Nonetheless, exosomes can evade the phagocyte

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49 system by their small size and show their superiority in intercellular communications [2, 3].
50 Exosomes are endocytic and heterogeneous membrane-derived vesicles which are actively
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51 secreted by various cell types. They have cup-shape structures, when visualized by transmission
52 electron microscopy, with a diameter of 30-100 nm [4]. Exosomes used to be considered as
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53 garbage cans for discarding redundant cell materials such as the transferrin receptors, caspase 3
54 and drugs [5-7]. The eminent feature of exosomes is their ability to carry content from the cell-
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55 of-origin to the target-cell very specifically, while this signal can be detected in blood [8]. Today,
56 other roles are suggested for exosomes including immune regulation [9], intercellular
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57 communications [10, 11] and biological events such as coagulation [12] and tissue
58 microenvironmental regulation [13] as well as their part in cancer development, metastasis and
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59 drug resistance [14, 15].

60 The process of exosome formation starts from inward budding of multivesicular endosomes
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61 (MVEs; also referred to as multivesicular bodies/MVBs); which are membrane-bound organelles

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62 that harbor small intraluminal vesicles, into the lumen of the compartment. Early endosomes are
63 formed via non-clathrin or clathrin-mediated endocytosis. The acidification of early endosomes
64 leads to the formation of late endosomes. Thus, MVEs, can fuse with the cell membrane or with
65 the lysosomes to either release the vesicles as exosomes or degrade its content. Next to the
66 classical pathway, a second route of exosome formation has been proposed by Pol and
67 colleagues (Figure 1) [16]. This pathway involves direct release of vesicles from the plasma
68 membrane. The content of the MVBs can vary from nucleic acids to proteins and lipids.
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69 Exosomes can be obtained from cell culture supernatants or various body fluids like plasma or
70 serum, saliva, breast milk and urine [17-19]. Commonly, exosome isolation methods rely on
71 ultracentrifugation at more than 100,000×g. However, there are other techniques like antibody-
72 coated magnetic beads, commercial precipitation kits, microfiltration techniques and the state-of-
73 the-art microfluidic technology for capturing exosomes.

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74 In cancer, exosomes play an important role in tumor initiation and growth [4]. Moreover, they

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75 are important players in the malignant progression of cancer with mechanisms, such as
76 immunosuppression via the inhibition of immune cell proliferation, induction of apoptosis of

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77 activated CD8+ T cells and suppression of natural killer cells activity [20, 21]. The new studies
78 have reported higher amount of exosomes in patients diagnosed with cancer compared to healthy
79 individuals [14]. In addition, exosomes are considered as a potential source of information for

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80 detection of cancer and evaluation of tumor progression and metastasis [14, 22]. Exosomal
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81 sorting complex responsible for transport (ESCRT) mechanism has been shown to provide
82 exosomes that resemble the content of a parent cell and make exosomes as attractive biomarker
83 candidates [23-25]. ESCRTs were characterized originally in a series of genetic screens as a
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84 protein sorting and membrane sculpting/scission machinery using yeast [26]. In fact, in the
85 absence of ESCRT function, transmembrane cargoes cannot efficiently reach the lumen of the
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86 lysosome. There are several exosome-based studies for diagnosis of pancreatic, prostate, breast
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87 and ovarian cancers; glioblastoma (GBM); and melanoma [27-32].

88 In this review, we have discussed some aspects of exosome biology including the content and the
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89 specific markers of exosomes that help in detection and monitoring cancers. Herein, the utility of
90 proteins and nucleic acids have been studied more than lipids and metabolites. Moreover, the
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91 techniques used for isolation and identification of exosomes, have also been reviewed.
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92 2. Exosomal content and cancer diagnosis

93 2.1. Proteins as exosomal biomarkers

94 Exosomes contain different molecules such as proteins, miRNAs and mRNAs which have
95 different roles in cancer (Figure 2) [33]. Tumor could be initiated through an intercellular
96 transfer of proteins by exosomes [34]. The proteomic studies showed a specific set of proteins
97 coming from the cytosol as well as the plasma membrane reflecting the cellular origin of the

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98 exosomes. Some proteins are specific to the cell-of-origin, while others are common across all
99 exosomes. Table 1 summarizes the major exosomal protein biomarkers which have been studied
100 in cancer diagnosis.

101 2.1.1. Tetraspanins

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102 Tetraspanins such as CD9, CD63, CD81 and CD151 are membrane proteins which are present in
103 exosomes originated from nearly any cell type [35, 36].

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104 CD9, one of the widely distributed members of tetraspanin family, is a 24 kDa cell surface
105 glycoprotein which is involved in cell adhesion and migration [37]. It is also expressed in some

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106 of tumors and normal human cells as well as exosomes [38-40]. The expression of CD9 is
107 reduced in metastatic lesions compared with primary tumors, and correlates with the bad

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108 prognosis of tumors at advanced stages as reviewed by Yang and colleagues [41]. However, the
109 increase of exosomal CD9, during the progression of prostate cancer (PCa), has been reported by
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110 Soekmadji and colleagues [42]. CD9 was firstly identified on exosomes from dendritic cells and
111 since then it is often used as an exosome biomarker [43, 44]. Using ExoScreen technique,
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112 Yoshioka and colleagues detected significantly higher levels of CD147/CD9 double positive
113 exosomes in sera from colorectal cancer patients (n =194) than healthy controls (n = 191) [45].
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114 In another study, Doldán and colleagues developed an electrochemical immunosensor for the
115 detection of exosomes based on CD9 antibodies functionalized gold electrodes [46]. The
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116 biosensor successfully detected CD9 on exosomes from MCF7 breast cancer cells. The role of
117 CD9 in modulating the activity of cellular signaling pathways through exosomes was studied by
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118 Chairoungdua and colleagues [47]. They suggested that down-regulation of the Wnt/β-catenin
119 signaling pathway mediated by CD9 expression, might suppress tumor metastasis.
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120 Also, Rana and colleagues showed that tetraspanins TSPAN8 and CD9 can regulate exosomal
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121 uptake by the target cell [48]. Chen and colleagues identified more than 2000 proteins in urinary
122 exosomes which revealed the high enrichment rate of CD9 in urinary exosomes [49].

123 CD63, lysosomal-associated membrane protein 3 (LAMP-3) or CD63, is a subfamily of

124 tetraspanin superfamily which is localized within the endosomal system as well as the cell
125 surface [50]. This antigen is identified as an exosomal biomarker which also has a role in
126 exosome biogenesis and secretion [51]. The expression of CD63 is down regulated upon the

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127 progression of some types of cancers including melanoma, ovarian and lung cancers as reviewed
128 by Pols and colleagues [52]. Liang and colleagues suggested the role of exosomes in ovarian
129 cancer progression and chemoresistance acquisition. They also reported the existence of CD63
130 on exosomes derived from two ovarian cancer cell lines, OVCAR-3 and IGROV1 [53]. In
131 another study, Logozzi and colleagues applied a sandwich ELISA to capture and quantify

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132 exosomes with high levels of CD63 in plasma sample of patients with melanoma [54]. In order
133 to predict future metastatic location of cancer cells, Suetsugu and colleagues developed a

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134 fluorescent protein-based imaging strategy using a GFP-tagged CD63 to determine the fate of the
135 breast cancer-cell derived exosomes [55].

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136 Another member of the tetraspanin superfamily with a broad tissue distribution and regulation
137 role in cell development, activation, growth and motility is CD81 protein [56, 57]. The

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138 expression of this antigen is essential in both T and B cells for T-cell activation [58]. CD81 is
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139 highly enriched in extracellular vesicles and has been identified as an exosomal biomarker [40].
140 Welker and colleagues studied patients with chronic hepatitis C and discovered the relation
141 between elevated CD81 in the exosomal serum fraction and inflammatory activity and severity
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142 of fibrosis [59]. Luga and colleagues found that exosomes secreted by fibroblasts lacking CD81,
143 can reduce the metastasis of MDA-MB-231 cancer cells to the lungs of immunocompromised
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144 mice [60]. Their findings suggested the key role of CD81 in metastasis.
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145 CD151 is a transmembrane protein which supports cell migration and motility through
146 association with laminin-binding integrins as well as proteases [61, 62]. This antigen has been
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147 proposed as a cancer marker associated with cancer progression and metastasis [63]. Elevated
148 CD151 expression in different types of cancers correlates with poor prognosis [64, 65]. Sadej
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149 and colleagues reported that the tumorigenicity and communication between tumor and
150 endothelial cells can be decreased through down-regulation of CD151 which proposed this
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151 antigen as a potential prognostic biomarker [66]. CD151 is a well-known tetraspanin engaged in
152 metastasis which exists on the surface of exosomes [61]. Sandfeld-Paulsen and colleagues
153 conducted a study to evaluate the role of exosomes in cancer diagnosis which showed that
154 CD151 is highly expressed in exosomes from patients with lung cancer [67]. Moreover, this
155 antigen has been correlated with the increase in aggressiveness of the tumor.

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156 2.1.2. Rab proteins

157 Like other GTPases, Rab proteins have two conformations; the inactive GDP and the active GTP
158 bound forms. There are more than 60 kinds of Rab proteins in human. Rab proteins are key
159 proteins in regulation of membrane trafficking, intracellular transport, lipid remodeling, vesicle
160 tethering and fusion and exosome release [68, 69]. Depending on the cell type, Rab5, Rab7,

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161 Rab11, Rab27 and Rab35 are involved in the vesicle secretion and therefore, cancer prognosis
162 [70-73].

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163 Rab5 is involved in early endosome transport, biogenesis and retrieval of synaptic vesicle

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164 components as well as mediating endocytosis pathway [74-76]. Therefore, interfering with the
165 levels and activation of Rab5 can influence the endocytosed materials in early endosomes. It has
166 been suggested that hypoxia stimulates tumor cell migration and invasion through altering

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167 endosome dynamics in cancer cells [77]. Therefore, Silva and colleagues studied the roll of
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168 hypoxia-induced Rab5 overexpression in tumor cell migration, invasion and metastasis in lung,
169 breast and melanoma cancers [78]. They showed that Rab5 is a key mediator of hypoxia-induced
170 tumor cell invasion.
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171 Rab proteins play a role in exosome release [68]. Therefore, dysregulation of Rab levels may
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172 influence cancer development. Baietti and colleagues found that chronic activation of Rab5 can
173 reduce exosomal release of biomarkers such as syndecan, CD63, and Alix [79]. In another study,
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174 Ostrowski and colleagues showed that knocking out of five Rab proteins, including Rab5, may
175 lead to inhibition of exosome secretion [80].
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176 The role of Rab7 in cellular processes is rather perplexing, since it regulates endosomal
177 maturation and directs the late endocytic pathway as well as the fusion activity of MVBs[81].
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178 Moreover, this protein is known to be involved in endocytic trafficking, phagocytosis and
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179 autophagy [82]. Alonso-Curbelo and colleagues demonstrated that RAB7 can induce tumor
180 invasion in melanoma patients [83]. It has been shown that Rab7 can regulate the release of
181 exosomes in MCF-7 tumor cells [79].

182 Rab11 and Rab35 are involved in recycling and early sorting of endosomes in exosome secretion
183 process [72, 84, 85]. They are widely expressed in different cell types [86]. It has been shown
184 that impairing the docking/fusion of MBVs to the plasma membrane which leads to hindrance

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185 the exosomal release, may be due to the inhibition of these GTPases [87]. Abrami and colleagues
186 showed that the depletion of Rab11 as well as Rab35 in RPE1 cells, reduced secretion of flotilin-
187 and anthrax toxin-loaded exosomes to the extracellular medium [88]. Chung and colleagues
188 studied the overexpression of Rab11 in colorectal cancer [89]. They suggested Rab11 as a
189 potential biomarker for monitoring the progression and treatment process of colorectal

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190 carcinoma. Recently, He and colleagues described the role of Rab11 overexpression in poor
191 prognosis of pancreatic cancer [90].

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192 The 2 isoforms of Rab27, Rab27A and Rab27B, are involved in the late secretory pathway of

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193 exosomes in multiple cancer types [91]. The relationship between the amount of Rab27
194 expression and the invasiveness of cancer cells has been found in several studies [92-94].
195 Ostrowski and colleagues showed that these isoforms can control different steps of the exosome

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196 secretion pathway [80]. Moreover, they showed that silencing of Rab27A and Rab27B can
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197 reduce the exosome content of the supernatant of the HeLa cell culture. Worst and colleagues
198 studied the expression of Rab27A, Rab27B and VPS36 in PCa. They found that these three are
199 down regulated in metastatic PCa tissue samples [95]. Recently, Li and colleagues applied a
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200 specific small hairpin RNA for Rab27A which leads to the decrease in the secretion of exosome
201 by A549 cells [96].
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202 2.1.3. Annexins

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203 Annexins are a group of calcium- and phospholipid-binding proteins which are enriched in
204 exosomes for membrane trafficking and fusion events [97, 98]. Their role in modulating cell
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205 adhesion and affecting cancer progression has been identified in several studies. Boudhraa and
206 colleagues published a review article discussing the role of annexin A1 in cancer [99]. Recently,
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207 Mallawaaratchy and colleagues suggested exosomal annexin A1 as a potential prognostic

208 biomarker for GBM [100].
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209 Annexin A2 is another member of this family which is up-regulated in different cancers and
210 plays an important role in many cancer-associated functions [101]. Maji and colleagues
211 demonstrated that exosomal annexin A2 is highly expressed in malignant cells and is implicated
212 in the metastatic breast cancer microenvironment signaling [102]. In another study, Zhang and
213 colleagues found that knockdown of annexin A2 can inhibit migration and invasion of

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214 hepatocellular carcinoma cells through regulating the trafficking of CD147-harboring membrane
215 microvesicles [103].

216 Annexin A6 is closely associated with a variety of tumors, acting either as a tumor suppressor or
217 motility promoting factor [104]. Its role depends on the type of cancer and the level of

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218 malignancy. This protein contributes to the invasiveness of breast cancer cells by affecting the
219 location of cadherins through influencing the functional focal adhesions [105].

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220 2.1.4. Flotillins
221 Flotillins, including flotillin 1 and flotillin 2, are membrane-associated proteins which are

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222 involved in scaffolding, signaling, and endocytosis functions [106]. They are enriched in
223 exosomes and can be used as an exosomal biomarkers [107, 108]. Strauss and colleagues found

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224 that treatment of oligodendrocytes with cholesterol can increase the release of exosomes which
225 are enriched in flotillin 2 [109]. Phuyal and colleagues suggested that flotillins can mediate
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226 exosomal release in androgen-resistant prostatic cancer cell line, PC-3 [110]. In another study,
227 Wang and colleagues designed a set of Western blot experiments which revealed that the level of
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228 flotillin 2 is higher in urinary exosome samples of PCa patients compared to healthy ones [111].

229 2.1.5. Proteins involved in ESCRT complex

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230 Programmed cell death 6-interacting protein (PDCD6IP) also known as ALIX is a cytoplasmic
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231 protein which is known for its role in apoptosis. Although the role of ALIX in biogenesis of
232 exosomes appears to be cell type dependent, it is involved in this process as a binding protein in
233 ESCRT complexes [112]. However, diagnostic and prognostic significance of ALIX initiated
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234 several studies on exosomes in different tumors [113-115].

235 Tumor susceptibility protein (TSG101) is an essential part of ESCRT complexes which mediates
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236 the biogenesis of MVBs [116]. Villarroya-Beltri and colleagues applied an ubiquitin-like
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237 modification named ISGylation which could induce aggregation and degradation of TSG101
238 protein in order to impair exosome secretion [117]. TSG101 is an exosomal marker which helps
239 to assess the number of exosomes in tumors [118]. Yang and colleagues found that the level of
240 TSG101 remarkably increased in the cytoplasm of tissue samples from breast cancer patients
241 with failed chemotherapy [119].

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242 2.1.6. Heat shock proteins

243 Exosomes also contain heat shock proteins (HSPs). These ubiquitous proteins are produced by
244 cells in stressful conditions. However, some of these proteins, such as isoforms of HSP70 and
245 HSP90 are expressed constitutively [120, 121]. HSPs have been found to play a role in antigen
246 presentation process and participate in loading peptides onto the major histocompatibility

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247 complex (MHC) molecules and thus have the capacity to stimulate antitumor immune responses
248 [122]. Lv and colleagues showed that exosomes from resistant human hepatocellular carcinoma

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249 cells can improve tumor immunogenicity with the induction of HSP-specific natural killer (NK)
250 cell responses [123]. The detection and quantification of exosomal HSPs can establish new

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251 diagnostic/prognostic biomarkers [124]. Gobbo and colleagues suggested HSP70 bearing
252 exosomes as a cancer marker [125]. They applied a peptide aptamer which could bind to the

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253 extracellular domain of HSP70 and blocks its interaction with toll-like receptor 2 (TLR2),
254 leading to the inhibition of myeloid-derived suppressor cells (MDSCs) activation which suggests
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255 the potential of targeting exosomes as a novel immunotherapy for cancer treatment. Campanella
256 and colleagues found that the level of exosome-associated HSP60 in colon cancer patients
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257 decreased after surgical removal of the tumor [126].

258 Table 1: Exosomal protein biomarkers in cancer diagnosis.

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Exosomal proteins Cancer type Clinical value Ref.

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Prostate The expression of CD9 is [127]

CD9 reduced in metastatic
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Bladder lesions. [49]

Melanoma [52]
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The expression of CD63 is

Tetraspanins
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CD63 Ovarian down regulated upon [53]

cancer progression.
Breast [55]

CD81 is overexpressed in
CD81 Breast stroma associated with [60]

human invasive ductal

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carcinoma.

Elevated CD151
expression in cancers
CD151 Lung [67]
correlates with poor

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prognosis.

Melanoma

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Rab5 overexpression can
Rab5 Lung induce tumor cell [78]

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migration.
Breast

Rab7 is an early-induced
Rab7
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Melanoma melanoma driver which [83]
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Rab shows patient prognosis.

GTPases Colorectal Rab11 overexpression [89]

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Rab11 relates with poor

Pancreatic prognosis. [90]
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Lung Blockage of Rab27A/B [96]

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expression reduces the

Rab27
Prostate number of exosomes [95]
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secreted.

Increased ANXA1
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A1 Glioblastoma transcript levels were also [100]

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observed in GBM.

Annexins Breast Annexin A2 [102]

overexpression has also
A2
Hepatocellular been demonstrated in [103]
different malignancies.

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Depending on the type of

cancer, it acts either as a
A6 Breast [105]
tumor suppressor or
motility promoting factor.

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The expression of flottilin
Flotillins Flotillin2 Prostate 2 relates to cancer [111]

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progression and prognosis.

ALIX expression is an

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Programmed cell death
ALIX colorectal important indicator of the [112]
6-interacting protein
metastatic potential.

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TSG101 helps to assess the
Tumor susceptibility
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TSG101 Breast number of exosomes in [119]
protein
tumors.
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HSP60 Colon The overexpression of [126]

Heat shock proteins HSPs signals a poor
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HSP70 Breast prognosis in cancers. [125]

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260 2.2. Nucleic acids

261 Nucleic acids are also present in exosomes and reflect the mutational status of the originated
262 cells. These molecules include double-stranded DNA (dsDNA), messenger RNAs (mRNAs), and
263 recently recognized micro RNAs (miRNAs) [33, 128]. Several other RNA species were also
264 recognized in exosomes, such as transfer RNAs (tRNAs), long noncoding RNAs (lncRNAs), and

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265 viral RNAs [129]. Recently, Chevillet and colleagues stoichiometrically analyzed the miRNA
266 content of exosomes. They suggested that most of miRNAs are present in far less than one copy

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267 per exosome, showing that most exosomes from standard preparations do not have the most
268 common sequences. [130]. Nevertheless, the role of miRNAs is highlighted as a marker in

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269 diagnosis and monitoring of the progression of different types of cancers [131, 132]. For
270 example, it has been shown that miR-21 is overexpressed in exosomes obtained from patients

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271 with GBM, esophageal squamous cell carcinoma and cancer [133-135]. However, in patients
272 with hepatocellular carcinoma, findings on circulating miR-21 are inconsistent and do not
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273 provide sufficient clinical evidence for other differential diagnosis [136]. In another study,
274 Fabbri and colleagues found that exosomal miR-21 and miR-29a can metastasize preferentially
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275 to the lung and reduce overall survival of lung cancer mouse models [137]. In order to find
276 whether the level of miR-21 is a valuable diagnostic biomarker for patients with breast cancer,
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277 Anfossi and colleagues developed a study and demonstrated high levels of miR-21 in the serum
278 of patients with non-metastatic breast cancer [138]. Moreover, Lee and colleagues found
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279 significantly higher miR-21 levels in MCF-7 exosomes in comparison to normal cell-derived
280 exosomes [139]. Nilsson and colleagues developed an approach to use the RNA content of tumor
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281 exosomes derived from urine in the search for PCa biomarkers. They analyzed the tumor
282 exosomes and detected two PCa mRNA biomarkers, PCA-3 and TMPRSS2:ERG [140].
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283 However, they carried out a pilot study and the concept needs more efforts to be considered
284 potentially for diagnosis and monitoring of the cancer patient’s status.
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285 miRNAs have also been shown to stimulate tumorigenesis and neoplastic transformation. In
286 cancers, mesenchymal stem cell (MSCs) derived exosomes could deliver unique miRNA species
287 to other adjacent cells in order to promote tumor progression [141]. Zakaria and colleagues
288 determined the presence of miR-125b,130b and 155 in prostate cancer-cell derived exosomes
289 and studied their involvement in induction of neoplastic transformation of adipose-derived stem

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290 cells [142]. Table 2 summarizes exosomes containing nucleic acids which have been studied as
291 biomarkers and tumorigenesis factors in cancers.

292 Table 2: Exosomal nucleic acid biomarkers in cancer diagnosis.

Exosomal nucleic acids Cancer type Clinical value Ref.

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Induces neoplastic transformation
miR-125b, 130b, 155 Prostate [142]

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in adipose derived stem cells.

Significantly upregulated in

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Multiple exosomes from HR-MM and
miR-135b [143]
Myeloma enhances endothelial tube

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formation.
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Associated with the acquisition of
miR-10b Breast [138, 144]
malignant characteristics.
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Upregulated in exosomes and

miR-210 Leukemia enhances endothelial migration [145]
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and tube formation.

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Promotes the invasion of breast

miR-223 Breast [146]
cancer cells.
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miR-29a Lung Tumor growth and metastasis. [137]

miR-19a Breast Represents a biomarker. [138]

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miR-105 Breast A potent regulator of migration [147]

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microRNAs associated
with the RISC Loading Breast Induces cell transformation. [22]
Complex

miR-1229,let-7a Colon Upregulated in primary colon [148]

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cancer patients.

miR-23b Bladder Acquires metastatic potential. [149]

Upregulated in ovarian cancer

miR-214 Ovarian [131]

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patients.

293

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294 2.3. Lipids

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295 The lipid composition of exosomes varies based on the characteristics of the cell-of-origin. They
296 may contain cholesterol, sphingomyelin, hexosylceramides, phosphatidylserine, and saturated
297 fatty acids [73]. Lysobisphosphatidic acid (LBPA) is another lipid that is found in the internal
298
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membrane of MVBs and has an important role in exosome biogenesis [150]. Beloribi and
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299 colleagues assessed the effects of exosomal lipids on tumor cell behavior. They created
300 exosome-like nanoparticles (SELNs) with no nucleic acids and proteins. Moreover, they showed
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301 SELNs could inhibit Notch signaling pathway and apoptosis in SOJ-6 cells [151]. In another
302 study by the same group, the contribution of the exosomal lipids in tumor progression as well as
303 development of drug resistance was studied in MiaPaCa-2 cells [152]. They showed that SELNs
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304 could activate NF-kB, resulting in the overexpression and secretion of SDF-1α and consequently
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305 increase in survival of MiaPaCa-2 cells. Phuyal and colleagues reported the doubled cellular
306 levels of ether lipids in PC-3 cells which was pretreated with ether lipid precursor
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307 hexadecylglycerol [153]. Moreover, they found that cells containing high levels of ether lipids
308 could release more exosomes than control cells. Generally, the role of exosomal lipid
309 composition on tumor progression and their biological and pharmacological effects seems to be
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310 far from being well understood.

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311 3. Exosome purification and identification techniques in cancer diagnosis

312 The biomarker potential of exosomes holds huge promise and may revolutionize the way in
313 which we diagnose and manage cancers. Accurately measuring and purifying exosomes are the
314 most important problems in biology of vesicles. This becomes a critical issue when evaluating
315 exosomes for achieving therapeutic efficacy. There are several current approaches including

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316 optical methods, such as nanoparticle tracking analysis (NTA), dynamic light scattering (DLS),
317 and flow cytometry, and non-optical methods, such as resistive pulse sensing, electron
318 microscopy, and Raman spectroscopy, that may help in purifying and characterizing the vesicles
319 [154]. Electron microscopy is an informative method with the ability to show the vesicular
320 morphology. However, the amount of soluble factors contaminating the sample cannot be

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321 determined with this approach and it is not suitable for routine daily use. Western blotting is
322 another approach which has been used to find proteins such as calnexin and gp96 that would not

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323 take part in vesicle preparation. Although this approach is informative, but it is not quantitative
324 and the selection of such proteins is difficult [155]. On the other hand, current particle tracking

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325 approaches are not able to evaluate the entire size range of sample vesicles, and to discriminate
326 between vesicular and non-vesicular materials. Moreover, these tools lack the sensitivity to show

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327 the heterogeneity reported for extracellular vesicles.
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328 Basically, the major methods which are served for exosome purification/isolation are: i)
329 Ultracentrifugation with/without sucrose gradient; ii) Immunoisolation employing magnetic
330 microbeads; iii) Extraction kits and iv) Microfluidics. However, there are other novel detection
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331 modalities, like biosensing and Basic proteomics approaches which still need further
332 improvements in order to be applied for routine clinical tests.
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333 3.1. Ultracentrifugation

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334 The most commonly used method for exosome isolation is ultracentrifugation which relies on
335 several centrifugation steps including 800 g for 10 min, then 2000 g for 10 min, followed by
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336 centrifugation forces of more than 100,000 g [22, 156]. This high speed alongside with the
337 longtime of centrifugation duration, about 16 h or longer, is mandatory for a complete vesicle
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338 precipitation [157]. However, the differential centrifugation sediments exosomes as well as other
339 extracellular vesicles and contaminating protein debris, which are responsible for unsatisfactory
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340 results and relatively low yields [158].

341 To achieve higher purity, a sucrose gradient centrifugation step was added to the classical
342 ultracentrifugation [156]. In this method, sucrose sediments protein aggregates, whereas
343 exosomes float upward in a position with the same density. Using the sucrose gradient
344 ultracentrifugation method, Melo and colleagues could isolate cancer-cell derived exosomes with
345 a cell surface proteoglycan glypican-1 (GPC-1). GPC-1 could be found on exosomes obtained

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346 from the serum of patients with pancreatic or breast cancers. Moreover, exosomes bearing GPC-
347 1 could discriminate patients with pancreatic ductal adenocarcinoma (PDAC) at all stages from
348 non-pancreatic cancer controls, indicating the exosomal GPC-1 as an early detection marker of
349 pancreatic cancer [27].

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350 The miRNA content of exosomes is a reflection of parent cancer cells which drew the attention
351 of scientists to explore the miRNA expression levels as biomarkers [159]. This concept has been

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352 discussed in a vast number of studies which utilized ultracentrifugation method for exosome
353 isolation [22, 33, 133, 147, 148, 160-164]. In a recent work, Lee and colleagues developed a

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354 fluorescent oligonucleotide probe, molecular beacon, for quantitative detection of miR-21 in
355 exosomes isolated from breast cancer cells using ultracentrifugation method [139]. However,
356 they used commercial techniques for exosome isolation including Total Exosome Isolation™ and

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357 ExoQuick-TC™ but the results showed that the detection can be achieved regardless of the
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358 method of isolation, although the molecular beacon and miR-21 hybridization showed higher
359 fluorescence intensity when exosomes were obtained by ultracentrifugation.
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360 3.2.Immuno-isolation of exosomes by magnetic microbeads

361 Antibody-coated magnetic beads are a simple, rapid and promising method for exosome isolation
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362 from either cell culture media or body fluids. Compared to ultracentrifugation, higher recovery
363 and purity are reported for this technique [165]. Rapid characterization of the exosome surface
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364 phenotype by flow cytometry is an advantage of using bead-exosome complexes [156].

365 However, since the direct monoclonal antibody is against a specific exosomal membrane surface
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366 protein, the beads can only bind to a certain number of exosomes which leads to a partial
367 exosome isolation. Immuno-isolation of exosomes using magnetic microbeads has been
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368 performed on a number of cancer cells, including ovarian, colon and lung [131, 132, 166]. This
369 technique was utilized to target the epithelial cell adhesion molecule (EpCAM), which is
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370 overexpressed on epithelial progenitors, carcinomas, and cancer-initiating cells [132, 165].
371 Moreover, Tauro and colleagues compared differential centrifugation, density gradient
372 centrifugation with affinity capture procedures using colorectal cancer cell line, LIM1863 culture
373 supernatant and demonstrated the superiority of the affinity capture method as it enriched
374 exosomal markers and exosome associated proteins by at least two folds more than the other 2
375 methods used for exosome isolation. According to Klein-Scory and colleagues, affinity-based

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376 methods would be more likely to detect cancer-specific biomarkers as well as other proteins that
377 are expressed on exosome surfaces for exosome isolation [167]. They concluded that the
378 heterogeneity of pancreatic cancer cells (Paca44 and Panc1), depending on the expression of
379 anchor proteins on exosome surfaces, may affect the efficiency of EpCAM-based affinity
380 purification for exosomes.

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381 3.3. Extraction Kits
Commercially available kits such as the Invitrogen Total Exosome Isolation KitTM (Life

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382
383 Technologies, USA), ExoSpinTM Exosome Purification Kit (Cell Guidance Systems, USA),
ExoQuickTM exosome precipitation solution (BioCat, Germany) and ExoEasy Maxi KitTM

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384
385 (Qiagen, Germany) have been widely used by several research groups for exosome extraction.
386 Generally, these kits use polyethylene glycol or other polymers through an overnight incubation

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387 period at 4 ̊ C followed by low-speed centrifugation (10,000–20,000g) to induce sedimentation
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388 of exosomes from solution [168]. The fact that these methods are relatively quick and do not
389 require laborious ultracentrifugation step, make them an attractive technique for exosome
390 isolation. However, the mode-of-action of these kits has not been validated and similar to
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391 ultracentrifugation method, they may also precipitate non-vesicular contaminants. Moreover, the
392 high price of these kits is an important issue, especially if a large number of biological samples
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393 are to be processed [169].

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394 Exosomes isolation using extraction kits have been used in a number of studies in order to detect
395 cancerous biomarkers. Bryant and colleagues performed a comprehensive analysis of the miRNA
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396 composition of exosomes using ExoMiR extraction kit (Bio Scientific, Austin, TX, USA) [170].
397 Their findings showed a relation between miR-141 as well as miR-375 and metastatic PCa.
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398 Moreover, they found that the concentrations of miR-107 and miR-574-3p were significantly
399 higher in men with PCa than in healthy men. These findings revealed the potential of these
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400 miRNAs as biomarkers for an early indication of PCa but they have to be verified in a larger
401 cohort of samples. Using ExoQuick precipitation kitTM, Tanaka and coworkers found an
402 overexpression of another miRNA, miR-21, in exosomes isolated from the serum of patients
403 with esophageal squamous cell carcinoma suggesting its potential application in cancer diagnosis
404 [171]. Eichelser and colleagues were another group who utilized ExoQuick precipitation
405 technique for exosome isolation from breast cancer patients and analyzed their exosomal miR-

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406 373 as a biomarker for detecting aggressive tumors [172]. They showed elevated serum levels of
407 miR-373 in estrogen receptor (ER)-negative and progesterone receptor (PR)-negative, compared
408 to receptor-positive breast cancers. Furthermore, they demonstrated the fact that survival rate
409 differed significantly between breast cancer subtypes. However, different miR-373 levels were
410 observed in both ER/PR-negative cases and receptor-positive patients, questioning its clinical

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411 application. The ExoQuick precipitation technique has been used to identify cancer associated
412 biomarkers rather than miRNAs for early cancer detection. Alegre and colleagues isolated

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413 exosomes from serum of patients suffering melanoma cancer and evaluated their exosomal
414 biomarkers, MIA, S100B and tyrosinase-related protein 2 (TYRP2), as diagnostic and prognostic

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415 markers [32]. However, TYRP2 did not meet the requirements as an early diagnostic marker.

416 3.4. Microfluidics

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417 Microfluidics is the technology of transporting very little volumes of fluids (from micro to
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418 picolitres) through a network of micro-sized channels for applying in-line sensing on a micro-
419 electromechanical system [173]. With the emerging interest in the use of exosomes as diagnostic
420 tools, new isolation [174, 175], quantification [176] and molecular profiling [177] platforms
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421 based on microfluidic technology have been developed. These platforms can be categorized into
422 active and passive modes. Active separation methods require an external force field like acoustic
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423 field, for operation. Lee and colleagues reported an acoustic nano-filter system for exosomes
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424 purification according to their size and density [178]. In this microfluidic platform, vesicles
425 under 200 nm can be isolated using acoustic nanofilters, moreover this system allows for
426 differentiation of multiple types of vesicles according to their size. However, the price of this
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427 chip seems to be higher than other methods for application in clinical settings. Wu and
428 colleagues have developed an acoustofluidic method to isolate exosomes and other EVs directly
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429 from undiluted blood samples [179]. The high purity and efficiency of this method are some of
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430 its advantages. Additionally, the authors claimed that this platform was cost-effective. Recently,
431 Zhao and colleagues developed a microfluidic “Exosearch cheap” for the diagnosis of ovarian
432 cancer based on three most common ovarian cancer exosomal tumor markers namely CA-125,
433 EpCAM and CD24 through a continuous magnetophoresis technique for in-situ exosome
434 isolation [30].

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435 The most common passive approach for exosome isolation involves immune-affinity based
436 trapping method, including Exochip, iMER, µNMR and nPLEX systems [180]. Chip-based
437 immune-affinity microfluidic approaches are widely sought among different groups, due to its
438 ability for quantitative and high throughput analysis of exosomes contents [181-184]. However,
439 further off-chip additional steps for sample preparation and complicated fabrication process are

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440 challenges for these platforms. The principals here include the use of chip-bound antibodies with
441 high affinity for exosomal surface proteins in order to separate them (Figure 3). There are other

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442 passive techniques rather than immune isolation approach for separation of exosomes based on
443 microfluidics. These techniques could be combined with two or more other separation techniques

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444 in one device, resulting in manufacturing of integrated microfluidic systems. For example, Kim
445 and colleagues applied an electric gradient over nano-porous membranes of microfluidic devices

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446 [185]. Thus, they could enhance the pre-concentration and purification efficacy of these
447 membranes on exosome samples. In another study, Davies and colleagues developed a physical
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448 sieving method which directly separated exosomes from whole blood either by means of
449 pressure or electrophoresis through a membrane [175]. Short processing time and high purity of
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450 isolated exosomes are the advantages of this method whereas biased data because of non-
451 selective isolation as well as low recovery yield of isolated vesicles are considered as drawbacks
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452 of sieving method. Another filtration platform which was developed by Wang and colleagues
453 utilized porous microstructure for isolation of exosome-like vesicles [186]. Although the
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454 trapping procedure is fast and requires a small sample volume, the imaging and characterization
455 steps are rather time-consuming. Moreover, no analysis of the platform in clinical samples has
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456 been reported.

457 3.5. Biosensors

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458 Biosensors are analytical devices which consist of a biological sensing component, such as
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459 antibodies, enzymes or aptamers, and a transducer that convert the sensing signal to a measurable
460 physicochemical, optical and electrochemical signal [187-189]. They have been applied in many
461 fields, including food analysis and medical diagnosis, considering that their signal is being
462 generated in real time [190, 191].

463 Electrochemical-based detection sensors produce an electrical signal upon reacting with the
464 target substance of interest. These sensors have an innate high sensitivity [192]and simplicity

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465 which can be coupled with miniaturized and automated hardware in order to be used in routine
466 analysis [46]. However, only few studies on exosome-specific electrochemical biosensors have
467 been reported. Table 3 lists the limit of detection (LOD) of current electrochemical-based
468 methods employed for the detection of exosomes.

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469

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470 Table 3: Biosensors for the detection of exosomes.

LOD
Type of biosensor Ref.
(particles/µL)

Electrochemical aptasensor 0.1×104 [193]

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EFIRM method - [194-196]

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Electrochemical detection method 0.47×106 [197]

0.5×102

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Electrochemical detection method using metal nanoparticles [198]

Electrochemical sandwich immunosensor 2×102 [46]

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Magneto-Electrochemical sensor 3×104 [199]
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471

472 Based on the idea of improving the sensitivity of the method compared to routine immune assay,
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473 Zhou and colleagues developed an aptamer-based electrochemical sensor with specificity for
474 tetraspanin CD63 [193]. The detection strategy was based on competition between redox-labeled
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475 complementary oligonucleotides and the exosomes for the immobilized aptamers on the surface
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476 of gold electrodes. Moreover, they incorporated the sensor into a microfluidic device to
477 minimize the sample volume. In order to provide an on-site detection of distal tumor-shed
478 biomarkers, Wei and colleagues established a liquid biopsy method called electric field induced
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479 release and measurement (EFIRM) in conjunction with magnetic beads to isolate exosomes and
480 detect their biomarkers in blood and saliva [194]. However, this technique needs further
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481 optimization as well as improvement of exosome stabilization. Lau and colleagues used the same
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482 EFIRM method to study tumor-derived exosomes in a pancreatic cancer mouse model [195]. In
483 order to use exosomes in clinic, more rapid and accurate methods for the detection and isolation
484 of exosomes are needed. Doldán and colleagues fabricated a sandwich mode amperometric
485 immunosensor for the detection of exosomes based on CD9 antibodies functionalized gold
486 electrodes [46]. Although their method had high sensitivity and specificity for the detection of
487 exosomes in real samples, but more optimizations are still needed to incorporate the sensor into
488 miniaturized and semiautomatic devices.

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489 3.6. Mass-spectrometry (MS)-based molecular profiling

490 3.6.1. Basic Proteomics Approaches

491 Exosome proteomics studies can help in understanding the biological roles of exosomes as well
492 as identifying new biomarkers in patients' fluids. Like exosome-derived miRNA, the proteomic

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493 profiling can help in identifying diagnostic indicators of cancers such as melanoma, PCa, brain
494 and lung cancer [200-203]. Recently, Duijvesz and colleagues utilized ultracentrifugation

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495 method for isolating exosomes containing novel candidate biomarkers including ALIX, FASN,
496 XPO1 and ENO1, as identified by LC-FTMS [201]. Although they confirmed exosomal presence

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497 and cancer tissue expression of a selected set of biomarkers, these biomarkers came from
498 exosomes derived from prostate cell line and the validation of such markers has to be done on a
499 large collection of samples. Two main strategies for exosomal protein characterization have been

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500 employed to examine the protein cargo of exosomes. The most widely used analytical approach
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501 is based on one-dimensional SDS-polyacrylamide gel electrophoresis, followed by liquid
502 chromatography (LC)‐MS/MS. Multi‐dimensional protein identification technology (MudPIT) is
503 another approach which is used to study the exosome proteome in a gel‐free method [204]. A
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504 major goal in using proteomics in the field of exosome research is to identify disease biomarkers
505 for the early diagnosis. For example, Mathivanan and colleagues developed proteomics analysis
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506 of a colorectal cancer cell-derived exosomes [166]. Using LC-MS/MS, they revealed 394 unique
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507 exosomal proteins of which 18% are localized in nuclear, 29% and 19% are cytoplasmic and
508 plasma membrane, respectively. Moreover, the presence of a subset of proteins common to all
509 exosomes as well as cell-specific ones was confirmed. In order to detect early signals of the
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510 cancer, Keller and colleagues studied on tumor progression related proteins (L1CAM, CD24,
511 ADAM10 and EMMPRIN) and found that they can be detected in malignant ascites-derived
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512 exosomes from ovarian carcinoma patients [205]. Therefore, they proposed that the detection of
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513 membrane proteins on circulating exosomes may be exploited for diagnostic purposes. Recently,
514 Wang and colleagues developed a study in order to explore the overall changes in exosomal
515 proteomes in metastatic and non‐metastatic non‐small‐cell lung cancers (NSCLC) and healthy
516 human serum samples using Tandem mass tags combined with multidimensional LC and MS
517 analysis [206]. Moreover, they hypothesized that serum exosomal biomarkers are potential
518 diagnostic markers in predicting NSCLC metastasis.

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519 Today, exosome samples from biofluids are related to the entire populations of cells. Therefore,
520 higher specificity in biomarker detection can be achieved through cell-specific exosome analysis.
521 Moreover, improved analytical sensitivity has led to the detection of new biomarkers as well as
522 new sensitive and specific assays in order to early disease detection and prognosis.

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523 3.6.2. Lipidomic characterization of exosomes
524 Lipidomics involves the characterization and quantitation of lipid species through different

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525 approaches, including LC and gas chromatography (GC) coupled to MS, in various biological
526 samples including exosomes [207]. Lydic and colleagues developed a broad exosome lipid

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527 analysis with lipid allocation at the entire composition level across a broad range of
528 glycerophospholipid, glycerolipid, sphingolipid, and sterol lipid classes and subclasses and
529 measured 500 lipid species in the colorectal cancer cell line LIM1215 and its exosomes using

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530 optimized sample extraction and novel sample derivatization techniques, coupled with high-
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531 resolution ‘shotgun’ MS and ‘targeted’ tandem MS methods [208]. Lipidomic characterization of
532 exosomes may reveal novel biomarkers in disease diagnosis, prognosis, and treatment. Llorente
533 and colleagues characterized 280 molecular lipid species from metastatic prostate cancer cell-
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534 line (PC-3)-derived exosomes. They found that exosomes were significantly enriched in
535 glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine (PS). Furthermore, they
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536 proposed hexosylceramides (HexCer) and lactosylceramide (LacCer) lipids from PC-3 exosomes
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537 which could be used as cancer biomarkers. In another study, Skotland and colleagues, reported
538 the lipid composition of exosomes purified from urine of 15 patients with PC and 13 healthy
539 controls [209]. Significantly different levels of nine individual exosomal lipid species were
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540 identified between the two groups. Therefore, they concluded that lipids in urinary exosomes
541 may be considered as promising PC biomarkers.
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542 4. Conclusion and Future prospective

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543 The full understanding of different interactions between components within tumors could pave
544 the way for a successful cancer treatment. The new emerging technologies hold the promise of
545 curing cancer by differentiating benign tumors from early- and late-stage cancers. Additionally,
546 the idea of applying a simple blood test in order to distinguish early malignant changes is
547 extremely appealing.

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548 Exosomes are cell derived particles with the ability to represent the state of their parenteral cells.
549 They are relatively stable in the blood with important roles in inter and intracellular
550 communications. Today, the young field of exosomes in cancer detection is gaining greater
551 interest within the research groups and medical communities. However, the way that exosomes
552 influence distant cell interactions within the tumor microenvironment has not yet been fully

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553 understood.

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554 In this review, we discussed some aspects of exosome biology focusing tumor related biomarkers
555 alongside the techniques used for the isolation and identification of exosomes. Due to the unique

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556 characteristics of exosomes and their wide distribution in the body fluids, these vesicles are
557 considered as ideal biomarkers in cancer diagnosis. However, there is still a long way to the
558 development of exosome-based assays. For example, the identification of clinically relevant

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559 exosomes among numerous other populations of exosomes secreted by almost all body cells is
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560 still a challenge mainly due to the lack of adequately sensitive and fast analysis platforms.
561 Therefore, the development of more accurate methods for the isolation and characterization of
562 different subpopulations of vesicles is highly in demand. Moreover, developing new strategies to
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563 obtain a large amount of exosomes for clinical evaluation as well as introducing the therapeutic
564 agents into exosomes and exosome targeted delivery may be the most important issues in the
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565 field of exosome-based cancer diagnosis and therapy in the future. Design of simple, cost-
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566 effective and efficient lab-on-a-chip devices could improve both clinical uses and facilitate
567 biological studies of exosomes.
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569 Conflict of interest

570 We declare there is no conflict of interest about this article.

571 Acknowledgment

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572 Financial support of this study was provided by Mashhad University of Medical Sciences.

573

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1223 Figure legends

Figure 1 Pathways of exosome formation. Reproduced from Ref. [210].

Figure 2 Exosome biomarkers. Reproduced from Ref. [211].

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A 3 step immune-affinity based procedure used for exosome counting in
Exochip system. Reproduced from Ref. [212]. In this method blood serum pre-
Figure 3

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coated with exosome-specific capture antibodies (anti-CD63) was stained using
a fluorescent dye and analyzed through conventional counting methods

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Figure 1

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* Exosomes are endosomal-derived vesicles, playing a major role in cell-to-cell
communication.

* Exosomes are considered as ideal biomarkers in cancer diagnosis, due to their unique
characteristics.

* In this review, we have discussed some aspects of exosomes including their contents,
applications and isolation techniques in the field of early cancer detection.

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