The versatile role of exosomes in cancer progression: diagnostic and therapeutic implications

Abstract

Background

Recent advances in cancer biology have highlighted the relevance of exosomes and nanovesicles as carriers of genetic and biological messages between cancer cells and their immediate and/or distant environments. It has been found that these molecular cues may play significant roles in cancer progression and metastasis. Cancer cells secrete exosomes containing diverse molecules that can be transferred to recipient cells and/or vice versa to induce a plethora of biological processes, including angiogenesis, metastasis formation, therapeutic resistance, epithelial-mesenchymal transition and epigenetic/stemness (re)programming. While exosomes interact with cells within the tumour microenvironment to promote tumour growth, these vesicles can also facilitate the process of distant metastasis by mediating the formation of pre-metastatic niches. Next to their tumour promoting effects, exosomes have been found to serve as potential tools for cancer diagnosis and therapy. The ease of isolating exosomes and their content from different body fluids has led to the identification of diagnostic and prognostic biomarker signatures, as well as to predictive biomarker signatures for therapeutic responses. Exosomes can also be used as cargos to deliver therapeutic anti-cancer drugs, and they can be engineered to serve as vaccines for immunotherapy. Additionally, it has been found that inhibition of exosome secretion, and thus the transfer of oncogenic molecules, holds promise for inhibiting tumour growth. Here we provide recent information on the diverse roles of exosomes in various cellular and systemic processes governing cancer progression, and discuss novel strategies to halt this progression using exosome-based targeted therapies and methods to inhibit exosome secretion and the transfer of pro-tumorigenic molecules.

Conclusions

This review highlights the important role of exosomes in cancer progression and its implications for (non-invasive) diagnostics and the development of novel therapeutic strategies, as well as its current and future applications in clinical trials.

Introduction

Cells use different methods to communicate with each other and with their surrounding environment through direct contact or the secretion of soluble factors [1]. During cancer progression, cancer cells communicate with neighbouring cancerous and non-cancerous cells, including mesenchymal stromal cells, fibroblasts and endothelial cells, that collectively play crucial roles in cancer development [2]. The three major types of communication that cells use effectively are: active transport, passive transport and vesicular transport. Of the modes of vesicular transport via exosomes, i.e., micro-vesicles and apoptotic bodies, the former has been intensely investigated in recent years [1, 3]. Exosomes with a diameter of 50 nm to 100 nm are secreted by a wide range of cell types [4], including cancer cells, to perform a myriad of functions, such as the modulation of immune function [5], the regulation of cell metabolism [6], conferring drug resistance, and promoting metastasis and angiogenesis [7]. The presence of multi-vesicular endocytic compartments determines whether cells are capable of mediating exosomal secretion [4]. Exosomes, which are clustered under extracellular vesicles (EVs), are secreted by almost all mammalian cell types, as well as by some lower eukaryotes and prokaryotes [1, 8, 9]. Cells may secrete exosomes in response to a range of stimuli, including p53 activation [10, 11], ceramide synthesis [12], intracellular calcium concentration alteration [13], pH changes [14], plasma membrane depolarization [15, 16], T-cell receptor activation [17], hypoxia and ionising radiation [18]. How exosome biogenesis is regulated and what their impact is on recipient cells, which is largely determined by their content, will be discussed in the next section.

Exosome structure, biogenesis and content

The term ‘exosome’ was first coined by Tram et al. in 1981 as it was found that cultured normal and neoplastic cells may secrete vesicles with 5′-nucleotidase activity [19]. The process of exosome secretion was subsequently described in further detail by Pan et al. and Harding et al. after studying endocytosis and the intracellular processing of transferrin receptors (TR) in reticulocytes. It was found that colloidal gold-TF was incorporated into multi-vesicular endosomes (MVEs) as well as inclusion vesicles, and that these inclusion vesicles contained TR-receptors that were shed from developing reticulocytes via exocytosis [20, 21]. Exosomes may contain various biomolecules such as mRNAs, microRNAs (miRNAs), long non-coding RNAs (lncRNAs), DNA and proteins [22]. Intriguingly, it has been found that the contents of exosomes from donor cells can be transferred to recipient cells and that the DNA/RNA and protein molecules derived from these exosomes can functionally modulate cellular processes within recipient cells [23]. This type of communication may involve a wide range of cell types, including normal and tumour cells, fibroblasts and endothelial cells. Currently, exosomes have been isolated and purified from various sources of body fluids, such as breast milk [24], blood plasma [25], cerebrospinal fluid [26], saliva [27], peritoneum lavage fluid [28], urine [29] and serum [30], which underscores the putative importance of exosomes and their roles as messengers between cells, tissues and organs.

Exosome structure

Exosomes are cup-shaped vesicles with a lipid bilayer that is enriched in cholesterol, sphingomyelin and ceramide [12, 31]. Unlike apoptotic bodies and other vesicles, the formation and release of exosomes follow distinct pathways. The first step in the formation of exosomes involves inward budding of the membranes of endosomes to form MVEs with intraluminal vesicles (ILVs). The second step involves the release of exosomes. MVEs can either undergo degradation in lysosomes or fuse with the plasma membrane to release the ILVs as exosomes [4, 32]. Additional evidence strengthening the endosomal origin of exosomes comes from the observation that the proteins found in the exosomes have cytosolic origins, especially from endocytic compartments. Endocytic proteins that have been found in exosomes include annexin II, RAB5/RAB7 and TSG101 [4]. An important component required for the budding of endosomes into MVEs is the sphingolipid ceramide [32]. Trajkovic et al. found that exosomes contain high amounts of ceramide and the enzyme sphingomyelinase (SMase), which facilitates the formation of ceramide. They also found that treatment of cells with a neutral SMase inhibitor significantly inhibited exosome biogenesis and subsequent release [32], suggesting an important role of this enzyme in exosome biogenesis, which will be discussed in the next section.

Exosome biogenesis

The formation of MVEs can occur either in an Endosomal Sorting Complex Required for Transport (ESCRT)-dependent or an ESCRT-independent manner. The ESCRT machinery is composed of the ESRT-0, -I, -II and -III complexes. These complexes sort ubiquitinated intracellular cargos, such as internalised receptors, into MVEs and, by doing so, prevent their degradation in lysosomes [33]. The lysosomal/late endosomal degradation of the epidermal growth factor receptor (EGFR) is, for example, catalysed by ubiquitination of the EGFR upon its exit from early endosomes to lysosome/late endosomes by the E3 ligase c-Cbl [34, 35]. The release of MVEs from a cell begins with the binding of ESCRT-0 to endosomes and its subsequent interaction with the ubiquitin moieties of the cargos that would normally undergo lysosomal degradation [36]. Whereas ESCRT-0 forms domains of clustered cargos, ESCRT-I and ESCRT-II facilitate the formation of buds in the membranes of endosomes. The ESCRT-0-ubiquitin domains are recruited to the buds, followed by ESCRT-III which cleaves the buds to form ILVs [33]. Although ESCRT is considered to be important for the formation of ILVs, it has been found that ILV synthesis may also occur after ESCRT inhibition. The components of the endocytic pathway were found to exhibit morphological changes in ESCRT-depleted cells, but clearly maintained differences between early and late endosomes [37]. Important components of the ESCRT-independent pathway include activated sphingosine 1-phosphate [38] and the tetraspanin-enriched microdomain (TEM) interactome [39].

Exosome content

Since cancer cells communicate with each other and other cell types via exosomes through the messages that they carry, the exosome content must be variable and specific in order to convey different messages. The exosome database (www.exocarta.org) lists 9769 proteins, 3408 mRNAs and 2838 miRNAs in its latest update, several of which play important roles in tumour progression and metastasis through the creation of a vicious tumour microenvironment. Some of the most commonly found proteins in exosomes, which reflect their endosomal origin, include membrane transport and fusion proteins (GTPases, annexins, flotillin), tetraspanins (CD9, CD63, CD81, CD82), heat shock proteins (Hsp70, Hsp90), multi-vesicular body synthesis proteins (Alix, TSG101) and lipid related proteins [40]. Exosomes may also transport different lipids such as sphingolipids, cholesterol and ceramide, which are components of lipid rafts. They have also been reported to transport signalling molecules such as phospholipase A2 (PLA2), arachidonic acid and prostaglandins [7, 41]. Some of the lipids secreted by exosomes, such as lysophosphatidylcholine and PLA2, are known to modulate immune responses by favouring dendritic cell (DC) maturation [42, 43]. In addition, exosomes are known to contain nucleotides such as mRNAs, lncRNAs and miRNAs. Highly stable RNAse resistant nucleic acids, especially miRNAs [44], can be transferred to recipient cells to confer oncogenic and non-oncogenic functions, which will be discussed in further detail in Section 4.2.2. Internalization of exosomal contents in recipient cells may occur via their fusion with target cell membranes, clathrin-mediated endocytosis, caveolin-mediated endocytosis, lipid raft-mediated endocytosis, macro-pinocytosis, receptor-ligand interactions and/or phagocytosis [45–47].

There is ambiguity regarding the question whether any cell can take up exosomes or whether this process is cell-type specific. It has, for example, been reported that exosomes secreted by glioblastoma cells can be taken up by various normal and transformed cells [48]. Although exosomes secreted by rat pancreatic adenocarcinoma cells were found to be taken up in vitro and in vivo by a variety of leukocytes, this uptake was found to be associated with the presence of ligands on leukocytes that could interact with exosomal receptors [49]. Also, others reported a cell-type specific uptake of exosomes. Rana et al., for example, found that exosomes secreted by normal lymph node stromal cells expressing the Tspan8-alpha4 complex were most readily taken up by endothelial and pancreatic cells [50]. It has also been reported that cell-type specific uptake of exosomes may require the presence of ligands, such as MUC1, on their surface, which can interact with specific receptors, such as the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN), present on the surface of target cells [51]. The cell-type specific uptake of exosomes may be employed for the design of novel targeted therapeutic approaches and warrants further in-depth investigation on how exosomes can be tailored to express ligands on their surface corresponding to receptors that are (over)expressed by cancer cells.

Role of Rab GTPases and other regulators in exosome secretion

Dysregulation of Rab GTPases has been encountered in several cancers [52]. This family of enzymes is also known for its role in exosome secretion and transport, as well as in membrane trafficking. Different family members are known to regulate the secretion of small- and/or larger-sized exosomes and the transport of specific biomolecules, including miRNAs. It has been found, for example, that the transport of miR-143 by exosomes from endothelial cells, induced by the shear stress responsive transcription factor Krüppel-like factor 2 (KLF2), depends on the expression of Rab7a and Rab27b. Interestingly, it was also found that the KLF2-induced exosomal export of miR-143 was not dependent on Rab27a, a homologue of Rab27b, which is in accordance with the notion that Rab family GTPases exhibit cell type-specific functions [53]. Whereas it has been found that Rab5B, Rab9A and Rab17 expression is associated with the production of small-sized exosomal vesicles, Rab32 has been found to mediate the secretion of larger-sized exosomal vesicles [54]. It has also been found that inhibition of Rab35 may lead to a reduction of the mean size of the readily releasable pool (RRP) of exomes. Rab35 is a target of TBC1D10A-C, and its inhibition has been found to lead to impairment of exosome secretion [55].

It has been reported that the activities of different Rab GTPases may be regulated by different factors, including intracellular Ca²⁺ concentration [35], hypoxia-inducible factor-1α (HIF-1α) expression [37], heparanase expression [56] and/or microenvironmental pH changes [14]. The release of exosomes from the RRP mediated by Rab11 and Rab35 has, for example, been found to be stimulated by intracellular Ca²⁺ [55]. Rab11 plays an important role in the docking and fusion of multi-vesicular bodies (MVBs), but the release of exosomes has been observed only in the presence of increased intracellular Ca²⁺ concentrations [57]. The release of exosomes may also be accelerated under hypoxic conditions, and this effect has been found to be mediated by HIF-1α [58]. It has been reported that under hypoxic conditions tumour masses may undergo several changes, including the formation of cancer stem cells (CSCs), increased angiogenesis and the activation of cancer survival mechanisms [59]. Hence, cancer cells need to modulate their microenvironment in order to facilitate their growth and further transformation. This modulation may include the recruitment of cancer-associated cells and (excreted) stimulatory factors. These reciprocal interactions have been shown to be mediated by exosomes. Hence, the targeting of exosome-based communication mechanisms may serve as a therapeutic strategy. The validity of this notion has already been shown in some clinical trials, which will be discussed below.

Role of exosomes in cancer progression

Exosomes secreted by both tumour cells and tumour-associated cells play predominant roles in favouring tumour growth via the transfer of pro-tumorigenic factors (Fig. 1). Glioma cells, for example, may secrete exosomes containing EGFRvIII that can promote the growth of recipient cells that lack this truncated epidermal growth factor receptor variant through the activation of transforming signalling pathways, such as the Akt and mitogen-activated protein kinase (MAPK) pathways. In addition, it has been found that exosomes containing EGFRvIII may promote angiogenesis by enhancing the expression of the vascular endothelial growth factor (VEGF), as well as cellular proliferation and survival by increasing the expression of the anti-apoptotic protein Bcl-xL and by downregulating the expression of p27, a cyclin-dependent kinase (CDK) inhibitor [60]. The presence of inhibitors of apoptosis (IAP), such as survivin, XIAP, cIAP1 and cIAP2, in exosomes secreted from cancer cells may also protect cancer cells from apoptosis and favour tumour growth [61]. More recently, it has been found that exosomes may confer aggressive phenotypes to cancer cells through stemness regulators such as miRNAs. Breast cancer cells treated with exosomes secreted by cancer-associated fibroblasts (CAFs) containing miR-21, -378e and -143 were, for example, found to exhibit increased anchorage independent growth and mammosphere forming abilities concomitant with upregulation of the expression of stem cell markers (Oct3/4, Nanog, Sox2) and epithelial-mesenchymal transition (EMT) regulators (Snail and Zeb) [62]. These results suggest a role of the tumour microenvironment in modulating cancer cell fate and aggressiveness, resulting in tumour metastasis and relapse. Similarly, exosomes secreted by LNCaP and PC3 prostate cancer cells under hypoxic conditions have been reported to increase the stemness of naïve LNCaP and PC3 cells. When cultured in the presence of exosomes, significant increases in prostasphere formation were observed in these cells. This was due to the phenotypic conversion of fibroblasts into CAFs. Importantly, it was found that exosomes derived from hypoxic prostate cancer cells may contain more proteins such as tetraspanins, HSP and annexin II, matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9, cytokines and signalling molecules such as TGF-β2, tumour necrosis factor-(TNF)-1α, interleukin (IL) 6 (IL6), Akt, integrin-linked kinase 1 (ILK1), and β-catenin, than those derived from normoxic cells, and the authors postulated that loss of E-cadherin expression and concomitant increases in cytoplasmic and nuclear β-catenin levels might be responsible for the observed increased motility, invasion and stemness of these cells [63]. It has also been shown that tumour cells may adopt dormancy or the acquisition of a quiescent state at metastatic sites for certain periods of time until cues for tumour re-growth and progression become available. Exosomes released by cancer cells may promote tumour growth and metastasis at distant sites. They may also determine the site of metastasis based on the presence of specific factors, such as integrins, on their surface. In the following sections different mechanisms of exosome-mediated cancer progression will be discussed.

Fig. 1.

Schematic representation of exosomal molecules secreted by cancer cells and cells of the tumour microenvironment that drive different events leading to therapy failure, distant metastasis and the acquisition of aggressive phenotypes. Inhibition of exosomal secretion may represent a novel targeted strategy to treat drug-resistant tumours as well as those with a high metastatic capacity

Role of exosomes in cancer growth

Angiogenesis

Tumours require a continuous nutrient and oxygen supply through blood circulation. In a hypoxic milieu CSCs favour angiogenesis via the secretion of VEGF, which promotes endothelial cell migration and tube formation [64]. In addition, CSCs may contribute to the growth of new blood vessels by differentiating into endothelial cells [65] and by secreting pro-angiogenic factors such as angiopoietin [66]. It has also been reported that exosomes secreted by both tumour cells and CSCs may promote angiogenesis, but that these exosomes differ significantly in their content and their ability to promote tumour growth. It has been found, for example, that exosomes secreted by CD105⁺ CSCs contain high levels of 24 miRNAs regulating transcription, metabolic processing, proliferation, nucleic acid binding and cell adhesion molecules [67]. In addition, the authors found that only exosomes secreted by CD105⁺ CSCs contained pro-angiogeneic mRNAs such as those encoding VEGF, fibroblast growth factor (FGF), angiopoietin1, ephrin A3, MMP-2, and MMP-9 and growth factors. CD105⁺ CSC-derived exosomes were also found to be more effective than exosomes derived from CD105⁻ tumour cells in favouring angiogenesis. Others found that CD90⁺ liver CSCs secrete exosomes containing lncRNA H19, which may stimulate angiogenesis by favouring tube formation and enhancing the adhesions between CD90⁺ cells and endothelial cells by upregulating the expression of intercellular adhesion molecule 1 (ICAM-1) [68]. Since only a limited number of studies has been published on the occurrence of differences in composition and function of exosomes secreted by CSCs and tumour cells, and the notion that CSC-derived exosomes may be more tumorigenic, additional studies are warranted. Nevertheless, it can be postulated that it may be promising to develop therapeutic strategies aimed at both inhibiting exosome secretion by CSCs and targeted elimination of CSCs.

WNT5A signalling in melanoma cells may trigger Ca²⁺-dependent release of exosomes, and it has been found that these exosomes contain immunomodulatory and pro-angiogenic factors, such as IL6, VEGF and MMP-2. These factors, in turn, may enhance the immunosuppressive capacity of tumours as well as endothelial cell branching to confer more aggressive and metastatic phenotypes [69]. A recent study has shown that cancer cells undergoing EMT may secrete exosomes containing Rac1/p21-activated kinase-2 (PAK-2) and, by doing so, communicate with endothelial cells to promote angiogenesis [70]. Co-treatment of endothelial 2F-2B cells with exosomes secreted by tumour cells undergoing EMT increased the motility of these cells, enhanced the number of vessels formed and increased their length, again indicating that exosome-mediated communication is imperative for cancer progression. Conversely, it was found that inhibition of downstream targets of Rac1, such as PAK-2, reduced tube formation, tube length and branch points of endothelial cells, indicating that exosomal Rac1 signalling in endothelial cells may serve as a therapeutic target. Cigarette smoke extract (CSE)-transformed human bronchial epithelial (HBE) cells have been found to secrete signal inducer and activator of transcription-3 (STAT-3)-regulated exosomal miR-21, which can be transferred to normal HBE cells to change their phenotype towards a transformed state and, subsequently, increase their VEGF expression to promote angiogenesis. Additionally, it was found that treatment of human umbilical vein endothelial cells (HUVECs) with exosomes derived from CSE-transformed HBE cells containing miR-21 increased their tube formation, and that their relative tube length was increased in a dose-dependent manner [71]. The control over angiogenesis by switching to higher pro-angiogenic and lower anti-angiogenic protein levels has been underscored by studies in which exosomes derived from nasopharyngeal cancer cells were investigated. These exosomes were found to contain elevated levels of pro-angiogenic proteins, such as ICAM-1 and CD44v5, and decreased levels of the anti-angiogenic protein thrombospondin-1 (TSP-1). Internalization of these exosomes by HUVECs altered the levels of angiogenic proteins in these cells and, subsequently, elicited increased tube formation, migration and invasion [72]. Taken together, it appears that cancer cells may secrete several factors through exosomes that can modulate a variety of their own cellular activities and those of their surrounding cells, thereby harnessing tumour-associated cellular activities supporting tumour progression.

Additional lines of research have shown that hypoxia may favour angiogenesis by promoting the secretion of exosomes containing pro-angiogenic factors [73]. It has, for example, been found that exosomes secreted by leukaemia cells under hypoxic conditions may contain elevated levels of miR-210, and that these exosomes enhance tube formation in HUVECs by eliciting expression downregulation of Ephrin A3 (EFNA3), an anti-angiogenic factor [74]. Similarly, multiple myeloma cells under hypoxic conditions have been found to secrete exosomes containing high levels of miR-135b, which favours tube formation in endothelial cells through inhibition of HIF-1α [75]. Thus, exosomes secreted by tumour cells may secrete pro-angiogenic factors and miRNAs that modulate the phenotype of endothelial cells to promote angiogenesis.

Epithelial-mesenchymal transition

A number of processes, such as the transition of epithelial cells to mesenchymal cells, extracellular matrix (ECM) remodelling, the acquisition of unique molecular signatures (including stemness and resistance markers) and cytoskeletal reorganization take place during EMT. A classical hallmark of EMT is a decrease in E-cadherin expression and a concomitant increase in the expression of several transcription factors, such as Snail, Slug and Twist, and N-cadherin [76]. It has also been reported that the process of EMT requires support from tumour-associated cells. The treatment of nasopharyngeal carcinoma (NPC) cells with exosomes secreted by mesenchymal stem/stromal cells (MSCs) enriched in FGF19 isolated from bone marrow of healthy donors has, for example, been found to result in EMT-like changes in cellular characteristics such as an elongated morphology and an increased expression of the mesenchymal markers N-cadherin and vimentin, thereby conferring increased migratory abilities to these cells. Mechanistically, FGF19 increased the expression of pERK and phosphorylated fibroblast growth factor receptor-(FGFR)-4 (pFGFR4), which subsequently modulated the expression of EMT markers [77]. Knowing that MSCs secrete factors that favour tissue regeneration and repair via the induction of proliferation, migration and survival signals, and by suppressing apoptosis, it seems plausible to assume that those same exosomes may accelerate tumour growth, a notion that should be taken into account in future studies. Moreover, as discussed below, there are studies suggesting a role of MSCs in inhibiting the growth of cancer cells. This raises the question whether MSCs may play differential roles depending on the niche in which they reside and the presence of local environmental cues. Further in-depth studies aimed at understanding the positive and negative roles of MSCs in tumour progression may provide novel leads to the development of effective therapeutic strategies.

The phenomena of exosome-mediated increases in migration and invasion have been observed in many other studies, including those dealing with the effects of exosomes secreted by bladder cancer cells on urothelial cells. It was found that these urothelial cells exhibited enhanced invasive and migrative capacities, which could subsequently be inhibited by blocking exosome uptake via heparin pre-treatment [78]. Epstein-Barr virus (EBV)-positive NPC cells contain increased levels of HIF-1α in their secreted exosomes. The secretion of HIF-1α into the exosomes of these cells was augmented by the principal oncoprotein of EBV, latent membrane protein 1 (LMP1). HIF-1α secreted into these exosomes was found to be stable, as it retained its DNA-binding ability and transcriptional activity. The uptake of these exosomes by EBV-negative NPC cells was found to result in an increased migration and invasion resulting from decreased E-cadherin and increased N-cadherin expression [79]. Intriguingly, exosomes can also be secreted into human breast milk, and it has been reported that exosomes from benign as well as malignant breast tumours contain high levels of transforming growth factor beta (TGF-β), a key factor involved in EMT induction, which may upregulate the expression of the mesenchymal markers α-smooth muscle actin (α-SMA) and vimentin, and downregulate the expression of E-cadherin [80].

It has also been found that miRNAs may play important roles in the process of EMT through modulating the expression of different EMT markers [81]. Primary melanocytes may acquire metastatic capabilities and become aggressive as a result of communication between melanoma cell-derived exosomes and primary melanocytes, and it has been found that the change towards a EMT-like phenotype results from activation of the MAPK pathway and a decreased expression of miRNA let-7i. Exogenous re-expression of let-7i in primary melanocytes using a let-7i mimic and its subsequent co-culture with exosomes derived from melanoma cells was found to result in modulation of the expression of the let-7i targets LIN28B and HMGA2, and a concomitant decrease in the migration and invasion of primary melanocytes due to E-cadherin up-regulation and vimentin down-regulation [82]. Thus, these studies underscore the role of exosomes in enhancing the invasive and metastatic capacities of tumour cells through EMT, and provide additional indications for therapeutically targeting exosome uptake in recipient cells to reverse EMT.

Metastasis

The treatment of metastasised tumours is a major clinical challenge, which may at least in part be attributed to vast heterogeneities in both tumour cell populations and their environmental tissue niches [83]. It has amply been shown that cancer cell derived-exosomes may play a role in the metastasis of tumour cells by enhancing their migratory ability. Breast cancer cells that overexpress CXCR4 exhibit, for example, stem cell-like/migratory inducing properties, and exosomes secreted by these cells have been found to be internalised by T47D breast cancer cells. This internalisation led, subsequently, to increased cancer cell invasion and migration in vitro, whereas inoculation of the exosomes in mice led to an enhanced tumour growth and an increased metastatic potential [84]. Exosomes derived from gastric cancer cells and malignant pleural effusions have been found to promote peritoneal metastasis of gastric cancer cells by up-regulating the expression of adhesion molecules in mesothelial cells and, concomitantly, increasing the migratory ability of gastric cancer cells [85]. These studies underscore the importance of tumour-derived exosomes in the metastasis of tumour cells and provide new directions for the treatment of metastases.

It has amply been shown that exosomal miR-21 performs diverse tumour-related functions, as discussed in various sections in this review. Specifically, it has been found that miR-21 secreted via exosomes may promote the invasion and metastasis of cancer cells. Under hypoxic conditions, oral squamous cell carcinoma (OSCC) cells secrete exosomes containing elevated miR-21 levels, and these exosomes have been found to increase the migration and invasion of normoxic OSCC cells in a HIF-1α and HIF-2α-dependent manner [86]. It has also been found that exosomes secreted by oesophageal cancer cells containing miR-21 can be taken up by oesophageal cells in a nSMase2-dependent manner [87]. Subsequent co-culture experiments showed that miR-21 shuttling into recipient oesophageal cancer cells increased their invasive and migrative capacities. Mechanistically, miR-21 was found to downregulate the expression of its target gene programmed cell death 4 (PCD4), and to activate c-Jun N-terminal kinase (JNK) downstream signalling, resulting in enhanced expression of MMP-2 and MMP-9. Thus, exosomal miR-21 may serve as a therapeutic exosomal target that warrants further investigation.

The importance of interactions between macrophages and tumour cells during metastasis development has been investigated by Yang et al. They reported the transfer of exosomal miR-223 from IL4 activated macrophages to co-cultured breast cancer cells. In addition, they reported that transfection of a miR-223 mimetic into breast cancer cells increased their invasiveness through modulation of the myocyte enhancer factor 2C (Mef2c)-β-catenin pathway. After miR-223 mimetic transfection a decreased Mef2C expression was observed which, in turn, favoured nuclear translocation of β-catenin resulting in increased breast cancer cell invasion [88]. Mef2C is a co-transcription factor that plays an important role in both normal cell differentiation and in cancer development [89]. In hepatocellular carcinoma (HCC) cells, Mef2C has been found to act either as a tumour suppressor or as a tumour enhancer, depending on its subcellular localisation and its interaction with either VEGF or β-catenin. A nuclear sub-localisation of Mef2C favours angiogenesis via its interaction with VEGF, whereas a cytosolic sub-localisation suppresses tumour growth by inhibiting the nuclear translocation of β-catenin. In order to confirm the tumour inhibitory activity of Mef2C, its expression was upregulated in in vitro and in vivo mouse HCC xenograft models. By doing so, it was indeed found that Mef2C interacts with β-catenin and inhibits its nuclear translocation, resulting in decreased cell proliferation in vitro and reduced tumour growth in vivo [89].

Another recently discovered mechanism of metastasis promotion by breast cancer cell-secreted exosomes has been reported by Fong et al. They found that exosome-secreted miR-122 led to decreases in glucose uptake by non-tumour cells, such as lung fibroblasts, brain astrocytes and neurons, in pre-metastatic niches resulting in enhanced metastasis. By decreasing the expression of the glucose transporter GLUT1 and that of pyruvate kinase M (PKM), an enzyme involved in glucose metabolism, nutrient availability was increased facilitating the colonization of circulating cancer cells in lung and brain [90].

Drug resistance

The development of resistance to chemotherapeutic drugs is one of the most common causes of treatment failure. The underlying molecular mechanisms include decreased uptake of anti-cancer drugs, increased expression of efflux transporters, enhanced DNA repair and detoxification of drugs [91]. These intrinsic mechanisms are linked to extrinsic mechanisms favouring the survival of cancer cells. These extrinsic mechanisms are represented by, at least in part, exosome-based signalling. The major mechanisms involved in exosome mediated drug resistance in cancer cells include (i) conferring resistant phenotypes to drug sensitive cancer cells and (ii) recruiting tumour-associated cells that regulate protective mechanisms in cancer cells. It has been reported that transfer of miR-100, miR-222 and miR-30a from drug resistant breast cancer cells to drug sensitive breast cancer cells can modulate drug-induced apoptosis and confer chemo-resistance. Specifically, it was found that exosome-borne miR-222 of docetaxel-resistant breast cancer cells can downregulate the expression of its target gene phosphatase and tensin homolog (PTEN) in recipient cells [92]. Another mechanism of conferring drug resistance to drug sensitive breast cancer cells involves exosomal transfer of the efflux pump P-glycoprotein (P-gp) [93]. Others have found that exosomes secreted by cancer-associated adipocytes (CAAs) and CAFs in ovarian cancer patients may exhibit elevated miR-21 levels which, when transferred to ovarian cancer cells, may inhibit their apoptosis and confer chemo-resistance through downregulating the expression of its target APAF1 [94]. Hu et al. reported yet another mechanism that may underlie chemo-resistance in a colon cancer model, wherein exosomes secreted by CAFs primed CSCs to promote their clonogenicity and growth when treated with anti-cancer drugs such as 5-flurouracil (5-FU) or oxaliplatin [95]. Gastric cancer cells have been found to exhibit enhanced resistance to 5-FU through upregulation of multi-drug resistance (MDR) proteins and activation of the CaM-Ks/Raf/MEK/ERK pathway via exosomes secreted by MSCs containing P-gp/MDR. Mechanistic studies revealed that this drug resistance was due to influx of Ca²⁺ via P-gp, followed by activation of calcium/calmodulin kinases and, finally, activation of the downstream Raf/MEK/ERK kinase cascade [96]. Since CSCs exhibit resistance to chemotherapeutic pressure, priming of CSCs with CAF-secreted exosomes may further increase drug resistance. Hence, targeting the secretion of exosomes from both CSCs and CAFs may provide new avenues to overcome drug resistance [95].

Targeting cancer cells using antibodies directed against antigens present on their surface forms the basis of immunotherapy, and acquired resistance to such therapies has delineated a role of exosomes in this resistance. It has been found, for example, that B-cell lymphoma-secreted exosomes containing CD20 in an ATP-binding cassette (ABC) transporter A3 (ABCA3)-dependent manner can protect lymphoma cells from antibody attack by binding anti-CD20 antibodies [97]. Similarly, it has been found that the effect of Trastuzumab can be negated by human epidermal growth factor receptor 2 (HER2) secreted in exosomes from HER2-overexpressing breast cancer cells [98]. Drug-resistant tumour cells can also reprogram lysosomal trafficking and secretion to enhance exosome-mediated drug effluxes. Drug-resistant ovarian cancer cells may, for example, abnormally sort lysosomal proteins and secrete exosomes containing more cisplatin than drug sensitive cells. Such cells have been found to exhibit a significant upregulation of genes associated with membrane fusion, vesicle trafficking and the cisplatin efflux transporters MRP2, ATP7A and ATP7B [99].

Another mechanism by which exosomes may confer drug resistance to cancer cells is by secreting lncRNAs that can elicit epigenetic changes. LincRNA-ROR (linc-ROR) is a long non-coding RNA that was first described in 2010 for its role in the induction of pluripotent stem cells [100]. Subsequently, it was found that linc-ROR expression is associated with a poor prognosis in breast, pancreas, liver, endometrium, colon and nasopharyngeal cancer patients as it modulates the expression of target genes governing tumour progression-associated mechanisms such as proliferation, invasion, metastasis and apoptosis [101]. Takahashi et al. reported that TGF-β can mediate drug resistance in sorafenib-treated HCC cells by increasing linc-ROR levels in exosomes secreted by these cells. This, in turn, may repress p53 to inhibit apoptosis and enhance tumorigenic capacities of recipient cells, including the induction of increases in tumour-initiating cells expressing CD133 and their ability to form colonies [102]. Although no linc-ROR elicited epigenetic changes were reported in this study, in another study by Fan et al. it was reported that linc-ROR can promote tumour growth and metastasis by acting as a decoy oncoRNA to inhibit methylation of the TESC gene promoter through histone G9A methyltransferase and promoting the release of histone H3K9 methylation [103]. So, chemo-resistance in HCC cells may be due to epigenetic modifications induced by exosome-secreted linc-ROR. Taken together, these studies highlight multiple mechanisms involving exosomes in conferring drug resistance. This information may be instrumental for the development of novel therapeutic approaches aimed at sensitising drug-resistant cells to chemo- and/or immunotherapy.

Quiescence and senescence

As a result of a high therapeutic pressure and an unfavourable environment, cancer cells may adopt multiple survival modes, including drug efflux and the acquisition of a quiescent or dormant state. The latter mechanism refers to a reversible state by which cancer cells arrest in the G0 phase of the cell cycle with the ability to re-enter the cell cycle in the presence of favourable external stimuli. Cells can be maintained in this state for a long time through the regulation of genes associated with cell cycle progression, DNA replication, mitochondrial function, transcription regulation and RNA processing [104]. When the tumour environment becomes favourable again, these genes may be activated to cause tumour growth, relapse and metastasis. A key problem is the variability of the dormancy periods and, thus, the unpredictable occurrence of relapses. Another problem is the usually more aggressive nature of the recurrences. The regulation of dormancy and quiescence is multifactorial and may, at least partially, be exosome mediated. It has, for example, been reported that dormant breast cancer cells in a bone environment may interact with MSCs, causing the release of exosomes containing miR-222/223. These exosomes lead to entry of the breast cancer cells into a quiescent state by downregulating the expression of the cell cycle regulatory proteins CDK4, cyclin D1 and p21^WAF1. This downregulation may lead to a cell cycle arrest and, subsequently, a reduction in sensitivity to chemotherapy. Similarly, it has been found that exosomal transport of, amongst others, miR-127, miR-197, miR-222 and miR-223 by bone marrow stroma cells to breast cancer cells, and miR-23b from bone marrow MSCs to metastatic breast cancer cells, may induce breast cancer dormancy within the bone [105, 106]. Interestingly, it has been reported that targeting these dormant breast cancer cells with antagomiR-222/223 may re-sensitise them to chemotherapy [107]. Similar approaches may be relevant for therapeutically targeting other dormant cancer cells.

In contrast to quiescence, senescence refers to a presumably irreversible arrest of cells in response to Hayflick factors, i.e., telomere shortening, transcriptional depression of the INK4a/ARF locus and DNA damage. Senescence is used as a protective mechanism to limit cell proliferation, usually due to aging and cellular stress, and to limit tumour growth [108]. Senescence induction facilitates cancer treatment as senescent cells become arrested in the G1 phase of the cell cycle and cell proliferation is inhibited in response to anti-cancer drugs and/or radiation [109]. Treatment failure in head and neck cancer has, for example, been associated with inhibition of radiation-induced senescence due to mutations in the TP53 gene [110], as also in invasive colon cancer through escape from senescence [111]. Interestingly, it has been found that both senescence-induced cellular aging and cancer are regulated through multiple common regulators such as sirtuins and p53 [112], in which both these factors are known to play a role in regulating exosome secretion.

It has also been shown that senescence induction due to Hayflick factors may result in an increased secretion of exosomes in a p53-dependent manner via upregulation of target genes such as the tumour suppressor-activated pathway 6 (TSAP6), CHMP4C and hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) genes [10, 113, 114]. The increased secretion of exosomes by senescent cells is referred to as senescence-associated secretome phenotype (SASP). Senescent cells secrete, via exosomes, various factors such as growth factors, cytokines and extracellular proteases, which may act as signals to modulate the functions of neighbouring cells in the tumour environment [115]. In addition, senescent cells may release exosomes as a means to communicate their state and function with neighbouring cells and to favour tumour progression. It has, for example, been found that senescent fibroblasts may secrete increased levels of VEGF to cause in vivo tumour vascularisation in mice and to promote in vitro basement membrane invasion by HUVECs [116]. TSAP6 is an essential component of the p53-dependent exosome secretion pathway as TSAP6−/− mice are unable to secrete exosomes following p53 activation [11]. In addition, it has been found that exosomes secreted by p53 deficient cancer cells are smaller in size due to downregulation of its target gene HGS, compared to those secreted by wild-type p53 cancer cells [114].

Irradiation of prostate cancer cells with clinically relevant doses may lead to premature senescence accompanied by an enhanced secretion of exosomes containing B7-H3, a protein that elicits anti-tumour immunity in a p53-dependent manner [117]. As such, detecting the levels of this protein may help to predict responses to radiotherapy [118]. In a recent study, the development of resistance to paclitaxel in ovarian cancer cells was attributed to senescence induction by exosomal miR-433 secreted by high miR-433 expressing cancer cells. These senescent ovarian cancer cells were found to exhibit altered cell cycle states and to secrete significantly higher levels of the pro-inflammatory cytokines IL6 and IL8. Cell cycle progression in ovarian cancer cells with increased miR-433 expression was affected due to decreased hyperphosphorylation of Rb resulting from decreased CDK6 expression, a cyclin-dependent kinase involved in Rb phosphorylation [119]. It has also been reported that miR-433-mediated decreases in mitotic arrest deficiency protein 2 (MAD2) levels may contribute to resistance to paclitaxel in patients with high-grade serous epithelial ovarian cancer [120]. Consistent with these results, the expression of two miR-433 target genes, HDAC6 and MAD2, was found to be decreased in cancer cells overexpressing miR-433, which may be responsible for the development of drug resistance in these cells [119]. Conversely, exosome cargos may also repress senescence as reported by van Balkom et al. They found that endothelial cell-derived exosomes containing miR-214 may repress senescence by silencing the expression of a DNA damage-induced tumour suppressor, ataxia telangiectasia mutated (ATM), in recipient endothelial cells to stimulate migration and angiogenesis [121]. From the limited studies available, it is becoming evident that targeting exosomal transport to reverse dormancy and quiescence may help to prevent the resurgence of tumours. Since the exosomal contents may either activate or repress senescence, further studies are warranted to delineate the exact role of exosome-induced senescence and to explore its potential for the development of novel therapeutic strategies.

Role of exosomes in modulating the tumour microenvironment and the formation of pre-metastatic niches

Modulation of the tumour microenvironment

An important aspect of tumour progression is communication between tumour cells and cells within the tumour microenvironment, such as fibroblasts, adipocytes and cells of the vascular and immune system (Fig. 2). Baroni et al. found, for example, that miR-9 secreted via exosomes by breast cancer cells have the ability to induce cancer-associated fibroblast (CAF)-like properties in normal human breast fibroblasts by modulating the expression of genes involved in cell motility and extracellular matrix remodelling. In addition, they found that miR-9 inhibition could affect the migratory properties of CAFs [122]. Others have found that exosomal transfer of human telomerase reverse transcriptase (hTERT) mRNA from cancer cells to fibroblasts conferred several phenotypic changes to these fibroblasts, such as increased proliferation, delayed senescence and resistance to DNA damaging drugs [123], eventually leading to a sustained support of CAFs to tumour growth and progression.

Fig. 2.

Schematic representation of exosome-mediated communication between tumour cells and cells in the tumour microenvironment. Exosome-borne factors, such as microRNAs, receptors and a myriad of other proteins, may confer varying phenotypes, such as survival, evasion from immune surveillance, metastasis and relapse, to tumour cells

Activation of signalling pathways is one of the mechanisms by which exosomes may modulate the tumour microenvironment to promote tumour growth. Gu et al. reported exosomal transfer of TGF-β and subsequent activation of the TGF-β/Smad pathway in human umbilical cord-derived mesenchymal cells (hucMSCs), through which gastric cancer cells can trigger the differentiation of hucMSCs into CAFs. It was also found that co-culture of hucMSCs with tumour cell-derived exosomes increased the migratory capacity of hucMSCs and, interestingly, that the treatment of cells with a TGF-β inhibitor effectively blocked both hucMSC migration and their differentiation into CAFs [124]. These results reiterate the mechanism underlying the modulatory effect of cancer cells on tumour-associated cells favouring tumour growth. Moreover, Webber et al. found that exosomal transfer of TGF-β from cancer cells to fibroblasts resulted in their conversion to myofibroblasts, as indicated by a strong induction of α-SMA [125]. Myofibroblasts present in tumour stroma play important roles in the secretion of growth factors, such as activin A, VEGF, insulin-like growth factor 1 (IGF-1) and, subsequently, inducing tumour cell proliferation, tumour progression, angiogenesis and invasion [126–128]. Therefore, targeting the differentiation of fibroblasts into myofibroblasts via exosomal transfer may have important implications for the future designing of anti-cancer therapies. Bi-directional crosstalk between chronic myelogenous leukemia (CML) cells and stromal cells is known to play an important role in the survival and proliferation of CML cells by activation of EGFR signalling in stromal cells through amphiregulin that is present in CML-derived exosomes. EGFR signalling leads to IL8 upregulation in stromal cells which favours CML cell survival. It has also been found that pre-treatment of stromal cells with CML-derived exosomes may result in annexin A2 upregulation in the stromal cells, which in turn facilitates the attachment of leukemic cells to stromal monolayers [129]. Wu et al. reported that through activating the nuclear factor kappa B (NF-κB) pathway in macrophages, gastric cancer cell-derived exosomes enhanced the secretion of pro-inflammatory cytokines, which subsequently increased the proliferation and migration of gastric cancer cells [130].

Also, tumour microenvironment-associated cells may secrete factors that facilitate tumour growth [131], and recent work has shown that exosomes secreted by these cells may not only play a major role in sustaining tumour growth, but also in acquiring drug resistance under hypoxic conditions. Languino et al. have, for example, reported transfer of TGF-β receptor II (TβRII) from exosomes isolated from stromal fibroblasts of patients with OSCC to OSCC-associated keratinocytes, which in turn led to increased TGF-β signalling in OSCC cells through re-activation of their response to TGF-β ligand [132]. Others have reported activation of STAT1-dependent paracrine antiviral signalling in breast cancer cells through the transfer of exosomes from stromal cells, and found juxtacrine NOTCH3 activation within the breast cancer cells. They also found that the two pathways converged and that STAT1-mediated transcriptional changes resulted in the establishment of drug-resistant breast cancer cells [133]. Specialised adherent cells present in soft tissues and body fluids such as bone marrow, adipose tissue, peripheral blood, foetal liver, lung, amniotic fluid and umbilical cord blood are termed MSCs. These cells possess the ability to differentiate into mesenchymal tissue cells such as osteoblasts, chondrocytes and adipocytes [134, 135]. MSCs can both suppress and enhance tumour growth. MSCs may suppress tumour growth by inducing G1-phase cell cycle arrest [136], dickkopf-1 (Dkk-1)-mediated inhibition of Wnt signalling [137] and reduction of IGF-1R/PI3K/Akt signalling by sequestering free insulin-like growth factors (IGFs) [138]. In addition, it has been found that downregulation of the expression of the translation initiation factors eIF4E and eIF4GI may lead to a reduced migration and proliferation of cancer cells [139] and that exosomal miR-16-mediated downregulation of VEGF may lead to inhibition of angiogenesis [140] and downregulation of Akt [141]. MSCs may migrate to sites of injury [142] or tumour formation [143] in response to chemokines, such as stromal cell-derived factor 1 (SDF-1) [142], growth factors, such as basic fibroblast growth factor (bFGF) [144], platelet-derived growth factor (PDGF), epidermal growth factor (EGF) [143] and VEGF [145]. Once MSCs become part of the tumour microenvironment, they are ‘educated’ through various factors including exosomes and, consequently, converted into tumour-associated MSCs. These tumour-associated MSCs can promote tumour growth through multiple mechanisms, including chemokine-mediated macrophage recruitment to tumour sites [146], aberrant miRNA expression resulting in downregulation of the forkhead transcription factor FOXP2 to induce stem cell-like features [147], interaction with endothelial cells to promote angiogenesis [143], activation of various cellular signalling pathways [148], modulation of immune responses [149] and increasing the population of CSCs [150], ultimately resulting in treatment failure due to the acquisition of drug resistance [151].

Lin et al. showed that tumour-cell derived exosomes may be internalised by bone marrow-derived MSCs and that this internalisation may subsequently alter these MSCs into tumour promoting cells. These cells were found to favour tumour growth by promoting macrophage infiltration via enhanced production of CCR2 ligands in B16-F0 melanoma cells and EL-4 lymphoma cells in mice, and by increasing the number of circulating monocytes. It was also found that treatment with a CCR2-specific inhibitor and macrophage ablation by clodronate liposomes reversed the phenotype to normal MSCs [152]. The implication of MSC-promoted macrophage infiltration is an increase in monocyte-derived tumour-associated macrophages (TAMs). Monocytes generally survive beyond their normal life span of two days under inflammatory conditions in the tumour microenvironment and, hence, they may give rise to TAMs. Intriguingly, their survival is mediated by cancer cell-derived exosome-induced inactivation of caspase-dependent apoptosis. MAPK pathway activation through exosomal transfer of receptor tyrosine kinases, such as phosphorylated EGFR and phosphorylated HER2, may be favoured by this event [153].

TAMs are important constituents of the tumour microenvironment and favour tumour progression by activating several important events such as angiogenesis, drug resistance, immune evasion, activation of CSCs and promoting its self-renewal, and extracellular matrix degradation [154]. These cells can exhibit either a M1 phenotype that suppresses tumour growth or a M2 phenotype that favours tumour growth [73]. Tumours with a high M2/M1 ratio usually exhibit a poor prognosis, whereas polarization of TAMs towards a M1 phenotype may increase the efficacy of anti-cancer drugs [154, 155]. A recent study has shown that TAMs in glioblastomas may constitute 50% of the tumour mass, and that 85% of these TAMs may be accounted for by infiltrating macrophages/monocytes from the blood circulation. It was also found that bone marrow-derived monocytes may infiltrate the tumours during early stages in response to an increased secretion of the chemokine CCL2 by the tumour cells, and that these monocytes may subsequently differentiate into macrophages and microglia-like cells. Inhibition of monocyte infiltration was found to improve survival in mouse models. Resident microglial cells and TAMs exhibit differentially expressed genes and it was found that among them cell migration was most enriched in TAMs [156]. It has also been reported that TAMs may favour ovarian tumour growth by inducing spheroid formation via an increased adhesion between tumour cells and TAMs resulting from EGF secretion by TAMs and activation of VEGF/vascular endothelial growth factor receptor (VEGFR) signalling through EGFR activation in the tumour cells. This activation was found to culminate in enhanced tumour cell proliferation, migration and trans-coelomic spread [157]. Recently, a role of exosomes in the polarization of macrophages towards the M2 phenotype was reported by Shinohara et al., and they found that miR-145 present in exosomes secreted by colorectal cancer cells may induce M2 macrophage polarization through histone deacetylase 11 (HDAC11) expression down-regulation and IL10 production up-regulation. M2 macrophage polarization was also found to cause significant increases in tumour volumes in xenograft models co-injected with exosome-treated macrophage-like cells [158].

Multiple myeloma (MM)-derived exosomes may also establish a favourable bone microenvironment for tumour growth by enhancing immunosuppression and regulating angiogenesis in bone marrow myeloid-derived suppressor cells (MDSCs) and bone marrow endothelial cells. These MM-derived exosomes may modulate key angiogenic pathways via several angiogenesis-related proteins/regulators contained in them, such as angiogenin, tissue inhibitor of metalloproteinase-1 (TIMP-1), Serpin E1, Serpin F1 and VEGF-B. In addition, it was found that these exosomes could up-regulate the proliferation of MDSCs by activating the STAT3 pathway (associated with self-renewal), and up-regulating the pro-survival proteins Bcl-xL and Mcl-1. These activated MDSCs exhibited enhanced inducible nitric oxide synthase (iNOS) production to favour the growth of MDSCs by suppressing the function of T-cells, thus facilitating MM progression [159]. Taken together, these studies highlight a role of exosomes in mediating tumour-stroma communication. Interfering with exosomal transfer may, hence, represent a novel strategy to target the tumour microenvironment and, by doing so, to halt tumour progression.

From anti-oncogenic to pro-oncogenic exosome-mediated mechanisms

Although different cells within the tumour microenvironment, such as MSCs, TAMs and fibroblasts, may secrete exosomes that promote tumour growth, they may also possess intrinsic anti-oncogenic functions. Only after they are recruited to tumour sites and reprogrammed by the cancer cells, they may favour tumour growth via their exosomal components. It has, for example, been found that exosomes secreted by mouse embryonic fibroblasts (MEFs) may contain functional PTEN that, when internalised by recipient MEFs, may reduce the rate of cell proliferation and the amount of pAKT [160]. In another recent study, it was reported that treatment of A2780 and SKOV-3 ovarian cancer cells with fresh or protease-digested exosomes derived from human adipose MSCs (hAMSC)-conditioned media could downregulate the proliferation, as well as the wound-repair and colony forming capacities, of these cells, due to the presence of three potential new miRNAs, seven potential candidate miRNAs and several known miRNAs in the exosomes, such as hsa-miR-4792, hsa-miR-320a, hsa-miR-320b, hsa-miR-7704, hsa-miR-181a-5p, hsa-miR-127-3p and hsa-miR-6087s. The ovarian cancer cells were also found to be susceptible to apoptosis due to the fact that the exosomal miRNAs could upregulate pro-apoptotic proteins, downregulate anti-apoptotic proteins and target important molecules associated with cell cycle progression and survival pathways [161]. The anti-oncogenic role of exosomes secreted by hAMSC was further underscored by their ability to upregulate the level of circulating and intra-tumoural natural killer T-cells (NKT-cells) in HCC rat models, leading to an increased anti-tumour activity and significantly smaller and lower grade-tumours compared to controls [162]. Another anti-oncogenic function of mouse bone marrow-derived MSCs is the transfer of, next to other exosomal components, miR-16, leading to inhibition of angiogenesis as a result of decreased VEGF expression in recipient breast cancer cells [140]. Alcayaga-Miranda et al. reported that exosomes derived from menstrual stem cells (MenSCs) may exhibit anti-oncogenic activity against PC3 prostate cancer cells resulting from a reduction in VEGF secretion, a decrease in reactive oxygen species (ROS) production, downregulation of NF-κB expression and the suppression of pro-angiogenic factors. Treatment of mice with MenSCs-derived exosomes led to in vivo regression of tumours exhibiting a decreased vascular density, reduced haemoglobin levels and reduced VEGF and HIF-1α expression levels [163]. Human macrophages may also possess intrinsic anti-tumour activities before they are recruited to tumour sites, as reported by Lee et al. Specifically, they found that exosomal disintegrin and metalloproteinase 15 (ADAM15) via its disintegrin-like domain may block interactions between integrin αvβ3 and vitronectin, and inhibit vitronectin- and fibronectin-induced growth and migration of breast, lung and ovarian cancer cells. The authors triggered the differentiation of monocytes to macrophages and found that these monocyte-derived macrophages abundantly secreted ADAM15 in their exosomes. These ADAM15⁺ exosomes were subsequently found to inhibit the in vitro growth and migration of MDAH-2774 ovarian cancer cells, as well as to suppress their in vivo growth and to increase the survival of xenografted mice [164].

Formation of pre-metastatic niches

The establishment of pre-metastatic niches represents an important phase in the metastatic process of cancer cells in which exosomes play an important role [59]. Primary tumour cells have been found to secrete several factors, such as VEGF, TGF-β and lysyl oxidase (LOX), that may recruit bone marrow-derived cells (BMDCs) to prepare suitable microenvironments at secondary sites for the attachment and survival of tumour cells [165–167]. The site of niche establishment depends upon various factors, including the expression and type of integrin present on the exosome surface, the exosomal microRNAs content and the presence of several supporting soluble factors. Below, we will discuss the role of various exosomal factors in preparing pre-metastatic niches, as well as in determining the organ(s) of choice for metastasis.

Even though exosomes, independent of their origin from poorly or highly metastatic cells, are considered to be the primary regulators and modulators that govern pre-metastatic niche formation, Jung et al. reported that this event requires a soluble matrix, which depends on CD44v. Specifically, they found that CD44v6 expression knockdown reduced the metastasizing capacity of pancreatic adenocarcinoma cells and that this reduction could be rescued by supplementation of soluble matrix secreted by wild-type adenocarcinoma cells [168]. In another study, it was found that exosomes released by human renal carcinoma cells expressing CD105 could promote angiogenesis in normal endothelial cells. CD105⁺ exosomes could also increase the adhesion of normal endothelial cells and, by doing so, facilitate the formation of pre-metastatic niches in lungs by upregulating the expression of MMP-2, MMP-9, and VEGFR1 [67].

Costa-Silva et al. reported, for the first time, a role of pancreatic ductal adenocarcinoma (PDAC)-derived exosomes in the formation of pre-metastatic niches in the liver. An increased production of TGF-β and an upregulation of fibronectin production were seen after uptake of PDAC-derived exosomes by Kupffer cells in the liver. The deposition of fibronectin stimulated the migration of BMDCs, such as macrophages and neutrophils, into the liver for the establishment of pre-metastatic niches. It was found that macrophage migration inhibitory factor (MIF) was the exosomal component responsible tor triggering these changes and that MIF inhibition abolished the preparation of pre-metastatic niches, as well as the metastasis of PDAC cells to the liver [165]. Hood et al. reported that melanoma-derived exosomes homed to ipsilateral sentinel lymph nodes in vivo and, subsequently, recruited melanoma cells to these lymph nodes. Additional mechanistic studies revealed that melanoma-derived exosomes may activate multiple metastatic pathways involved in basement membrane invasion, migration, vascular organisation and the induction of angiogenic factors required for the survival and growth of melanoma cells within lymph nodes [59, 169].

Others reported on the role of differentially expressed miRNAs in exosomes derived from bulk cancer cells and CSC sub-populations contributing to metastasis and pre-metastatic niche formation in primary prostate cancers. Comparative exosomal miRNA profiling revealed 19 miRNAs, including miR-100-5p (in both bulk and CSC exosomes), miR-21-5p (in bulk exosomes) and miR-139-5p (in CSC exosomes) that may enhance prostate cancer proliferation and migration. Transfection of these miRNAs in normal prostate WPMY-1 fibroblasts resulted in invasive and metastatic characteristics through expression upregulation of MMP-2, -9, -13, as well as the receptor activator of nuclear factor kappa-B ligand (RANKL) in the pre-metastatic niche, thereby promoting osteoclast recruitment and differentiation [170]. Peinado et al. found that melanoma cell-derived exosomes could transform the phenotype of BMDCs to favour metastasis by transfer of the MET oncoprotein. These exosomes could also induce vascular leakiness at pre-metastatic sites and educate BMDCs towards a pro-vasculogenic phenotype [167].

Role of exosomes in cancer diagnosis and treatment

Although it is at present not fully understood how the sites of pre-metastatic niches and, subsequently, metastasis are determined, Hoshino et al. provided new insights by focusing on the expression of cell adhesion receptors, such as integrins, on exosomal surfaces. They noted that these integrins may serve to prepare pre-metastatic niches by interacting with components of the extracellular matrices of the respective target organs. They found, for example, that exosomes expressing integrins α₆β₄ and α₆β₁ on their surface co-localised with S100A4⁺ cells in laminin-rich lung environments to cause lung metastases, whereas exosomes expressing integrins α_vβ₅ led to the formation of liver metastases [171]. A further deciphering of the role of cell adhesion receptors on exosomes in organ-specific metastasis may hold promise for the development of new therapeutic strategies.

Exosome signatures for diagnosis and treatment

Since exosomes may carry a variety of biomolecules, such as mRNAs, miRNA and proteins, and since these biomolecules can be isolated from various body fluids with ease, it has been suggested that they may be used as diagnostic/prognostic signatures using minimally invasive techniques [172]. Chen et al. found, for example, that of 107 proteins that were differentially expressed, 24 differed significantly in urinary exosomes isolated from bladder cancer patients and non-cancer patients. They also found that urinary exosome-derived calcium-signal transducer 2 (TACSTD2), a cell surface glycoprotein that is overexpressed in numerous cancers, may serve as a biomarker for the diagnosis of bladder cancer. Specifically, they found that TACSTD2 levels were higher in microparticles secreted by bladder cancer patients compared to those secreted by controls, i.e., hernia and urinary tract infection cases [173]. Others have reported that loss of two proteins that regulate mitochondrial function (SH3GL2 and MFN2) in serum exosomes correlated with the presence of breast cancer and the occurrence of lymph node metastases [174]. The diagnosis of prostate cancer is challenged by unnecessary biopsies in patients with benign disease and unequivocal prostate specific antigen (PSA) levels. To overcome these challenges, exosomal RNAs were isolated from urine after which a novel molecular signature, called EXO106 score, was deduced using the sum of normalised PCA3 and ERG RNA levels. Using the EXO106 score, fewer biopsies were needed for men with unequivocal PSA scores, i.e., the EXO106 score provided a negative predictive value of 97.5% for the diagnosis of high-grade prostate cancer [175]. He et al. performed proteomic analyses of three HCC-derived cell lines and found that metastasis-inducing factors, such as MET, S100 family members and caveolins, were present only in exosomes derived from metastatic HCC cells [176].

Cancer may also be diagnosed through the measurement of miRNA and mRNA levels in blood-derived exosomes [73]. Joshi et al. found, for example, that high levels of miRNA-10b isolated from plasma-derived exosomes corresponded to the occurrence of pancreatic cancer and/or chronic pancreatitis [177]. Others revealed, through miRNA profiling of serum-derived exosomes from glioblastoma multiforme (GBM) patients, a diagnostic signature consisting of either the small noncoding RNA RNU6-1 alone or a combination of miR-320, miR-574-3p and RNU6-1 that may differentiate GBM patients from healthy individuals [178]. Similarly, exosomal signatures have been identified for the diagnosis and monitoring of melanoma [167] and breast cancer [84] patients. Skog et al. reported that mRNA mutants and glioblastoma-specific miRNAs can be detected in serum-derived exosomes of GBM patients. Specifically, they found that EGFRvIII variant mRNA could be detected and used as a non-invasive diagnostic marker [179].

Molecular exosome profiling can also be used to predict responses to anti-cancer therapies, such as docetaxel in the treatment of castration-resistant prostate cancer (CRPC). It has been found that DU145 prostate cancer cells that are either sensitive (DU145 Tax-Sen) or resistant (DU145 Tax-Res) to docetaxel differ in the amounts of exosomes secreted. Exosomes isolated from DU145 Tax-Res cells were additionally found to be enriched in drug efflux proteins such as MDR-1 and MDR-3. The same was observed in sera of CRPC patients and it was found that the presence of these proteins was indicative of resistance to docetaxel treatment [180]. Similarly, integrin β4 and vinculin levels were found to be elevated in exosomes isolated from taxane-resistant PC-3 prostate cancer cells and could be used as exosome-based diagnostic tools to predict taxane resistance in prostate cancer patients [181].

The amount of exosomes released in blood and their protein content may differ between healthy and cancer patients, which can be used as a diagnostic tool. It has, for example, been found that plasma from patients with chronic lymphoid leukaemia (CLL) may have significantly increased exosome levels, and miRNA profiling revealed a CLL-specific signature characterised by miR-150, miR-155 and miR-29a-c upregulation [182]. It has also been revealed by plasma-derived exosome analysis of melanoma patients of different stages that stage IV disease patients may exhibit a higher content of several (onco)proteins that correlate with a poor survival [167]. Others found that high exosome levels in plasma from colorectal cancer patients was associated with poorly differentiated tumours and high carcinoembryonic antigen (CEA) plasma levels [183]. Taken together, it can be concluded that exosome levels and their contents may be associated with multiple disease parameters. As such, they may be used as non-invasive diagnostic/prognostic signatures.

Exosomes: potential diagnostic biomarkers

As mentioned above, exosomes can be isolated from a variety of body fluids such as serum, plasma, saliva, urine and cerebrospinal fluid (CSF). Biomolecules contained within these exosomes include cell surface glycoproteins, double stranded DNA, miRNAs, lncRNAs, drug efflux proteins and other proteins that are associated with various processes of cancer development and progression. The levels of these biomolecules may be either upregulated or downregulated, depending on the stage and aggressiveness of the cancer, compared to those of healthy subjects. This information may facilitate the non-invasive diagnosis of cancer [73]. In addition, the respective biomolecules may be employed to monitor disease recurrence and response to therapy [73]. Several of these biomolecules and their diagnostic/prognostic significances are listed in Table 1.

Table 1.

Exosomal biomolecules and their clinical significance

Type of cancer	Biomolecule	Exosome source	Clinical significance	Reference
Acute myeloid leukaemia	CD34	Plasma	Presence of elevated levels of CD34(+) exosomes in cancer patients	[213]
Bladder cancer	EDIL-3	Urine	Increased EDIL-3 levels in high grade bladder cancer patients alone. EDIL-3 favours angiogenesis and endothelial cell migration by EGFR signalling	[214]
	lncRNAs HOTAIR, HYMA1, LINC00477, LOC100506688, and OTX2-AS1	Urine	Urinary exosomes of patients with high-grade muscle-invasive disease enriched in these lncRNAs	[215]
Breast cancer	miR-101 and miR-373	Serum	Elevated levels of miR-373 in triple negative, ER(−) and PR(−) cancer patients. miR-101 increased only in breast cancer patients when compared to benign and healthy cases	[216]
	Survivin	Serum	Survivin and its splice variant survivin-∆Ex3 were elevated in all cancer patients, whereas survivin-2B exhibited a differential expression in breast cancer patients	[217]
	Periostin	Plasma	Compared to patients with localised disease, patients with lymph node metastasis have higher levels of exosomal periostin	[218]
Cervical cancer	miRNA-21 and miRNA-146a	Cervicovaginal lavage	Abnormally elevated only in cervical cancer patients	[219]
Colon cancer	miR-19a	Serum	Elevated expression in cancer patients alone. High expression was correlated with a poor prognosis and tumour recurrence	[220]
	miRNAs let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a	Serum	Compared to healthy controls, colon cancer patients had increased levels of these miRNAs	[221]
Gastric cancer	LINC00152	Plasma	Elevated levels in gastric cancer patients alone	[222]
	miR-21 and miR-1225-5p	Peritoneum lavage fluid	Highly elevated in T4 metastatic stage patients compared to T1-T3 stages. High expression was correlated with peritoneal recurrence after gastric cancer resection	[28]
Glioma	miR-21	Cerebrospinal fluid	CSF of glioma patients contained high levels of miR-21. Its expression was correlated with metastasis and recurrence	[26]
Hepatocellular carcinoma	miR-21	Serum	miR-21 levels were elevated in patients with HCC compared to healthy volunteers and chronic hepatitis B patients. Higher levels were correlated with cirrhosis and advanced tumour stage	[223]
	miR-718 and miR-1246	Serum	Downregulation of these miRNAs were correlated with recurrence after liver transplantation. Decreased miR-718 levels were associated with tumour aggressiveness	[224]
Laryngeal squamous cell carcinoma	miR-21 and HOTAIR	Serum	Higher expression in patients with LSCC and lymph node metastasis than in those with benign disease	[225]
Melanoma	MDA-9 and GRP78	Serum	Only metastatic melanoma patients exhibited elevated expression levels of these markers	[226]
	miR-125b	Serum	Significantly downregulated in patients with advanced melanoma compared to disease-free melanoma patients and healthy controls	[227]
Nasopharyngeal carcinoma	Galectin-9	Serum	Exosome-derived galectin-9 was detectable only in cancer patients	[228]
Non-small cell lung cancer	LRG1	Urine	LRG1 expression was significantly up-regulated in NSCLC patients compared to controls	[229]
Oesophageal squamous cell carcinoma	miR-21	Serum	Elevated only in ESCC patients; correlated positively with tumour progression and aggressiveness	[230]
Ovarian cancer	miR-21, miR-141, miR-200a, miR-200c, miR-200b, miR-203, miR-205, and miR-214	Serum	Elevated levels of these 8 miRNAs in sera of ovarian cancer patients compared to controls	[231]
	miR-222-3p	Serum	Higher levels in sera of cancer patients compared to controls	[232]
Pancreatic cancer	Double-stranded genomic DNA with mutated KRAS and p53	Serum	Compared to healthy controls, only pancreatic cancer patients exhibited mutations in KRAS and p53 DNA	[233]
	Glypican-1 (GPC1)	Serum	GPC1(+) exosome levels were higher in cancer patients than in healthy control and benign prostatic disease patients. Patients with distant metastases exhibited higher GPC1(+) exosome levels compared to those with lymph node metastasis or no metastasis	[234]
Prostate cancer	PSA, PSMA	Urine	Present only in cancer patients	[235]
	P-glycoprotein	Serum	Elevated levels in docetaxel-resistant prostate cancer patients	[236]
	miR-1290 and miR-375	Plasma	Higher levels were correlated with a poor overall survival in castration-resistant prostate cancer patients	[237]

Exosome-based targeted therapy

Inhibition of exosome secretion and transfer of oncogenic molecules

As discussed above, SMase has been found to be involved in the secretion of exosomes by regulating the synthesis of ceramide. Kosaka et al. found that GW4689, a chemical inhibitor that targets SMase, could reduce the secretion of miRNAs [184]. It was also found that GW4689-based exosome secretion targeting effectively reduced the in vivo occurrence of lung metastases in mice injected with Lewis lung carcinoma (LLC) cells. Exosomes derived from injected LLC cells could overcome this inhibition, thereby underscoring the importance of exosome-mediated cancer promotion [185]. Since intracellular calcium levels may play important roles in exosome secretion (see above), the targeting of calcium channels to modulate exosome secretion has been investigated as a therapeutic option. The effect of inhibiting H⁺/Na⁺ and Na⁺/Ca²⁺ channels, using dimethyl amiloride (DMA), a drug that blocks exosome formation, and K⁺/H⁺ ATPase using omeprazole, on exosome secretion has been studied by Chalmin et al. They found that both the drugs reduced exosome secretion in vitro and in vivo, and enhanced the efficacy of the anti-cancer drug cyclophosphamide in mouse models. In addition, they found that treatment with DMA or omeprazole inhibited the phosphorylation of STAT3 in MDSCs, thereby reducing its immunosuppressive ability and its ability to elicit immune evasion of cancer cells [186].

Also, Rab GTPases that play important roles in exosome secretion (see above) and other proteins that are known to be involved in exosome secretory pathways have been tested as putative therapeutic targets. Using a mouse model, Bobrie et al. found that Rab27 inhibition resulted in reduced exosome secretion. As a consequence, they observed a decrease in primary 4T1 breast cancer growth and its metastasis to lungs. In addition, they found that Rab27a-dependent secretion of exosomes resulting in the mobilization of neutrophils was required for 4T1 tumour growth [187]. A recent study showed that inhibition of YKT6, a SNARE protein that is involved in exosome synthesis and secretion, using siRNA and pre-miRNAs, led to a reduction in exosome secretion by approximately 80%. Since YKT6 expression is inversely correlated with the survival of non-small cell lung cancer (NSCLC) patients, inhibiting its activity and decreasing exosome synthesis may have important clinical implications [188]. Through a comparative proteome analysis of exosomes secreted by normal human T-cell blasts and acute leukaemia Jurkat T-cells, it was found that Jurkat cell-derived exosomes were enriched in 14 membrane proteins, the most abundant being valosin-containing protein (VCP). VCP is a membrane ATPase that acts to maintain endoplasmic reticulum (ER) homeostasis and ubiquitination, and may serve as a prognostic biomarker for gastric carcinoma. Treatment with a VCP inhibitor (DBeQ) was found to result in inhibition of exosome secretion only in Jurkat cells. Based on their results, the authors concluded that VCP targeting may be a novel therapeutic approach that interferes with exosome release [189].

It has also been reported that inhibition of exosome secretion may help to overcome drug resistance. Next to a low pH, cisplatin resistance in human melanoma cells was, for example, found to be due to the secretion of cisplatin-containing exosomes. Treatment of these cells with the proton pump inhibitor (PPI) lansoprazole resulted in an enhanced uptake of cisplatin and an inhibition of exosome release. In addition, it was found that tumours obtained from xenograft models contained more cisplatin when the animals were exposed to PPI and cisplatin. Subsequent plasma analysis revealed a decrease in the number of exosomes as well as in their cisplatin content [190]. Recently, Li et al. found that exosomes may contribute to drug resistance when tyrosine kinase inhibitors (TKIs), such as gefitinib, and chemotherapeutic agents, such as cisplatin, were co-administered. Gefitinib-treated PC9 NSCLC cells may acquire cisplatin resistance by upregulating autophagy and downregulating key apoptotic proteins. The authors also found that GW4869-mediated inhibition of exosome secretion in gefitinib-treated NSCLC cells not only reduced antagonistic effects when TKIs were co-administered with cisplatin, but also induced synergistic effects [191].

Other studies have been aimed at suppressing the pro-tumorigenic effects of exosomes by natural compounds and nutraceuticals. Zhang et al., for example, found that curcumin could reverse exosome-mediated inhibition of NK cell cytotoxicity. Specifically, they found that exosomes isolated from TS/A breast cancer cells could inhibit IL2-induced NK cell cytotoxicity by inhibiting the activation of STAT5. Treatment with curcumin caused a dose-dependent increase in ubiquitinated exosomal proteins and Jak3-mediated phosphorylation of STAT5 [192]. Interestingly, curcumin and its derivatives are known to target not only the cancer cells, but also CSCs through multiple regulatory mechanisms, hence sensitising resistant populations for a better treatment efficacy [193]. Taken together, curcumin may be able to reverse immunosuppressive effects induced by tumour cell-derived exosomes and increase the sensitivity of cancer cells to standard chemotherapy.

So, cancer cell sensitisation towards chemotherapy may be achieved via two approaches: (i) concomitant administration of drugs that inhibit exosome secretion and anti-cancer drugs and (ii) initial administration of exosome inhibitors to suppress cell-cell communication and subsequent anti-cancer drug administration to potentiate the chemotherapeutic effect. The latter approach seems to be more effective than the former, since it inhibits the communication between cancer cells as well as between cancer cells and other cells in the tumour microenvironment which, in turn, may sensitise cancer cells to chemotherapy.

Exosome-based immunotherapy

In recent years, exosomes have gained interest for their ability to modulate immune functions, especially exosomes derived from dendritic cells (DCs). These DC-derived exosomes (DC-Exo) carry antigens that can activate the immune system and stimulate the activity of immune cells, such as CD8⁺ T-cells, which may lead to tumour eradication. In addition, it has been found that tumour cells, when co-cultured with DC-Exo, may exhibit enhanced abilities to activate T-cells [194, 195].

DCs pulsed with tumour-derived exosomes (TEXs) may exhibit increased efficacies in eliciting immune responses both in in vitro and in vivo models. Rao et al. showed, for example, that DCs pulsed with hepatocellular carcinoma (HCC) TEXs exhibited potent immune responses in in vitro and in vivo mouse models by modulating the tumour immune microenvironment. In orthotopic models, significant tumour suppression was observed and improvements in immune functions were evident based on increased numbers of T lymphocytes, increased interferon-γ and decreased IL10 and TGF-β expression levels at tumour sites [196]. Anti-tumour immune functions elicited by exosomes can be further enhanced by the addition of ligands to tumour cells that stimulate DCs by using exosomes secreted by tumour cells. Wang et al. used exosomes derived from CD40 ligand gene-modified lung tumour cells (CD40L-Exo) and found that these exosomes promoted DC maturation and upregulated the secretion of cytokines, such as interferon-γ and IL12, in mouse splenocytes. In addition, they observed an increased ex vivo proliferation of CD4⁺ T-cells and that DCs pulsed with CD40L-Exo effectively inhibited tumour growth and increased the survival of mice inoculated with LLC cells [197]. Another way to improve the efficacy of DC-Exo is by culturing them in the presence of DC maturating agents. Such matured DCs are more effective in eliciting anti-tumour responses than immature DCs. Damo et al. reported the use of DC-Exo loaded with antigens and poly I:C as a DC maturating agent along with ovalbumin (OVA) to boost anti-tumour immunity in mouse melanoma models. The DC-Exo vaccines thus obtained were found to be more effective in stimulating OVA-specific CD8⁺ and CD4⁺ T-cells to acquire effector functions [198]. Heat shock proteins (HSPs), which represent important constituents of exosomes and serve as stress-induced molecular chaperones [199], can stimulate immune functions by augmenting the ability to target cancer cells. Li-Hong et al. reported that exosomes containing hsp60, hsp70 and hsp90 secreted by drug-resistant HepG2 HCC cells increased the activity of cytotoxic NK cells, upregulated the expression of the inhibitory receptor CD94 and downregulated the expression of the activating receptors CD69, NKG2D and Nkp44 [200]. In addition, it has been found that the location of HSPs in exosomes plays a crucial role in determining their anti-tumour immunity. It has, for example, been found that exosomes derived from myeloma cells with membrane bound Hsp70 may exhibit enhanced DC maturation capacities [199]. Thus, exosomes derived from DCs and other sources may serve as excellent vaccines for immunotherapy, and clinical trials have already been initiated to explore their safety and efficacy in humans (see below).

Engineered exosomes as therapeutic cargos

In recent years, exosomes have also received considerable attention as putative drug delivery vehicles due to several unique properties that they possess. Since exosomes are naturally derived vesicles, they do not trigger undesirable immune reactions and they remain undetected by the complement system, thus providing them with a better stability [201]. Recently, Aqil et al. investigated the efficacy of celastrol (CEL), a natural compound encapsulated in exosomes, against NSCLC cells. They found that CEL in a time- and concentration-dependent manner inhibited the proliferation of both lung carcinoma A549 (IC₅₀ 1.8 μM) and H1299 (IC₅₀ 1.4 μM) cells. Mechanistically, they found that CEL inhibited TNFα-induced NF-κB activation, upregulated the expression of ER stress chaperones and induced apoptosis. They also noted that exosome encapsulated CEL exhibited significant anti-tumour activity in xenograft models without systemic toxicity [202]. Others investigated an exosomal formulation of paclitaxel (ExoPTX) to overcome multidrug resistance in cancer cells. Exosomes shed by macrophages were used and subsequent encapsulation of paclitaxel produced ExoPTX with a high loading capacity and a sustained release rate. By using a drug-resistant kidney cell line (MDCK_MDR1) it was found that ExoPTX was 50 times more cytotoxic. Importantly, the authors found that encapsulation of paclitaxel in exosomes enabled it to bypass the P-gp efflux protein either by endocytosis-mediated transport or by fusion with the plasma membrane. In LLC-derived pulmonary metastatic mouse models, intranasal administration of ExoPTX resulted in its localization to cancer cells and a significant inhibition of the growth of lung metastases [203].

Some of the work related to exosomes as drug delivery vehicles has been aimed at strategies to produce exosomes at large scale that are devoid of harmful side-effects. In a recent study Mungala et al. investigated options to develop cost effective and biocompatible means of producing exosomes using bovine milk. Different drugs, such as withaferin A (WFA), bilberry-derived anthocyanidins, curcumin, paclitaxel and docetaxel were encapsulated in the exosomes. By doing so, they found that drugs loaded in bovine milk-derived exosomes exhibited increased efficacies compared to the free drugs and Exo-WFA was found to be effective in in vitro lung and breast cancer cell models, as well as in in vivo xenograft lung cancer models. It turned out that the exosomes remained in circulation until day 6. In addition, it was found that animals treated with empty exosomes did not exhibit any abnormal effects, indicating the safety of bovine milk-derived exosomes. Conjugation of folate to the exosomes further increased the tumour targeting ability of Exo-WFA [204]. In order to overcome limitations associated with exosome production and scalability, Jang et al. synthesised exosome-mimetic nanovesicles by the breakdown of monocytes/macrophages for targeted delivery of anti-cancer drugs. These exosome-mimetics had a higher production yield (100-fold). Through in vitro studies it was found that these nanovesicles loaded with doxorubicin retained their ability to selectively deliver drugs and induce tumour necrosis factor alpha (TNF-α)-stimulated endothelial cell death. In a mouse tumour model, the exosome mimetics were found to be effective in reducing tumour growth without causing any adverse side effects [205].

As of yet, exosomes have been successfully used as cargos for the delivery of miRNAs, anti-miRNAs and siRNAs to inhibit tumour growth and drug resistance. Breast cancer Hs578T cells and its aggressive triple negative clonal variant Hs578T(i)₈ exhibit enhanced invasion, migration and resistance to cisplatin due to miR-134 downregulation. It was found that exosome encapsulated miR-134 delivered to these breast cancer cells reduced their migrative and invasive capacities, and sensitised them to anti-hsp90 drugs. Upon miR-134 expression in the cells, significant decreases in STAT5B, Hsp90 and Bcl-2 protein levels were found [206]. Ohno et al. investigated the effect of let-7a miRNA-containing exosomes decorated with GE11 peptide to target EGFR expressing breast cancer cells through an EGFR-dependent internalization process. They injected luciferase-expressing HCC70 tumour cells into mammary fat pads of RAG2^−/− mice and found that, compared to control exosomes, the GE11 decorated exosomes bound 3 times more efficiently to the tumour cells. GE11 decorated exosomes containing let-7a miRNA also significantly inhibited HCC70 tumour growth, but the genes that are normally repressed upon let-7a expression were found to be unaffected. The authors concluded that let-7a may act by modulating other, as yet unknown, genes [207]. In another study, exosomes obtained from human embryonic kidney 293 (HEK293) cells were used to deliver siRNA against polo-like kinase 1 (PLK-1) in UMUC3 bladder cancer cells. PLK-1 is an important regulator of mitotic cell cycle progression and its upregulation has been found to result in increased tumour recurrence and metastasis. Delivery of this siRNA resulted in PLK-1 mRNA and protein expression knockdown and, concomitantly, apoptosis and necrosis induction [208]. In temozolomide (TMZ)-resistant glioblastoma cells it was found that anti-miR9 delivery via MSCs by either gap junction communication or by exosome transfer downregulated the expression of MDR-1, thereby causing a reversal of drug resistance and a re-sensitisation to TMZ [209].

In order to improve the targeting abilities of exosomes and to overcome limitations such as large scale production and identification of suitable cell types for the production of exosomes, Qi et al. developed a dual-functional exosome-based superparamagnetic nanoparticle cluster for drug delivery. In the presence of an external magnetic field, these exosomes (SMNC-EXO) exhibit superparamagnetic properties at room temperature enabling their efficient targeting to cancer cells. SMNC-Exo was found to exhibit excellent stability in fresh serum and to be biocompatible without adverse side effects in mice. SMNC-EXO loaded with doxorubicin (D-SMNC-EXO) accumulated passively in vivo at tumour sites to release doxorubicin via the enhanced permeability and retention (EPR) effect. The application of a magnetic field favoured active exosome accumulation. Among different treatment groups tested, mice dosed with D-SMNC-EXO in the presence of a magnetic field exhibited the highest tumour suppressive activities by decreasing Bcl-2 expression and increasing caspase-3 expression [210]. To further reduce toxicity, Tian et al. used immature mouse dendritic cells (imDCs) for exosome production. These exosomes were engineered to express Lamp2b fused to an iRGD peptide specific for αγ integrin and loaded with doxorubicin (iRGD-Exos-Dox). The authors found that these exosomes had a high encapsulation efficiency and delivered the drug to αγ integrin-positive breast cancer cells with an efficiency of approximately 95%. It was also found that iRGD-Exos-Dox inhibited the proliferation of different cancer cell types and delivered the drug efficiently to tumours in a triple-negative breast cancer mouse model. Mice treated with control exosomes exhibited a 15-fold increase in tumour volume, whereas iRGD-Exos-Dox treated mice exhibited only a 4-fold increase in tumour volume, with no mortality, morbidity, cardiotoxicity, hepatotoxicity and/or damage to other organs, such as spleen and lungs, thus suggesting a potential improvement in therapeutic efficacy [211].

Therapeutic approaches aimed at delivering drugs to malignant conditions in the brain often fail because of their inability to cross the blood-brain barrier (BBB). To overcome this problem, exosomes isolated from brain endothelial cells were used as vehicles to deliver paclitaxel or doxorubicin to brain cancer cells. It was found that the drugs encapsulated in endothelial cell-derived exosomes efficiently penetrated the BBB and resulted in smaller U-87 MG tumour masses in xenografted zebrafish embryos [212].

Taken together, these studies highlight the efficacy of exosomes as novel drug delivery vehicles. As such, they may be instrumental for the design of targeted therapeutic approaches.

Clinical Trials

Extensive research regarding the role of exosomes in cancer development and progression, as well as their putative relevance as diagnostic and/or therapeutic targets, has led to their inclusion into a variety of clinical trials. Most of the trials that are currently active or completed have focussed on the use of exosomes as drug delivery vehicles for cancer therapy, the identification of biomarkers for cancer diagnosis and exosomes derived from dendritic cells as cancer vaccines. No adverse effects or severe toxicities have been reported in the clinical trials conducted so far. Based on current knowledge, exosomes hold promise as cancer biomarkers, drug cargo vehicles and cancer vaccines. In Table 2 several clinical trials that are currently active, completed and/or not yet open for recruitment are listed.

Table 2.

Summary of completed, active and yet to be completed clinical trials using exosomes for the diagnosis and treatment of cancer

Clinical trial	Status	Result	Reference
Drug delivery & therapeutics:  Phase I clinical trial of the ability of plant exosomes to deliver curcumin to normal and colon cancer tissue	Unknown	–	[238]
Investigation of the ability of grape exosomes to prevent oral mucositis in patients with head and neck cancer undergoing chemo- and radiotherapy	Recruiting	–	[239]
Immunotherapy:  Phase II clinical trial to investigate the effect of second generation DC-derived exosomes (Dex) (IFN-γ-Dex) loaded with MHC class I and class II restricted cancer antigens to boost NK and T cell immune responses in inoperable NSCLC patients after first line chemotherapy	Completed	32% of the patients exhibited disease stabilization of more than four months; an increase in NK cell function was found in some patients with defective NKp30 expression	[240]
Phase I clinical trial to investigate the mechanism of activation of NK cells by Dex in melanoma patients	Completed	Dex enhanced the number of circulating NK cells and restored NKG2D ligand expression in circulating T and NK cells to elicit NKG2D-dependent NK cell cytotoxicity in 7/14 patients. NKG2D ligand present on the surface was required for NK cell activation. IL-15Rα was important for NK cell proliferation and IFN-γ secretion by NK cells	[241]
Phase I clinical trial to investigate the safety and feasibility of autologous exosomes pulsed with MAGE3 peptide as immunotherapy in stage III/IV melanoma patients	Completed	No grade II toxicity was found and the maximum tolerated dose was not achieved. According to the RECIST criteria, one patient exhibited a partial response. Large scale exosome production was feasible and exosome administration was safe	[242]
Diagnosis:  To correlate urinary exosome gene signatures with the presence or absence of high grade prostate cancer in prostate needle biopsies	Completed	Not available	[243]
Investigation of gastric cancer-derived exosomes as biomarkers and to study the prognostic efficacy of such exosomes in gastric cancer patients	Recruiting	–	[244]
Pilot study investigating the use of exosomes as a screening modality to diagnose oropharyngeal squamous cell carcinoma in human papilloma virus-affected patients	Recruiting	–	[245]
Pilot study to investigate whether Hsp70 exosomes in blood and urine samples of patients with malignant solid tumours may be used as diagnostic biomarkers	Recruiting	–	[246]
Analysis of urinary exosomal proteins to identify prognostic markers in patients with papillary, follicular or anaplastic thyroid cancer before surgery, immediately after surgery and at different time points post-surgery	Not yet open for recruitment	–	[247]
Safety and efficacy:  Phase I clinical trial to investigate the safety of autologous malignant glioma cells derived from malignant glioma patients treated with insulin-like growth factor receptor-1 antisense molecules encapsulated in diffusion chambers implanted in rectus sheath and exosomes released from dying tumour cells in activating the immune system against the tumour	Completed	Not available. However, based on the favourable safety profile, currently participants are recruited for a dose escalation study	[248] [249]
Phase I clinical trial to investigate the efficacy of ascites-derived exosomes (Aex) combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) as immunotherapy in colorectal cancer patients	Completed	Combination of Aex with GM-CSF was safe, well tolerated and induced anti-tumour cytotoxic T lymphocyte responses	[250]
Pilot study investigating the effect of the BRAF inhibitor vemurafenib on exosomes produced by patients with advanced unresectable or metastatic melanoma	Recruiting	–	[251]
Molecular studies:  Evaluation of miRNA expression in serum, bile, oesophageal cells, and miRNA expression within biliary exosomes to differentiate between gastroesophageal reflux, Barrett’s oesophagus and cancer	Recruiting	–	[252]
To investigate the consistency of PD-L1 expression in cancer tissue and plasma exosome in NSCLC patients	Not yet open for recruitment	–	[253]
To investigate the consistency of PD-L1 expression in lung cancer tissues and plasma exosomes before and after radiotherapy at different time points to probe the best timing at which PD-L1 is expressed	Not yet open for recruitment	–	[254]
Disease recurrence and response to treatment:  Role of exosome-mediated intercellular signalling and disease recurrence in pancreatic cancer patients	Recruiting	–	[255]
To investigate the efficacy of high dose hypofractionated radiotherapy in different cancers by quantifying the number of immune cells, as well as the amount of secreted factors and exosomes released in blood before, during and after high dose radiotherapy	Recruiting	–	[256]

Conclusions and future perspectives

In spite of being a relatively new field of research, exosomes have gained ample interest due to their multifaceted role in tumour biology, and their perspective as comprehensive tools for cancer eradication. Since exosomes mimic the contents of the tumour cells from which they originate, they possess the ability to affect neighbouring tumour cells as well as distant tumour and normal cells via circulation in different body fluids. It has been well documented now that transfer of exosomal components, such as mRNAs, miRNAs, lipids, proteins, signalling/epigenetic regulators and DNA molecules, may affect the behaviour of recipient cells. This exosome-mediated transfer of molecules may, for example, result in uncontrolled proliferation, epithelial-mesenchymal transition, angiogenesis and distant metastasis through modulation of the tumour microenvironment, expulsion of anti-cancer drugs to cause drug resistance, preparation of pre-metastatic niches, enhanced migration of tumour cells and re-emergence of tumours at metastatic sites after a prolonged period of dormancy. Together, these phenomena impose major challenges on cancer treatment. Apart from its tumour promoting effects, exosomes have also been found to serve as potential diagnostic/prognostic biomarkers. Current knowledge enables their use, not only as biomarkers for early cancer diagnosis, but also as useful biomarkers for predicting anti-cancer drug responses. The high stability of biomarkers within exosomes turns them into particularly suitable tools for these purposes. It is anticipated that these approaches will improve future cancer care and foster better treatment outcomes.

The ability of exosomes to carry a diverse range of molecules has enabled their use as depots to deliver conventional anti-cancer drugs, as well as natural compounds, miRNAs, siRNAs and anti-miRNAs. In vitro and in vivo studies have provided promising results and they have laid a foundation for ongoing and future clinical trials. Another promising aspect is the use of exosomes as vaccines for the treatment of cancer. Exosomes derived from dendritic cells and other sources have shown the ability to boost the capacity of immune cells to eradicate tumour cells, and they are currently in clinical trials to test their safety.

Though several studies performed so far have succeeded in using exosomes for cancer diagnosis, prognosis and therapy, it is important for future studies to take into account some of the challenges that still lie ahead related to large scale exosome production, biocompatibility, biodistribution and the efficient isolation of exosomal biomarkers from body fluids. It is also important to mention here that although some studies have already reported large scale exosome production, cost effective protocols, prolonged circulating times and biocompatible exosomes, further controlled pre-clinical and clinical studies are warranted.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.