Exosomes in cancer: small particle, big player

Exosomes are small, lipid bilayer membrane vesicles of endocytic origin. Exosomes
can be defined by several common characteristics, including size (50–100 nm in diameter),
density (1.13–1.19 g/ml), morphology (“cup” or “dish” shaped in transmission electron
microscopy), and certain enriched protein markers (tetraspanins, TSG101, Hsp70). Initially
discovered as the garbage bags for removal of unwanted material from cells, the role
of exosomes in immune response is gradually recognized as they function in antigen
presentation. More recently, the researchers reveal that exosomes contain proteins
and nucleic acids that are functional when transferred into recipient cells. Exosomes
have been shown to act as shuttles between cells by transmitting signals (referred
to as communicasomes). In this review, we highlight the recent advances in the roles
of exosomes in cancer with an emphasis on the potential of exosomes as diagnosis biomarker
and therapy target.

Biogenesis, release, and uptake of exosomes

Exosome formation is a fine-tuned process which includes four stages: initiation,
endocytosis, multivesicular bodies (MVBs) formation, and exosome secretion 1]. Multivesicular bodies (MVBs) are endocytic structures formed by the budding of an
endosomal membrane into the lumen of the compartment. After vesicular accumulation,
the MVBs are either sorted for cargo degradation in the lysosome or released into
the extracellular space as exosomes by fusing with the plasma membrane (Fig. 1). The mechanisms underlying the sorting of cargo into the intraluminal vesicles (ILVs)
are not yet fully elucidated. Both endosomal sorting complex required for transport
(ESCRT)-dependent and independent signals have been suggested to determine the sorting
of exosomes 2]. The formation of exosomes has been shown to be controlled by the syndecan heparan
sulfate proteoglycans and their cytoplasmic adaptor syntenin 3].

Fig. 1. Biogenesis, release, structure, and uptake of exosomes. Exosomes are produced from
the multivesicular bodies (MVBs) (also known as late endosomes). The membrane of the
MVBs bulges inward to form exosomes. During this process, proteins (e.g., receptor,
cytoplasmic proteins, tetraspanin), nucleic acids (e.g., DNA, mRNA, miRNA), and lipids
(e.g., cholesterol, ceramide) are packed into exosomes in a cell type-dependent manner.
MVBs fuse with the cellular membrane to release exosomes into the extracellular space.
Several mechanisms have been suggested to mediate the uptake of exosomes, including
a exosome fusion with the cellular membrane of the recipient cell, leading to the release
of the exosomal cargo into the cytoplasm, b juxtracrine signaling through receptor-ligand interactions, c and endocytosis by phagocytosis

The Rab guanosine triphosphatases (GTPases) have been found to critically regulate
exosome secretion. Ostrowski et al. have identified that Rab27a/b affects the size
and localization of MVBs 4]. Hsu et al. suggest that Rab3 regulates MVBs docking to tethering at the plasma membrane
5]. The accumulation of intracellular Ca
²⁺
results in increased exosome secretion 6]. In addition, intracellular and intercellular pH has been shown to affect exosome
release. When the microenvironmental pH is low, exosome secretion and uptake by recipient
cells increases 7]. There is evidence that oncogenes and tumor suppressors regulate exosome secretion
in cancer 8]. Yu et al. demonstrate that p53-regulated protein tumor suppressor-activated pathway
6 (TSAP6) induces exosome secretion under stressed conditions 9], 10]. Heparanase is an enzyme with elevated level in cancer. Overexpression of heparanase
promotes exosome secretion 11]. Intriguingly, exosomes from normal mammary epithelial cells inhibit exosome secretion
by breast cancer cells, implicating a feedback control to maintain dynamic equilibrium
12].

Exosomes transfer information to the target cells through three main ways: (1) receptor-ligand
interaction; (2) direct fusion with plasma membrane; (3) endocytosis by phagocytosis
(Fig. 1). Although the specific receptors that mediate the uptake of exosomes have not been
found, there are several proteins that may act as potential receptors for exosome
uptake, such as Tim1/4 for B cells 13] and ICAM-1 for APCs 14]. The uptake of exosomes by direct plasma membrane fusion mode has not been well studied.
Melanoma cells could take up exosomes by fusion and low pH facilitates this process
15]. Phagocytosis is an efficient way of exosome uptake. Phagocytic cells have a greater
uptake of exosomes than non-phagocytic cells 16]. The uptake of exosomes by recipient cells is energy dependent 17]. Heparan sulfate proteoglycans (HSPGs) function as internalizing receptors of cancer
cell-derived exosomes. Enzymatic depletion of cell-surface HSPG or pharmacological
inhibition of endogenous proteoglycan biosynthesis significantly attenuates exosome
uptake 18].

Structure and contents of exosomes

Exosomes consist of a lipid bilayer membrane surrounding a small cytosol (Fig. 1). The structured lipids not only mold the exosomes but are also involved in exosome
function. In addition to lipids, nucleic acids and proteins have also been detected
in exosomes. Thakur et al. demonstrate that double-stranded DNA is present in exosomes from cancer cells and
reflects the mutational status of the originated cells 19]. Valadi et al. demonstrate that exosomes contain mRNA and miRNA 20]. Exosome-carried RNA can shuttle between cells and thus is called “exosomal shuttle
RNA” (esRNA). The protein composition of tumor cell-derived exosomes has been well
characterized for a number of cancers by using different proteomic methods. The most
common proteins, mRNA, and miRNAs found in exosomes have been deposited in ExoCarta
(www.exocarta.org). To date, 4563 proteins, 1639 mRNAs, and 764 miRNAs have been identified in exosomes
from different species and tissues by independent examinations. The exosomal contents
vary between different physiological and pathological conditions and original cell
types. Moreover, the composition of exosomes can be distinct from the originated cells
due to the selective sorting of the cargo into exosomes.

Isolation, detection, and analysis of exosomes

Exosomes have been isolated and characterized from distinct cells under normal and
stressed conditions. At present, the most commonly used methods for exosome isolation
include ultracentrifugation, combined with sucrose gradient, and the immune-bead isolation
(e.g., magnetic activated cell sorting; MACS). There are many commercial kits available
for the extraction of exosomes. Transmission electron microscopy (TEM), Western blot,
and FACS are frequently used to characterize the isolated exosomes based on their
biochemical properties (e.g., morphology, size, exosomal markers). There is a lack
of the accurate method to determine the concentration of exosomes. The researchers
have to rely on inaccurate measurements of protein concentration or nanoparticle tracking
analysis. Quantitative RT-PCR, nucleic acid sequencing, Western blot, or ELISA are
used for exosome RNA and protein identification. The International Society for Extracellular
Vesicles (ISEV) has recently released minimal experimental requirements for definition
of extracellular vesicles and their functions 21].

Roles of exosomes in cancer

Accumulating evidence indicates that exosomes play important roles in cancer. Exosomes
transfer oncogenic proteins and nucleic acids to modulate the activity of recipient
cells and play decisive roles in tumorigenesis, growth, progression, metastasis, and
drug resistance (Fig. 2). Exosomes can act on various recipient cells. The uptake of exosomes may induce
a persistent and efficient modulation of recipient cells. In this section, we will
discuss about the roles of exosomes in cancer and the molecular mechanisms (Table 1).

Fig. 2. Roles of exosomes in cancer. Exosomes are critically involved in tumor initiation,
growth, progression, metastasis, and drug resistance by transferring oncogenic proteins
and nucleic acids. Tumor-derived exosomes can activate endothelial cells to support
tumor angiogenesis and thrombosis. Tumor-derived exosomes can convert fibroblasts
and MSCs into myofibroblasts to facilitate tumor angiogenesis and metastasis. Tumor-derived
exosomes contribute to create an immunosuppressive microenvironment by inducing apoptosis
and impairing the function of effector T cells and NK cells, inhibiting DC differentiation,
expanding MDSCs, as well as promoting Treg cell activity. Tumor-derived exosomes can
mobilize neutrophils and skew M2 polarization of macrophages to promote tumor progression.
Moreover, tumor-derived exosomes can help tumor cells develop drug resistance by transferring
multidrug-resistant proteins and miRNAs, exporting tumoricidal drugs, and neutralizing
antibody-based drugs. In turn, exosomes from activated T cells, macrophages, and stromal
cells can promote tumor metastasis and drug resistance

Table 1. Overview on the function of exosomes in cancer

Tumorigenesis

Normal cells are transformed into cancer cells in the process of tumorigenesis. Exosomes
from malignant cells have shown the potential to induce normal cell transformation.
For instance, prostate cancer cell-derived exosomes could induce neoplastic transformation
of adipose-derived stem cells (ASCs) 22], which is associated with trafficking of oncogenic proteins (Ras superfamily of GTPases),
mRNA (K-ras and H-ras), as well as miRNAs (miR-125b, miR-130b, and miR-155) by exosomes.
In addition, Melo et al. suggest that breast cancer cell-derived exosomes contain
precursor microRNAs (pre-miRNAs) associated with RNA-induced silencing complex (RISC)-loading
complex proteins, which could induce a rapid and efficient silencing of mRNAs in nontumorigenic
epithelial cells, resulting in transcriptome reprogramming and oncogenic transformation
23]. They further demonstrate that the exosomes from serum specimen from breast cancer
patients but not those from healthy donors induce tumor formation in mice when co-injected
with the nontumorigenic epithelial cells, suggesting a potential mechanism for exosome
in tumorigenesis. Cancer is composed of heterogeneous cell populations. Side population
(SP) cells are a sub-population of cells that exhibit stem cell-like characteristics
and can be isolated in cancer by adapting the Hoechst33342 staining method. Koch et
al. demonstrate that in diffuse large B-cell lymphoma, side population cells could export
Wnt3a via exosomes to neighboring cells, thus modulating SP-non-SP transitions and
maintaining population equilibrium 24]. Altogether, these findings indicate that exosomes may contribute to tumor development
and uncontrolled tumor progression by acting as a mediator in the transformation of
normal cells to malignant cells and a modulator for the balance between cancer stem
cells (CSCs) and non-CSCs.

Tumor growth

The promoting effects of exosomes from distinct sources on tumor cell proliferation
have been widely reported. Cancer cells uptake exosomes that contain survivin, an
anti-apoptotic protein, to protect them from genotoxic stress-induced cell death 25]. Exosomes from serum of glioblastoma patients contain EGFRvIII mRNA, which stimulate
the proliferation of human glioma cells through a self-promoting way 26]. Colon cancer cell-derived exosomes are enriched in ?Np73 mRNA. The proliferation
potential of target cells is greatly enhanced by incubation with ?Np73-containing
exosomes 27]. The interaction between tumor stromal cells and tumor cells also efficiently promote
tumor growth. Exosomes from chronic myelogenous leukemia (CML) cells stimulate bone
marrow stromal cells to produce IL-8, which in turn promote the growth of leukemia
cells 28]. Bone marrow mesenchymal stromal cells (BM-MSCs) from multiple myeloma (MM) patients
release exosomes that express increased levels of oncogenic proteins, cytokines, and
adhesion molecules to facilitate the growth of MM cells 29]. Thus, exosomes from tumor cells and microenvironment could act coordinately to promote
tumor growth.

Tumor angiogenesis

The formation of new blood vessels is required for tumor growth and progression. Proteomic
analysis has revealed that abundant angiogenic factors are present in malignant mesothelioma-derived
exosomes 30]. Exosome uptake induces upregulation of angiogenesis-related genes and results in
enhanced endothelial cell proliferation, migration, and sprouting 31]. Exosomes derived from hypoxic glioblastoma cells are more potent to induce angiogenesis
32]. Exosomes from metastatic breast cancer cells contain miR-105. Exosome-mediated transfer
of miR-105 degrades ZO-1 protein, disturbs tight junctions, and induces vascular permeability
in distant organs 33]. Exosomal miR-92a from K562 leukemia cells targets integrin ?5 to enhance endothelial
cell migration and tube formation 34]. MiR-210 is significantly enriched in exosomes from hypoxic K562 cells, which promotes
the angiogenic activity of endothelial cells 35]. Multiple myeloma cells grown under hypoxic condition produce more exosomes containing
miR-135b, which directly suppresses FIH-1, an inhibitor of HIF-1, to enhance endothelial
tube formation in endothelial cells 36]. Exosomes are critically involved in tumor angiogenesis by directly delivering angiogenic
proteins into endothelial cells or modulating the angiogenic function of endothelial
cells by exosomal miRNAs.

Tumor metastasis

Exosomes contribute to tumor metastasis by enhancing tumor cell migration and invasion,
establishing pre-metastatic niche, and remodeling the extracellular matrix. EBV-positive
nasopharyngeal carcinoma (NPC) cell-derived exosomes contain HIF-1?, which increases
migration and invasiveness of EBV-negative NPC cells 37]. Metastatic cancer cells secrete increased level of miRNA with tumor-suppressor function,
which may suggest another mechanism for the role of exosomes in metastasis 38]. The formation of pre-metastatic niche is a prerequisite for tumor metastasis. Exosomes
from highly metastatic melanoma enhance the metastatic ability of primary tumors by
converting bone marrow progenitor cells to a pro-vasculogenic and pre-metastatic phenotype
via the MET receptor 39]. Gastrointestinal stromal tumor cells release exosomes containing protein tyrosine
kinase to convert progenitor smooth muscle cells to a pre-metastatic phenotype 40]. Suetsugu et al. show that highly metastatic breast cancer cells can transfer their
own exosomes to other cancer cells and normal lung tissue cells in vitro and in vivo
by using fluorescent protein imaging method 41], which provides direct evidence for the involvement of exosomes from highly metastatic
cancer cells in educating stromal cells. Luga and colleagues have shown that exosomes
produced by stromal cells are taken up by breast cancer cells and are then loaded
with Wnt11, which is associated with stimulation of the invasiveness and metastasis
of the breast cancer cells 42]. Exosomes from activated CD8+ T cells promote cancer cell invasion and lung metastasis
via the Fas/FasL pathway 43], which adds another layer of mechanism for the role of tumor-infiltrating lymphocytes
in cancer metastasis. Exosome-mediated transfer of oncogenic microRNAs into cancer
cells is associated with enhanced metastatic potential. IL-4-activated macrophage-derived
exosomes transfer miR-223 to co-cultivated breast cancer cells, leading to increase
of cell invasion 44]. Exosome-mediated delivery of miR-221/222 from MSCs to gastric cancer cells greatly
enhances gastric cancer cell migration 45]. Fabbri et al. suggest that miRNAs in tumor-secreted exosomes can directly bind toll-like
receptor (TLR) in immune cells to promote tumor metastasis 46]. Recently, Costa-Silva and colleagues demonstrate that MIF-containing exosomes from
pancreatic ductal adenocarcinoma (PDAC) cells induce TGF-? production in liver Kupffer
cells, which in turn upregulates fibronectin (FN) expression by hepatic stellate cells
and enhances recruitment of bone marrow-derived cells, finally leading to the formation
of liver pre-metastatic niche 47], suggesting a complicated network that involves cancer cells, stromal cells, and
immune cells in exosome-initiated pre-metastatic niche formation. Intriguingly, Zomer
et al. use the Cre-LoxP system to visualize extracellular vesicle (EV) exchange between
tumor cells in living mice 48]. They show that the less malignant tumor cells that take up EVs released by malignant
tumor cells display enhanced migratory behavior and metastatic capacity, indicating
that the metastatic behavior can be phenocopied through extracellular vesicle exchange.
Taken together, these findings reveal that the intercellular communication mediated
by exosomes may be an important mechanism for tumor metastasis.

Tumor drug resistance

Exosomes contribute to the development of therapy resistance in tumor cells through
a variety of mechanisms. Tumor-derived exosomes can transfer multi-drug resistance
(MDR)-associated proteins and miRNAs to target cells 49], 50]. In addition, exosomes participate in the process of tumor resistance by mediating
drug efflux. The drugs and their metabolites can be encapsulated and exported by exosomes
51], 52]. Melanosomal sequestration of cytotoxic drugs contributes to the intractability of
malignant melanomas 53]. Moreover, exosomes may counteract the effect of antibody drugs by modulating their
binding to tumor cells. Lymphoma exosomes carry CD20, which bind therapeutic anti-CD20
antibodies and protect target cells from antibody attack 54]. Exosomes from HER2-overexpressing breast cancer cells express active HER2 and can
bind to the HER2 antibody trastuzumab to inhibit its activity 55]. Exosomes secreted by stromal cells also contribute to tumor drug resistance. BM-MSC-derived
exosomes induce multiple myeloma cells resistant to bortezomib through the activation
of several survival relevant pathways 56]. Therefore, exosomes released by cancer cells and stromal cells may have a potential
to modulate sensitivity of cancer cells to distinct therapies.

Tumor immune escape

Initially reported as tumor-associated antigens and tumor immune response stimulators,
the recent studies have shown that tumor-derived exosomes might rather perform immunosuppressive
functions. Tumor exosomes block the differentiation of murine myeloid precursor cells
into dendritic cells (DC) 57]. Tumor exosome-carried TGF-?1 skews IL-2 responsiveness in favor of regulatory T
cells and away from cytotoxic cells 58]. Human nasopharyngeal carcinoma-derived exosomes recruit, expand, and regulate the
function of regulatory T cells through CCL20 59]. NPC cell-derived exosomes impair T cell function, which is associated with upregulated
miRNAs in the exosomes 60]. Tumor cell-derived exosomes switch the differentiation of myeloid cells to myeloid-derived
suppressor cells (MDSCs) and induce accelerated lung metastasis in a MyD88-dependent
manner 61], 62]. Hsp72 on tumor-derived exosomes promotes the immunosuppressive activity of MDSCs
via autocrine activation of IL-6/STAT3 pathway 63]. Breast cancer cell-derived exosomes simulate the activation of NF-?B and enhance
the secretion of pro-inflammatory cytokines in macrophages 64]. Exosomes from human prostate cancer cells express ligands for NKG2D on their surface
and downregulate NKG2D expression on natural killer (NK) and CD8+ T cells, leading
to the impairment of their cytotoxic function 65]. Collectively, these data suggest that tumor-derived exosomes interfere on multiple
levels with the immune system to drive tumor immune evasion.

Tumor-stroma interaction

Tumor stroma is believed to be critically involved in tumor development and progression.
Webber et al. suggest that prostate cancer cells could trigger differentiation of fibroblasts into
myofibroblasts through exosomal TGF-? 66]. In addition, prostate cancer exosomes triggered TGF?1-dependent fibroblast differentiation
resemble stromal cells isolated from cancerous prostate tissue 67], which accelerates tumor growth by supporting angiogenesis. MSCs function as precursors
for tumor myofibroblast. The research from our lab suggests that tumor cell-derived
exosomes could induce differentiation of human MSCs to carcinoma-associated fibroblasts
(CAFs) 68]. Adipose tissue-derived MSCs treated with breast cancer-derived exosomes also display
the characteristics of myofibroblasts 69]. Moreover, stromal communication with cancer cells modulates therapy response. Boelens
et al. suggest that exosomes transferred from stromal cells to breast cancer cells
constitute a juxtacrine NOTCH3 pathway to expand therapy-resistant tumor-initiating
cells 70]. Luga et al. demonstrate that fibroblast-secreted exosomes mobilize autocrine Wnt-planar
cell polarity (PCP) signaling to drive breast cancer cell invasion and metastasis
42]. Therefore, exosomes may mediate a reciprocal interplay between tumor cells and stromal
cells to synergistically promote tumor progression.

Tumor thrombosis

Tissue factor (TF) overexpression is closely associated with tumor progression. TF
can get incorporated into tumor-derived exosomes. The hypercoagulable state in cancer
patients may be partially influenced by the release of TF-bearing exosomes from tumor
cells. Garnier et al. demonstrate that exosomes link the procoagulant status with
metastatic phenotype in cancer. Induction of EMT changes in epithelial cancer cells
results in the release of exosomes containing elevated level of tissue factor. Importantly,
TF-rich exosomes can be transferred to endothelial cells and cause their exaggerated
procoagulant conversion 71], suggesting that EMT influences tumor-vascular interaction through altered TF-containing
exosomes. However, the exact roles of exosomes in tumor thrombosis and consequent
impact on tumor growth, progression, and metastasis remain to be further explored.

Exosomes as cancer biomarkers and targets

The findings that exosomes play critical roles in almost all aspects of cancer provide
opportunities for the development of exosomes as ideal diagnostic biomarkers and therapeutic
targets. Exosome-shuttled proteins and nucleic acids have been suggested as novel
diagnostic and prognostic indicators for a variety of cancers. Moreover, utilizing
tumor-derived exosomes as vaccines and exosomes from distinct sources as carriers
for drugs and small molecules have been proved to be effective in pre-clinical studies
and clinical trials.

Exosomes as cancer diagnostic biomarkers

Exosomes are readily accessible in nearly all body fluids including blood, urine,
saliva, and ascites. Exosomes contain bioactive molecules that reflect the pathological
state of the originated cells, thus providing an enriched source of biomarkers (Table 2). The level of exosomes is elevated in the plasma of some cancer patients as compared
to healthy controls. There is a positive correlation between the abundance of tumor
exosomes and tumor stage in ovarian cancer patients 72]. Tumor is characterized by a specific miRNA profile. The majority of circulating
microRNAs is concentrated in exosomes 73]. Exosomal miRNAs have been suggested as diagnostic and prognostic indicators for
lung cancer, esophageal squamous cell carcinoma, prostate cancer, breast cancer, glioblastoma,
ovarian cancer, and other cancer types 74]–80]. Exosomal miRNAs are positively correlated with the stage and degree of cancer progression.
In addition to miRNAs, long non-coding RNAs (LncRNAs) are also detected in exosomes
81], 82]. LncRNA from serum of gastric cancer patients is defined as a novel exosomal biomarker
83], 84].

Table 2. Exosomes from distinct biofluids of cancer patients as biomarkers

Exosomes as cancer therapy targets

Exosome-based immunotherapy

Dendritic cell-derived exosomes (dexosomes) have been developed as immunotherapeutic
anticancer agents 85]. Tumor peptide-pulsed DC-derived exosomes suppress growth of established murine tumors
in a T cell-dependent manner 86]. Exosomes secreted by living tumor cells contain and transfer tumor antigens to dendritic
cells and induce potent CD8+ T cell-dependent antitumor effects on mouse tumors 87]. Dexosomes have entered clinical trials for colorectal cancer, metastatic melanoma,
and non-small cell lung cancer and have achieved modest therapeutic effects 88].

Exosome removal for cancer therapy

The removal of exosomes from advanced cancer patients is a novel strategy to treat
cancer 89]. Exosome depletion by dimethyl amiloride (DMA) in mice restores the anti-tumor efficacy
of cyclophosphamide (CTX) through the inhibition of MDSC functions. Amiloride, a drug
used to treat high blood pressure, inhibits exosome formation and blunts MDSC suppressor
functions in colorectal cancer patients 63]. The biotechnology company Aethlon Medical has developed an adjunct therapeutic method
HER2osome, which is able to reduce tumor-secreted HER2 positive exosomes in the circulation
and thus inhibit HER2-positive breast cancer progression. However, further work is
needed to evaluate the clinical safety of such a treatment strategy based on exosome
removal.

Exosomes as anti-cancer drug delivery vehicles

The use of exosomes as nucleic acid or drug delivery vehicles has gained considerable
interest due to their excellent biodistribution and biocompatibility 90]. Exosome-mediated delivery of therapeutic short interfering RNA (siRNA) to the target
cells has been tested. The exosome-delivered siRNA is effective at causing post-transcriptional
gene silencing and inducing cell death in recipient cancer cells 91]–93]. To improve drug delivery efficacy to tumors, the researchers have modified exosomes
with targeting ligands such as iRGD-Lamp2b. The modified exosomes show highly efficient
targeting to ?V integrin-positive breast cancer cells, and intravenous injection of
these exosomes obviously inhibits tumor growth 94]. In addition, exosomes have been utilized as effective vehicle for drug delivery
95]. Exosomes from MSCs have been tested as the vehicle to package and deliver active
drugs such as paclitaxel 96].