Exosome Mediated Communication within the Tumor Microenvironment
Lara Milane, Amit Singh, George Mattheolabakis, Megha Suresh, Mansoor M. Amiji
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S0168-3659(15)30001-8
doi: 10.1016/j.jconrel.2015.06.029
COREL 7734

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Journal of Controlled Release

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28 May 2015
19 June 2015

Please cite this article as: Lara Milane, Amit Singh, George Mattheolabakis, Megha
Suresh, Mansoor M. Amiji, Exosome Mediated Communication within the Tumor Microenvironment, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.06.029

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Exosome Mediated Communication within the Tumor
Microenvironment

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Lara Milane, Amit Singh, George Mattheolabakis, Megha Suresh, and Mansoor M. Amiji*

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Department of Pharmaceutical Sciences, School of Pharmacy, Bouve College

of Health Sciences, Northeastern University, Boston, MA 02115
(*Corresponding author: Tel. 617-373-3137, Fax: 617-373-8886, Email: m.amiji@neu.edu)

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Running title: Exosome Mediated Communication

Abstract

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It is clear that exosomes (endosome derived vesicles) serve important roles in cellular
communication both locally and distally and that the exosomal process is abnormal in cancer.
Cancer cells are not malicious cells; they are cells that represent survival of the fittest at its
finest.
All of the mutations, abnormalities, and phenomenal adaptations to a hostile
microenvironment, such as hypoxia and nutrient depletion, represent the astute ability of cancer
cells to adapt to their environment and to intracellular changes to achieve a single goal
survival. The aberrant exosomal process in cancer represents yet another adaptation that
promotes survival of cancer. Cancer cells can secrete more exosomes than healthy cells, but
more importantly, the content of cancer cells is distinct. An illustrative distinction is that
exosomes derived from cancer cells contain more microRNA than healthy cells and unlike
exosomes released from healthy cells, this microRNA can be associated with the RNA-induced
silencing complex (RISC) which is required for processing mature and biologically active
microRNA. Cancer derived exosomes have the ability to transfer metastatic potential to a
recipient cell and cancer exosomes function in the physical process of invasion. In this review
we conceptualize the aberrant exosomal process (formation, content selection, loading,
trafficking, and release) in cancer as being partially attributed to cancer specific differences in
the endocytotic process of receptor recycling/degradation and plasma membrane remodeling
and the function of the endosome as a signaling entity. We discuss this concept and, to
advance comprehension of exosomal function in cancer as mediators of communication, we
detail and discuss; exosome biology, formation, and communication in health and cancer;
exosomal content in cancer; exosomal biomarkers in cancer; exosome mediated
communication in cancer metastasis, drug resistance, and interfacing with the immune system;
and discuss the therapeutic manipulation of exosomal content for cancer treatment including
current clinical trials of exosomal therapeutics. Often referred to as cellular nanoparticles,
understanding exosomes, and how cancer cells use these cellular nanoparticles in
communication is at the cutting edge frontier of advancing cancer biology.
Keywords: exosomes, cancer, microRNA, signaling endosome, metastasis
1. Introduction
One of the most groundbreaking discoveries in cell biology in the past thirty years was
the discovery of exosomes; endosome derived vesicles (30-100 nm) shed by cells in processes
of cellular housekeeping and communication. Exosomes were discovered in 1983 as transferrin
associated 50 nm vesicles extruding from reticulocytes [1]. Although it has been thirty years
since the discovery of exosomes, we are still in the infancy of understanding exosome biology

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and function. It is clear that exosomes are an important avenue for intracellular communication
but some exciting and important questions remain to be answered. The most pivotal question
that remains to be answered is; is exosomal release a form of deliberate cell communication?
Considering how cancer cell exosomes can transform local healthy epithelial cells into
cancerous cells, function in invasion of the extracellular matrix, and contribute to distal
metastasis [2], it seems intuitive that this process would be deliberate; as if exosomes were the
cells elevator pitch to their surroundings here is a quick message, this is what I want you to
know. Our progressive understanding of the function of exosomes in health and disease
certainly implies that the exosomal process (formation, content selection, loading, trafficking,
and release) is an orchestrated and deliberate process. For example, a recent study using a
chorioallantoic membrane of chick embryos as an in vivo tissue model, demonstrated that tumor
cell xenografts within the membrane required exosome release for directional movement and
correlated exosome release to the speed of cell movement through the membrane [3].
Inhibiting exosome release from the tumor cells disabled directional movement in the membrane
[3]. Although we are gaining insight into both, how proteins are sorted into exosomes and
selected for secretion, and the astounding functions of exosomes in cancer, the pivotal question
still remains unanswered and naturally leads to a second question; is exosome release only
deliberate in cancer cells? Exosomes are of endosomal origin so to understand the exosomal
process and to explore these questions, we must turn to the endosomal process and the
distinctions between cancer cell exosomes and exosomes released from healthy cells to
investigate.
The difference between normal (healthy) cell exosomes and cancer cell exosomes has
been well noted in numerous studies indicating that both the rate of exosomal release and the
content (most notably microRNA) is increased and distinct. Various cancer cell lines have been
shown to produce more exosomes relative to normal cells. For example, a study published in
2014 compared exosome release from a normal human mammary epithelial cell line and a
breast cancer clone derived from the parent cell line. The study demonstrated that within a 24
hour period, exosome release from the normal (parent) cell line was (4.5 2.3) x 108 exosomes
per 106 cells whereas exosome release for the cancer cell line was (53.2 1.6) x 10 8 exosomes
per 106 cells [4]. Aligned with these, in vitro studies [4], in vivo studies [5] , and clinical analysis
[2] have revealed an increase in cancer cell and tumor derived exosomes in cells and in
patients. In addition to increased exosome release, the distinct content of exosomes has
clinical significance.
A seminal study by Melo, et al., demonstrated that, unlike exosomes released from
normal cells, exosomes released from breast cancer cells secrete microRNAs that are
associated with the RISC loading complex; this complex is essential for processing of premicroRNA into mature microRNA which can functionally silence target genes [2]. The
researchers demonstrated that exosomes from the sera of breast cancer patients as well as
exosomes from breast cancer cells in culture, were capable of transforming normal epithelial
cells into tumor forming cells and that this transformation was dependent on the RISC complex
protein, Dicer [2]. The study went on to illustrate that CD43 (associated with metastatic breast
cancer) is responsible for the increase of Dicer loading in breast cancer exosomes [2]. This
study not only identifies the significance of Dicer in breast cancer derived exosomes, it
demonstrates the potential of exosomes as mediators of tumorigenesis [2]. Until recently, the
most commonly accepted theory of exosome sorting and loading was that exosome content is
determined non-specifically under multivesicular formation and not through a deliberate sorting
and packaging process [6]. Although completely logical, explaining the difference in cancer
exosome content as a fundamental difference in cellular composition, this deduction fails to
address why the rate of exosomal release can be increased in cancer, why exosomal content
does not always parallel cellular content, and how exosomes seem to function in deliberate
processes such as invasion. The theory of passive exosome loading is beginning to be
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disproved as we learn more about the lipids, receptors, and sorting machinery such as the
endosomal sorting complex required for transcript (ESCRT) that coordinate to select and
package exosomal content [7].
Although we are beginning to decipher the molecular mechanisms of exosomal sorting
and release, and how the content of cancer exosomes differs from exosomes derived from
healthy cells, still, little progress has been made to explain and understand how the exosomal
process is distinct in cancer cells and why cancer cells can have an increased rate of exosome
release and distinct exosomal content. A 2009 study demonstrated that exosome release and
uptake is increased in an acidic pH, suggesting that the acidic microenvironment of tumors
contributes to the increased rate of exosomal release and uptake by cancer cells [8]. We
postulate that, in addition to factors such as microenvironmental pH, the aberrant exosomal
process in cancer cells can be partially attributed to cancer specific differences in the
endocytotic process of receptor recycling/degradation and plasma membrane remodeling and
the function of the endosome as a signaling entity (Figure 1). Abnormalities in cancer cell
surface receptors are well documented with notable over-expression of many different plasma
membrane receptors such as growth factor receptors, glucose and nutrient importers, ABC
transporters (dug efflux pumps), folate receptors, and various importers [9-12]. Cancer cells
also have type-specific mutations or protein alterations that result in increased endocytosis [13].
Although endocytosis does not imply a direct release of exosomes, under conditions of
increased endocytosis, paralleled exocytosis (and accompanying exosome release) is important
for maintaining cell volume and membrane integrity [14, 15]. Receptor dysregulation in cancer
can result in plasma membrane remodeling independent of ligand binding and receptor
clustering (dimerization), for example, through receptor cross regulation; HER2 overexpression
often characterizes types of lung, salivary, stomach, bladder, ovarian, and breast cancer and
has been shown to increase recycling of the EGFR (Epidermal Growth Factor Receptor) [16].
Mutations in the p53 tumor suppressor gene (175H and 273H) in lung cancer cells have been
shown to increase EGFR and integrin (51) recycling to the plasma membrane and studies
have demonstrated that this increase was due to association of integrin and EGFR with RCP
(Rab coupling protein; the Rab proteins function in intracellular trafficking) [17]. These studies
also demonstrated that knockdown of RCP only effected integrin and EGFR trafficking in p53
mutant cells (not in wild type p53 cells) and this knockdown in the mutant cells impaired
invasion and directional movement [17]. These results, combined with the results from the study
of exosome function in directional movement of cancer cells (tumor cell migration through the
chorioallantoic membrane of chick embryos) suggest that p53 mutations may enhance invasion
by promoting receptor recycling and exocytosis with subsequent exosome release and
directional movement. As well as cancer specific differences in endocytosis, receptor recycling,
and plasma remodeling resulting in subsequent differences in the exosomal process, the
function of the signaling endosome contributes to the formation of abnormal cancer exosomes.
The signaling endosome is an effector that works in concert with cellular pathways, actively
engaging with cytosolic components to load specific content through specific receptors or
channels, such as the loading of microRNA through ceramide-rich lipid raft regions [18-22]. The
function of the signaling endosome as a dynamic and responsive entity results in a cell specific
endosomal process including exosome formation, content selection and loading, endosomal
trafficking, and exosome release. As such, exosomes released from cells can function to
achieve specific cellular directives such as digestion of the extracellular matrix for invasion and
movement.
As exosomes are derived from the endocytotic pathway, endocytosis can be considered
the first step in the exosomal process. For a review of endocytosis in cancer, the reader is
directed to Mosesson, Mills, and Yardens review; briefly, endocytosis can be clathrin mediated
which is usually associated with clustered receptors, endocytosis can be cavaelae mediated
(cavoelae are specialized lipid rafts), and endocytosis can be clathrin and cavoelae independent
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Figure 1. The Exosomal Pathway in Cancer.

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The exosome process begins with endocytosis, follows the endosomal pathway, and ends
with exocytosis. (1) Genotypic and phenotypic alterations in cancer result in cell-specific
changes in the expression of plasma membrane receptors and plasma membrane
constituents. (2 & 3) Cancer plasticity, altered plasma membrane receptor expression,
characteristic changes in endocytosis (such as the overexpression of HER2 resulting in
increased EGFR recycling), and increased plasma membrane remodeling results in
endocytosis. (4) The endosome functions as a signaling entity; to coordinate with cellular
pathways and interact with cellular components. The signaling endosome responds to the
celluar environment and cellular signals. As such, endosomes can be recruited to achieve
specific cellular tasks such as promoting invasion through exosome release of proteases. (5)
For loading of specific content into endosomes, proteins, molecules, and microRNAs are
trafficked towards the endosome. (6) Intraluminal vesicles (ILV) begin to form. (7) The
endosome functions as a true signaling entity to select content for exosome packaging and
release, and for degradation and recycling (not shown). Lipid rafts in the endosome load
microRNA while proteins, receptor ligands, and transcription factors can be loaded via the
ESCRT machinery (endosomal sorting complex required for transcript), importers, channels,
or receptors. (8) The Rab GTPases function in trafficking of multivesicular bodies (MVB) to
the site of the plasma membrane. (9) SNARE (soluble NSF attachment protein receptors)
dependent or independent mechanisms (such as ceramide signaling) function to fuse MVBs
with the plasma membrane. (10) Exosomes exhibit an effect in their local microenvironment
(such as enhancing invasion) but they also enter the systemic circulation to influence distal
regions of the body.
[13]. Endocytosis occurs as a signaling response to internalize extracellular molecules (nonspecific), to internalize receptor ligands (receptor mediated), to recycle or degrade plasma
membrane receptors (ligand bound and inactivated receptors) and to recycle or degrade
membrane constituents [21, 23]. Cancer cells are associated with abnormal expression levels
of plasma membrane receptors and components and these distinctions are often used to
characterize cancer subtypes (biomarkers). For example, over expression of epidermal growth
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factor receptor (EGFR) is often associated with triple negative breast cancer that lacks estrogen
receptors, progesterone receptors, and HER2 receptors [24]. Also of note, the expression of
plasma membrane receptors and constituents is under a state of constant and dynamic
remodeling as the phenotype of the cancer cell changes; tumors are heterogeneous cell
populations but individual cancer cells can also be highly plastic cells capable of rapidly
adapting to hostile microenvironmental pressures [12]. This plasticity often results in changes in
the expression of plasma membrane receptors and subsequent plasma membrane remodeling
[12]. As receptors are marked for recycling or degradation they enter the endocytotic pathway,
transitioning from early endosomes to multivesicular bodies and can subsequently lead to
exosomal release. In support of our concept, a recent study demonstrated that endocytosis of
lipid rafts in mesenchymal stem cells directly correlates to increased secretion of exosomes
[25]. Lipid rafts are a scaffold for clathrin-independent endocytosis and plasma membrane
remodeling [21]. Cancer cell-specific characteristics such a lipid constituents of the plasma
membrane, expression of receptors, mutations in oncogenes, reactions to hypoxia, and
acquisition of drug resistance assuredly result in signaling endosome responses and
subsequent exosome alterations. Additional factors influence exosome release such as
microenvironmental interactions and feedback loops between cancer cells and the extracellular
matrix, stromal cells, other tumor cells, and healthy cells. [4].
To propel the field of exosomal biology and therapeutics in cancer we have compiled a
comprehensive review of cancer exosome biology and therapeutics. A key to understanding
exosome biology is understanding the endosome as a signaling entity or a transient organelle
(signaling endosome), not a passive vesicle in which exosomes are formed. This is perhaps the
first tenant to understanding exosomal communication; understanding the endosome as a
signaling entity that determines the contents of exosomes through its interaction with cytosolic
components. In this review we discuss the concept of the endosome as a signaling entity that
dictates exosomal fate and our concept of plasma membrane remodeling governing the
exosomal process in cancer (Figure 1), detail exosome biogenesis and function (Section 2),
discuss exosomal content in cancer (Section 3), detail the application of exosomes in cancer
biomarker screening (Section 4), discuss exosomal communication in cancer (Section 5), and
detail the application of exosomes as cancer therapeutics (Section 6). Exosomes are an
important constituent of the tumor microenvironment; as such, comprehension of cancer derived
and associated exosomes is a critical component of cancer biology.
2. Exosome Biology, Formation, and Communication in Health and Disease
2.1 Biogenesis
Cells release many different types of vesicles such as exosomes derived from endocytotic
multivesicular bodies, ectosomes formed from outward budding and pinching of the plasma
membrane, and secretory vesicles formed at the trans-Golgi network [26]. The two most
distinguishing features of exosomes are their endocytotic origin and their small size (from 30 100 nm); ectosomes are much larger (100 350 nm) while secretory vesicles can vary greatly
in size [26].
Exosomal release is one of three possible fates for multivesicular bodies (MVB).
Multivesicular bodies are formed from early endosomes. When plasma membrane receptors
are marked for recycling or degradation through ubiquitination and when invagination of the
plasma membrane occurs, endocytosis initiates and early endosomes are formed through
internalization of the plasma membrane. As internal vesicles form within the endosome, the
endosome transitions to become multivesicular bodies [6]. Multivesicular bodies are also
referred to as late endosomes or multivesicular endosomes. The internal vesicles are termed
intraluminal vesicles (ILVs) and they are formed from inward budding of the MVBs, with
membrane orientations the same as the extracellular facing side of the cellular plasma
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membrane [27]. The three fates for multivesicular bodies are; recycling through the trans-Golgi
network, lysosomal degradation, or secretion through exocytosis (exosome release) [6]. The
endosomal origin of exosomes is confirmed by proteomic analysis; exosomes contain proteins
from endosomes, the plasma membrane and the cytosol [28]. Exosomes also have high levels
of lipids and constituents associated with lipid rafts such as phosphatidylserine, sphingomyelin,
ceramide, cholesterol [27].

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2.2. Sorting
Much investigation has focused on determining how exosome content is selected and if
this is a deliberate process; exosome content is rich in enzymes, microRNA, transcription
factors, heat shock proteins, MHCs, cytoskeleton components, signal transducers, and
tetraspanins (transmembrane proteins). The function of ESCRT in sorting ubiquitinated proteins
provides insight to the sorting processes that govern exosomal content [7].
The early endosome recruits the ESCRT machinery for the formation of ILVs; the ILVs are
pre-exosomes [27, 29]. The ESCRT machinery consists of four ESCRT protein complexes and
associated proteins; ESCRT-0 functions in a ubiquitin-dependent fashion to cluster cargo,
ESCRT-I and ESCRT-II are involved in budding, while ESCRT-III functions in vesicle scission
[27, 29]. Accessory proteins include ALIX and VPS4; ALIX is involved in vesicle budding and
VPS4 assists with scission [27, 29].
There are also ESCRT independent mechanisms of MVB, ILV, and exosome formation;
these mechanisms can involve lipids such as ceramide and cholesterol, phospholipase D2, or
tetraspanins [27, 29]. Thinking of the early endosome as a signaling entity, it is easy to
conceive how exosomal content can be determined by the membrane constituents of the early
endosome. The early endosome has receptors that can facilitate the encapsulation of cytosolic
components; for example, tetraspanins have been demonstrated to facilitate the incorporation of
specific cargo into exosomes. Tetraspanin CD9 has been shown to mediate exosomal loading
of the metalloproteinase CD10 while CD63 has been shown to control the loading of LMP1
Epstein-Barr virus protein into exosomes [30-32]. Likewise, the lipid composition of the early
endosome and MVB as well as membrane dynamics have been shown to govern exosomal
content. For example, the formation of lipid rafts by ceramide has been demonstrated to
mediate microRNA loading into exosomes [18, 19].
Additional studies suggest a dominating role of ceramide and nSMase2 (neutral
sphingomyelinase 2; the rate limiting enzyme of ceramide biosynthesis) in the exosomal
process as ceramide orchestrates an ESCRT independent process of exosome formation,
loading, and release [19, 33]. This could have powerful implications for cancer therapeutics;
would disrupting the ceramide content of exosomal membranes disable the ability of cancer
cells to form and secrete exosomes and subsequently reduce metastasis? Or perhaps only
impair microRNA loading into exosomes? The contents of cancer cell exosomes are distinct
from exosomes released from healthy cells; cancer exosomes deliver functional complexes
including excessive microRNA that can exert biological activity (without further activation) upon
uptake by recipient cells exosome content in cancer is the topic of Section 3 below.
2.3 Exocytosis and Secretion
Exosome secretion through exocytosis is mediated through Rab GTPases, molecular
motors, cytoskeletal proteins, and SNAREs (soluble NSF attachment protein receptors);
secretion can also be effected by factors such as intracellular Ca2+ levels (increased Ca2+
results in increased exosome secretion) and extracellular/intracellular pH gradients [8, 34].
The Rab GTPases are responsible for trafficking of late endosomes/MVBs to the site of
the plasma membrane [27, 35]. Different Rab isoforms appear to dominate MVB trafficking to
the plasma membrane in different cell types; for example in HeLa cells, silencing (RNAi) of the
Rab27a isoform increased the size of MVBs while silencing of the Rab27b isoform diverted the
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MVBs to the perinuclear region of the cell instead of towards the plasma membrane [35].
Following plasma membrane docking, the SNARE protein complexes facilitate membrane fusion
of the MVBs with the plasma membrane although SNAREs do not appear essential for exosome
release [27].

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2.4 Exosome Uptake

Exosomes are internalized by recipient cells through receptor-mediated endocytosis,
pinocytosis, phagocytosis, or through fusion with the cell membrane resulting in direct release of
contents into the cytoplasm. As exosomes can really be thought of as cellular derived
nanoparticles, it is logical to expect that that these cellular nanoparticles can target specific cells
depending on their surface modification. This has been an area of intense research for drug
delivery applications and therapeutic development, but this targeting ability has also been
demonstrated by endogenous exosomes. For example, a 2010 study exploring the effects of
the tetraspanin Tspan8 on angiogenesis in a rat adenocarcinoma model, demonstrated that
Tspan8 in exosomes recruited specific proteins for loading that facilitated endothelial cell
binding and uptake (CD106 and CD49d), Tspan8 exosomes targeted endothelial cells, and
induced angiogenesis [36]. This study depicts how the endosome is a signaling entity that
dictates exosome fate; Tspan8 incorporated in the endosome and subsequently into exosomes
selected proteins to ensure target cell uptake (endothelial cells) to exert a biological effect
(angiogenesis) in the target cell. Although the paracrine effects of exosomes are well
documented, numerous studies are beginning to emerge documenting distal uptake of
exosomes. A seminal exosomal study (discussed in the later metastasis section) by Lieberman
et al details that distal sites of animal models (the lungs of treated mice) can be transformed
upon systemic administration of exosomes [5]. Although it is unclear how far endogenous
exosomes can travel in the body and how long they can maintain stability in systemic
circulation, studies of exogenous therapeutic exosomes (detailed in Section 5) allude to the
tremendous stability of exosomes and their ability to target most tissues. Systemically
administered exosomes targeting the acetylcholine receptor have even been shown to deliver
siRNA into the brains of treated mice, demonstrating the clear therapeutic potential of exosomes
to target even the most difficult tissue to access while maintaining systemic stability [37].
Positive and negative feedback may also regulate exosome uptake and release in a cell
specific manner. In cell culture studies an interesting feedback loop has been noted; exposure
to self (same cell derived) exosomes promotes exosome release [4]. The study examined dose
responses (exosome release) from cells (a normal mammary epithelial cell line and a cancer
derived cell line) after exposure to self-derived exosomes and exosomes from the other cell line.
The results of the study demonstrated that self exosomes promote further exosome release
(positive feedback), whereas exposure of breast cancer cells to exosomes from normal
mammary epithelial cells inhibited exosome release and, likewise, exosome release from the
normal mammary epithelial cells was inhibited upon exposure to cancer cell derived exosomes
[4]. This feedback control most likely does not translate to all cell types but does reveal that
feedback processes can influence exosome secretion and in this scenario, implies a process of
self propagation of exosome secretion and non-self inhibition of exosome secretion.
2.5 The Plasma Membrane Remodeling/Endosome/Exosome Axis in Cancer
The concept of the endosome as a signaling entity (Figure 1) is not a new concept; for a
comprehensive review on endocytosis, endosomes, and signaling, the reader is directed to
Sorkin and Zastrows 2009 Nature Review in Molecular Cell Biology [22] and for a focused
review of multivesicular bodies in signaling, the reader is directed to Dobrowolski and De
Robertiss 2012 Perspective [20]. The endosome functions in cellular signaling in many
important ways; the most obvious function of endosomes is in signal housekeeping as
endocytosis can function to recycle or degrade receptors, the kinetics of receptor return to the
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membrane can be important for signal propagation and transduction [20]. The endosome can
function to generate signals, as with some receptor tyrosine kinase (RTK) signaling and SMAD
signaling [20, 21]. Endosomes enriched in APPL (adaptor protein containing phosphotyrosineinteraction domain, pH domain, and leucine zipper motif) are involved in RTK signaling while
endosomes enriched in SARA (SMAD anchor for receptor activation) are involved in TGF-
(transforming growth factor-) signaling [38, 39]. For example, when TGF- receptor is
endocytosed into endosomes with SARA, TGF- receptor binds to SARA and forms a complex
that recruits SMAD2; SMAD2 is then phosphorylated and released into the cytoplasm where it
joins with SMAD4 forming an active transcription factor complex that can translocate to the
nucleus [38].
The endosome also behaves as a signaling entity as it interacts with cytosolic
components to select content for exocytosis or lysosomal degradation [21]. Through signaling
interactions, endosomal constituents can determine exosome content. For example, a study
examining the interaction between the tetraspanin protein CD9 and the metalloprotease CD10
demonstrated that CD9 controls the loading of CD10 into exosomes [30]. The study used K562
erythromyeloid cells that have a low level of basal CD9 expression, transfected the cells with a
plasmid conferring CD10 expression and neomycin resistance via a lentiviral vector and
selected for CD10 expressing (neomycin resistant) cells [30]. These cells were further
transfected with puromycin resistant vectors containing either an empty plasmid, wild-type CD9,
or a mutant CD9 with an altered binding site for CD10 (substitution at residue 82 of the Cterminal tail) [30]. The expression of both wild-type CD9 and mutant CD9 did not alter the total
cellular expression of CD10; the level of wild-type CD9 and mutant CD9 was similar in
exosomes [30]. The wild-type CD9 associated with CD10 in the exosomal fraction and
increased the level of CD10 recruitment into exosomes 5-fold relative to the K562 with basal
(low) CD9 expression, and 2.5 fold relative to the mutant CD9 cells [30]. Additional experiments
in Nalm-6 cells demonstrated that knockdown of CD9 with short hairpin RNA reduced the level
of CD9 expression and CD10 recruitment in exosomes [30]. CD9 is associated with the plasma
membrane, cytosol, endosomes, and exosomes and is often used as an exosome biomarker
[40]. This study is a demonstration of how endosomal tetraspanins function to load specific
content into exosomes.
A study that provides further insight into the connection between endocytosis, exosome
release, and cancer is a 2011 study of the receptor tyrosine kinase, Met [41]. The study used
two previously validated oncogenic Met mutants in NIH3T3 cells and demonstrated that Met
mutants had a higher intracellular concentration of Met (52%) compared to wild-type cells (18%)
and this intracellular concentration co-localized with early endosomes and late endosomes, but
not with lysosomes, resulting in receptor return to the plasma membrane and resistance of the
receptor to degradation [41]. The Met mutants had defective actin fibers and required
endocytosis to remodel the cytoskeleton (remodeling was inhibited upon treatment with
endocytotic inhibitors); the Met mutants also had a > 3 fold higher rate of migration through a
membrane compared to wild-type cells, migration of the Met mutants was inhibited by blocking
endocytosis [41]. Inhibiting endocytosis inhibited the oncogenic activity of the Met mutants.
This was demonstrated by first, validating the ongogenic activity of the mutants in vivo; grafting
the Met mutant cells into nude mice resulted in rapid 500 mm3 tumors (within 12 days), whereas
at this time point, wild-type grafts did not result in palpable growths [41]. After validating the
oncogenic activity, in a new set of experiments, endocytotic inhibitors were administered when
tumor volumes were 50 mm3; 5 days after treatment tumor size decreased 40-50%
demonstrating the role of endocytosis in maintaining the oncogenic activity of the Met mutants
[41]. The collective results of these studies suggest that endocytosis and increased receptor
recycling is critical to maintaining the oncogenic activity of mutant tyrosine kinase receptors.
The release of exosomes may promote this oncogenic activity through enhancing motility (as
migration was inhibited when endocytosis was inhibited) and by establishing a suitable tumor
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microenvironment that promotes growth (in vivo tumor growth was inhibited when endocytosis
was inhibited).
In support of the concept that alterations in the expression of plasma membrane
receptors and plasma membrane remodeling in cancer contributes to the aberrant exosomal
process demonstrated by cancer cells; a recent study demonstrated that hypoxia increased the
release of exosomes from cancer cells [42]. Hypoxia results in translocation of Hypoxia
Inducible Factors (HIFs) from the cytoplasm to the nucleus of the cell where this alpha HIF
isoform (HIF-1, HIF-2, or HIF-3) complexes with a HIF beta isoform and ancillary proteins to
form an active transcription factor that binds to Hypoxia Responsive Elements (HREs) on target
genes. Target genes include numerous plasma membrane receptors such as GLUT-1 glucose
transporter, EGFR (Epidermal Growth Factor Receptor), and the drug efflux pumps; Pglycoprotein (P-gp, MDR1) and Multidrug Resistance Protein 1 (MRP1). There are over 100
genes with HREs; many of which are plasma membrane receptors including the transferrin
receptor, which was the first marker associated with exosome detection. The hypoxic induction
study examined three breast cancer cell lines (MCF7, SKBR, and MDB-MB-231 cells) and
demonstrated that exosome secretion increased in a dose response manner to increasing levels
of hypoxia [42]. Growth in 1% oxygen conditions resulted in an increase (1:1.5 relative to
normoxic cells) in exosome secretion for each cell line; dropping the oxygen concentration to
0.1% hypoxia further increased the production of exosomes for each cell line, almost doubling
the secretion of exosomes relative to normoxic cells [42]. Our own studies in a panel of cancer
cell lines have shown that hypoxia increases the expression and nucleic translocation of HIF-1
and HIF-2, resulting in a subsequent increase in the protein levels of the plasma membrane
receptors P-gp, MRP1, EGFR, and GLUT-1 [10]. As receptor expression changes (decreases
or increases) this can increase plasma membrane remodeling while increasing receptor
expression can also directly increase receptor activation and internalization or result in receptor
clustering which promotes endocytosis [21]. Numerous alterations in cancer cells, including
activation of the p53 oncogene, result in subsequent changes in plasma membrane proteins, as
well as changes in endocytosis (discussed in introduction). The most consistent trait of cancer
cells is their constant state of flux and ability to rapidly adapt to microenvironmental selection
pressure; this phenomena is often referred to as plasticity and ensures membrane remodeling
and associated exosome release [12, 43].
3. Exosomal Content in Cancer

Cancer derived exosomes have a vast array of content composed of microRNAs,

mRNAs, transcription factors, proteins, and lipids (Figure 2 [2, 44-49]). The content is highly
variable and dependent on cell origin, but regardless of origin, the contents of exosomes are
highly functional and exert powerful effects in recipient cells.
3.1 MicroRNAs and mRNAs
MicroRNA (miRNA) are small, non-coding RNA that are usually 20-25 nucleotide long
sequences, which upon entering the recipient cell bind to the target mRNA sequence and inhibit
translation. It has been found that miRNA expression is dysregulated in most cancers. MiRNA
signatures of exosomes of different cellular origin are distinct. Many miRNA are tumor-specific
and therefore can be used as diagnostic and prognostic biomarkers [50]. The shuttling of
miRNA molecules that may either be tumor-supportive (oncomiRs) or tumor-suppressive is
especially important for cancer. While mRNA enters the recipient cell to be translated into
proteins, miRNA exert silencing effects on genes through a process called RNA interference
(RNAi). Exosomal RNA content has also been shown to differ from donor cell RNA as it
contains high levels of small RNA and lacks 18s and 28s ribosomal RNA (rRNA). Recently, the
presence of single and double stranded DNA molecules was also discovered in tumor-derived
exosomes [50, 51].
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IL4 activated macrophages are known to secrete exosomes containing miR223, which
promotes invasiveness of breast cancer cells [52]. The mir200 family present in breast cancer
exosomes and miR105 present in breast, ovarian, gastric and prostate cancer exosomes cause
metastasis, invasion, tumorigenesis and tumor progression [5, 53]. MiR494 found in small cell
lung carcinoma and breast cancer exosomes promotes proliferation, survival and migration [54,
55] Mir34a, on the other hand, which is known to increase apoptosis and senescence is found in
tumor derived exosomes (TEX) of the breast, prostate, brain and bladder [56-58]. Therefore, not
all miRNA may be implicated in tumor supportive mechanisms.

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3.2 Transcription Factors

Specific transcription factors are often upregulated in cancer to activate the expression
of proteins that promote tumor progression, survival, and metastasis. HIF1 involved in
upregulating metastatic potential, angiogenesis and proliferation in tumor cells. As discussed
below, HIF1 released in nasopharyngeal carcinoma cell exosomes can increase metastasis in
recipient cells [59]. Tumor derived exosomes that contain FasL and TNF induce T cell
apoptosis, which in turn causes immune suppression, thereby supporting tumor progression
[60-62].
Exosomes isolated from body fluids of cancer patients have also been shown to have
up-regulated levels of vascular endothelial growth factor (VEGF), while TEX contain
transcription factors that modify the tumor stroma to create a tumor friendly environment that

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Figure 2. Exosome Content in Cancer.

Schematic illustrating components of an exosome including mRNA, microRNA, DNA, heat

shock proteins, enzymes, MHC receptors, tetraspanins, lipid rafts, cytoskeletal elements,
and membrane transport proteins.
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supports metastasis. These stroma modulating factors include TGF, matrix metalloprotease 2
(MMP2) and urokinase plasminogen activator [63-67]. TGF expressed on the surface of tumor
derived exosomes is capable of differentiating fibroblasts into myofibroblasts, which increases
vasculature, facilitates tumor progression and metastasis [68].
Exosomes that release c-Met can increase angiogenic and metastatic potential in
recipient tumor cells [69]. EGFR has been detected in lung cancer cell exosomes, and coculturing of these cells with dendritic cells and Th0 cells, results in an increase in regulatory T
cells that are tumor antigen specific, which downregulate immune responses towards tumors
[70].

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3.3 Proteins
Having originated from endosomes, exosomes express a number of proteins involved in
membrane related activities including RabGTPase and Annexin, cellular adhesion proteins
including integrins and tetraspanins, cytoskeletal proteins such as actin and myosin and heat
shock proteins such as Hsp70 [71, 72]. In certain disorders of the central nervous system,
numerous proteins including -amyloid protein, -synuclein and superoxidedismutase have also
been found to be released from exosomes and are implicated in the pathogenesis of these
diseases [73-77]. In patients with cancer, TEX represent a large portion of the circulating
exosome population and elevated levels of tumor specific proteins are found in the blood of
cancer patients [78].
Cell adhesion proteins and enzymes that degrade the extracellular matrix have a pivotal
role in aiding metastasis. Cancer associated fibroblasts secrete CD81 rich exosomes containing
Wnt1 into the tumor stroma, which is then taken up by breast cancer cells bringing about
metastasis and cell migration [40, 79, 80]. Numerous studies have pointed out that exosomes
regulate stromal cells in the pre-metastatic organ, simultaneously recruiting hematopoietic stem
cells from the bone marrow to help in creating a pre-metastatic niche. Exosomes expressing the
CD9-M2, L2 and X2 complexes selectively target bone marrow cells and those that
express Tspan8 and CD151-31 and 64 complex with lymph node stroma [81, 82]. In
addition, tetraspanins can also associate with MMPs such as MMP14 and bring about a
breakdown of the extracellular matrix facilitating metastasis [81, 83, 84].
By inhibiting NKG2D on natural killer (NK) cells and inhibiting their proliferation, TEX
decrease the cytotoxic effects of NK cells on tumor cells. Tumor cell exosomes also carry pglycoprotein causing drug resistance [85], MMPs to bring about matrix degradation [86],
truncated EGFR that enhances oncogenic signaling [87], and delta like4 (Dll4) [88] and VEGF
that causes angiogenesis [89, 90].
3.4 Lipids
As previously mentioned, exosomes have elevated sphingomyelin (SM), cholesterol,
glycerophospholipid and ceramide content [50, 73, 91-94]. Elevated levels of SM and
cholesterol were uncovered in exosomes from PC-3 prostate cancer cells. Prostate cancer cells
secrete exosomes with elevated levels of glycophospholipids including HexCer and LacCer,
making these potential biomarkers [95, 96].
In a study conducted by Toda et. al, it was demonstrated that, in addition to surface
protein ligands, lipids including SM and monosialodihexosylganglioside were also involved
heavily in intracellular trafficking of exosomes [96, 97]. Docosahexaenoic acid, which can be
found at millimolar concentrations in exosomes, aids in breast cancer suppression by acting on
the anti-estrogen receptor site, inducing apoptosis by modifying metabolism of cholesterol, this
occurrence could have powerful implications for designing exosome based therapeutics for
breast cancer [94, 96, 97]. Although the contents of exosomes are highly variable, they have
cell type specific trends correlating to their origin cells; as such, exosomes are proving to be
useful tools in cancer biomarker screening.
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4. Exosomes in Cancer Biomarker Screening

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Poor prognosis with existing therapeutic approaches against cancer is largely attributed
to late stage detection of the disease, which in part is due to lack of appropriate disease-specific
biomarkers. Therefore, considerable focus has shifted to studying and identifying cancerspecific molecular signatures that can be utilized for detection of the disease at an early stage.
To this end, much like circulating tumor cells, exosomes carry a wealth of information about the
disease characteristics in the form of proteins, lipids, nucleic acids and metabolites that are
shed from tumor cells into the exosomes [98]. Besides being secreted by different types of cells
in vitro, studies have confirmed the presence of exosomes from various biological fluids
including blood, urine, saliva, ascites, cerebrospinal fluid, synovial fluid, breast milk, and
bronchoalveolar lavage [99]. Compositional analysis of these exosomes demonstrates disease
and cell-specific unique molecular fingerprints alongside other conserved components that carry
valuable genomic and proteomic information. Biomarker mining from exosomes becomes an
even more attractive alternative because the components are well protected within the lipid
bilayer and therefore can be preserved without any significant loss of information. Kalra et al
stored the exosomes isolated from colorectal carcinoma cells LIM 1863 in plasma for up to 3
months at 4 C, 37 C, -20 C and -80 C followed by western blot analysis for the TSG101
marker to demonstrate the protein stability within the lumen of the vesicles [100]. Ge et al did a
more rigorous analysis on the stability of miRNA in plasma and exosomes stored at 4 C, -20 C
and -80 C for up to 5 years. Their results confirm that while in plasma, miRNA degrades at 4 C
and -20 C, the total miRNA in exosomes do not show any significant difference in their total
miRNA content or the individual miRNA concentration compared to a fresh sample. Further,
subjecting the stored sample to a freeze/thaw cycle also did not lead to considerable loss in
miRNA content of the exosomes [101]. Exosomes therefore are robust biological entities that
offer tremendous promise in studying cancer-specific molecular signatures to identify
biomarkers that could serve as potential diagnostic tools in monitoring disease onset, response
to therapy or prognosis. Figure 3 outlines the chronological steps involved in identifying and
validating exosome-derived proteins and nucleic acids as potential biomarkers for disease
diagnosis, prognosis, and response to therapy.
A systematic approach to plasma proteome and nucleic acid analysis has led to the
identification of several proteins that could serve as biomarkers for identifying and staging
cancer and developing personalized therapeutic approaches to achieve maximum treatment
responses [99, 102]. The complexity of the plasma protein content poses a considerable hurdle
in this developmental path since the useful biomarkers are often in a very low concentration and
are dominated by other abundant proteins [98]. Exosomes on the other hand provide a much
cleaner clinical sample devoid of plasma proteins and thus make the analysis and biomarker
validation easier. Analysis of exosomes is an avenue for developing novel protein biomarkers
for monitoring cancer as opposed to the existing standard-of-care biomarkers that have failed to
make a mark in the clinical environment [103].
Prostate-specific antigen is the benchmark FDA-approved biomarker for prostate cancer
but its specificity has been under scrutiny due to inconsistent results. Duijvesz et al compared
the protein content of exosomes derived from non-cancerous prostate epithelial cells (PNT2C2
and RWPE-1) and compared it with prostate cancer cell (PC346C and VcaP) derived exosomes
by using tryptic digestion followed by LC-MS/MS analysis [104]. Their results revealed at least
four potential prostate cancer biomarkers proteins (PDCD6IP, FASN, XPO1 and ENO1) that
were found in abundance in the exosomes from cancer cells among which, the latter two appear
to be prostate-specific biomarkers since they have not been reported in other exosomes [104].
In a similar study, Hosseini-Beheshti et al critically evaluated the exosomal content of six
different prostate cancer cell lines with a varying degree of expression of the androgen receptor
(AR) [105]. They found 3 proteins (ENPL, GRP78 and AXA2L) that were exclusively present
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Figure 3. Validating Biomarker Screening of Cancer Exosomes.

Schematic for biomarker validation in exosome screening. Endocytotic vesicles (EV) are
isolated followed by nucleic acid and protein isolation for biomarker discovery and
identification. The biomarkers are validated and submitted for FDA approval in diagnostics,
prognosis, or therapeutics.
only in the AR- cells-derived exosomes while two proteins signatures were exclusive to AR+
cells-derived exosomes [105]. Nilsson et al identified mRNAs for PCA-3 and TMPRSS2:ERG in
the exosomes isolated from urine samples collected from patients suffering from prostate
cancer and their finding suggests that these biomarkers can be utilized for the assessment of
tumor genotypic/phenotypic and metastatic potential as well as monitoring therapeutic
responses to androgen deprivation therapy [106]. Recently, Bryant et al did a comprehensive
analysis of the miRNA composition of exosomes isolated from the serum of patients suffering
from non-metastatic and metastatic prostate cancer and compared it with those from healthy
individuals [107]. Their findings reveal that 12 miRs were differentially expressed in exosomes
of prostate cancer patients compared to healthy individuals and 16 miRs were differentially
expressed in metastatic patients compared to non-metastatic patient with miR-141 and miR-375
showing significant association with metastasis [107]. These reports show an early positive
indication of potential biomarkers for prostate cancer but have to be verified from patient
samples in a larger cohort.
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In glioblastoma, a highly malignant form of brain tumors, reports have established that
the presence of EGFRvIII mutant splice variant is of tremendous significance and is useful in
monitoring responses in therapeutic and vaccine clinical trials [108, 109]. A detailed proteomic
profiling of exosomes from brain cancer cells further revealed the presence of EGFR along with
TGF-, a cytokine with immunosuppressive function. Surprisingly, the analysis of exosomes
from the serum of patients with brain tumors also showed an increased level of TGF- along
with EGFR and EGFRvIII, suggesting the potential role of these biomarkers in the diagnosis and
prognosis of disease [61]. Skog et al used a nested RT-PCR method and detected EGFRvIII
from microvesicles isolated from the serum of patients suffering from glioblastoma but not from
any of the 30 control samples [64]. More recently, Shao et al developed a more ingenious
method for isolating exosomes [110]. The exosomes isolated from clinical blood samples were
tagged with antibodies, which was further tagged with magnetic nanoparticles to be captured on
a microfluidic device by microfiltration. The advantage of using such nanotechnology-based
biosensors lies primarily in the requirement of a very low sample volume for analysis with
minimum pre-processing. Importantly, the captured exosomes could either be analyzed in situ
by -NMR with far superior sensitivity compared to common techniques such as ELISA or flow
cytometry; or could be isolated from the device for subsequent molecular profiling by alternate
methods. Based on the analysis of exosomes from GBM patients and healthy individuals,
authors could establish an increased presence of EGFR, EGFRvIII, PDPN and IDH1 R132H in
the exosomes of the patients and they were able to distinguish between the sources of the
exosomes with a 90% confidence. Most importantly, analyses of the exosomes from patients
undergoing a therapeutic regimen showed a distinct predictive capability of drug efficacy with
the studied biomarkers [110]. As an alternative, miR-21 has been reported as one of the
biomarkers that is secreted in high concentration in exosomes shed by gliobalstoma cells as
well as those isolated from patients cerebrospinal fluid (CSF). Among a cohort of 13 patients
suffering with the disease, the miR-21 level in the exosomes was found to be at least 10-fold
higher than in exosomes from healthy individuals. In a critical analysis in a cohort of 29 patients,
miR-21 level analysis from the exosomes could distinguish disease-carrying individuals from
healthy individuals with a 87% sensitivity and 93 % specificity [111]. Two independent reports
confirmed an overexpression of miR-21 in the serum of patients suffering from large B-cell
lymphoma [112] and in exosomes isolated from the serum of patients with esophageal
squamous cell carcinoma [113], which questions the validity of miR-21 as a cancer-specific
marker and warrants more in-depth assessment.
Taylor et al analyzed the exosomes shed into the sera by patients, to stage ovarian
cancer and reported 0.320 0.056, 0.640 0.053, 0.995 0.084 and 1.420 0.228 mg/ml of
EpCAM protein of total exosomal protein in patients with stage I, II, III and IV ovarian cancer
compared to benign tumor bearing patients with protein levels of 0.149 0.065 mg/ml and
healthy individuals with 0.039 0.030 mg/ml EpCAM levels. They further detected 218 miRs in
exosomes with 31 showing elevated levels including miR-21, miR-141, miR-200a, miR-200c,
miR-200b, miR-203, miR-205, and miR-214 [114]. Im et al developed a nano-plasmonic
exosome (nPLEX) assay for surface plasmon resonance-based, label-free detection of
exosome proteins. They determined as many as 71 ovarian cancer signature proteins markers
from cancer cells; further established that EpCAM and CD24 levels are highly elevated in the
exosomes derived from ascites of cancer patients and could be used as biomarkers to study the
patients response to chemotherapy [115]. Several other such studies have been undertaken in
past 5 years to assess various biomarkers in different cancer patients and have been
summarized in Table 1. One hallmark feature with the majority of these reports is that actual
clinical samples have been increasingly incorporated into the studies to develop a direct
correlation between the biomarker level and disease status. This change in the paradigm
promises a rigorous and robust approach to biomarker identification, analysis, and subsequent
validation in larger populations of patients to eliminate previous pitfalls and ensure an improved
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clinical translation [103].
Table 1: Exosome Biomarkers
Biomarker

Type

Source

Analysis

Cells
Urine
Serum
Serum

EGFRvIII

mRNA

Serum

EGFR, EGFRvIII, PDPN, IDH1 R132H

Protein

MiR-21

micro RNA

MiR-21
MiR-155, MiR-210, MiR-21

microRNA
microRNA

Blood
Serum

RT-PCR
RT-PCR

MiR-21, MiR-141, MiR-200a, MiR-200c,

MiR-200b, MiR-203, MiR-205, MiR-214
EpCAM, CD24
MiR-718
MiR-17-3p, MiR-21, MiR-106a, MiR-146,
MiR-155, MiR-191, MiR-192, MiR- 203,
MiR-205, MiR-210, MiR-212, MiR-214
EpCAM

microRNA

Serum

Microarray

Prostate [104]
Prostate [106]
Prostate [107]
Glioblastoma
[61]
Glioblastoma
[64]
Glioblastoma
[110]
Glioblastoma
[111]
ESCC [113]
Lymphoma
[112]
Ovarian [114]

Protein
microRNA
microRNA

Ascites
Serum
Plasma

nPLEX
Microarray
Microarray

Ovarian [115]
HCC [116]
Lung [117]

Protein

Plasma

LRG1
Apbb1ip, Daf2, Foxp1, Incenp, Aspn,
BC031781, Gng2
NT5E/CD73

Protein
mRNA

Urine
Saliva

Magnetic
sorting
LC-MS/MS
Microarray

Protein

ascites

MS/MS

NT5E/CD73
CD63, Caveolin-1

Protein
Protein

Cells
Plasma

MS/MS
Exotest (ELISA)

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Protein
mRNA
mRNA
Protein

RT-PCR
-NMR, ELISA

CSF

RT-PCR

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Blood

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LC-MS/MS
RT-PCR
RT-PCR
SDS-PAGE

Disease

PDCD6IP, FASN, XPO1 and ENO1

PCA-3, TMPRSS2:ERG
MiR-141, MiR-375
EGFR, EGFRvIII, TGF-

cell

Lung [117]
Lung [118]
Pancreatic
[119]
Pancreatic
[120]
CRC [121]
Melanoma
[122]

Table 1: A summary of reports on protein or RNA biomarker mining in exosomes isolated from
cells and clinical samples.
5. Exosome Mediated Communication
5.1 Tumor Metastasis
Exosomes promote metastasis through their direct role in invasion and through content
specific effects that promote metastasis, transformation, and the establishment of the premetastatic niche. Exosomes have been demonstrated to have a fundamental role in the
physical process of invasion, the first stage of metastasis. MVBs and exosomes are associated
with invadopodia, the actin-rich protrusions of cancer cells that initiate invasion through
degradation of the extracellular matrix [123]. A series of experiments have demonstrated that
MVBs and exosomes recruit to the plasma membrane of invadopodia and that knockdown of
Rab27a decreases exosome secretion and extracellular matrix digestion associated with
maturing invadopodia [123]. Further experiments have demonstrated that exosomes extracted
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from cells with maturing invadopodia have the ability to induce invadopodia formation in noninvading cells [123]. Exosomes appear critical for invasion, extracellular matrix digestion, and
metastatic induction. Similar studies have investigated the interaction of TEX with the
extracellular matrix and have identified critical roles of cancer exosomes in promoting invasion.
For example, a study using exosomes derived from metastatic rat pancreatic adenocarcinoma
cells demonstrated that these exosomes express high levels of CD49c and CD44 and by
examining binding of these exosomes as well as exosomes derived from a CD44 knockout cell
line, binding to hyaluronic acid in the extracellular matrix was demonstrated [124]. These CD44
exosomes were also loaded with a high level of proteases which functioned to degrade the
extracellular matrix in an in vivo rat model of matrix remodeling [124].
Exosomal content also promotes metastasis and transfers metastatic potential. The
metastatic process consists of a series of events starting with epithethial-mesenchymal
transition (EMT; mobilizing cells) and then mesenchymal-to-epithelial transition (MET;
establishing a secondary tumor site). Exosomes have been identified as contributing to the
formation of the pre-metastatic niche; a primed site distant from the primary tumor where
eventual metastasis occurs. Circulating exosomes from the primary tumor develop and prime
sites for metastasis and may even attract cancer cells to the niche, functioning as the first
mediators of metastatic node formation [125]. Cancer exosomes have been shown to deliver
functional complexes capable of promoting both EMT (such as HIF-1) and MET (such as miR200).
A seminal exosomal study performed an eloquent investigation of the metastatic
capabilities of exosomes in vitro and in vivo. Judy Lieberman and her group at Boston
Childrens Hospital demonstrated that exosomes and ectosomes released from metastatic
cancer cells can transfer metastatic capability to non-metastatic cells and this transformation is
governed by the microRNA-200 family, known mediators of MET [5]. The study used cells with
distinct metastatic capabilities (metastatic 4T1E mouse cells and metastatic human cells CA1a
and BPLER cells and poorly metastatic 4T07 mouse cells and poorly metastatic human
mesenchymal MB-231 cells) to study in vivo metastatic induction in mouse and human
xenograft models.
Exosomes from the highly metastatic cells transferred miR200 to
xenografted human breast cancer cells and promoted metastasis of the xenografts [5]. This
study is a pivotal demonstration that exosomes can not only promote metastasis, they can also
transform distal cells into metastasis cells. Many studies have identified other specific
microRNAs that promote metastasis. For example, a 2015 study examined exosomal miR-122,
a biomarker clinically associated with metastatic breast cancer [126]. The results confirmed a
high expression of miR-122 in all of the exosomes derived from a panel of breast cancer cell
lines whereas most parental cell lines had low intracellular levels of miR-122, suggesting that
miR-122 is targeted for exosomal secretion by breast cancer cells [126].
miR-122
downregulates pyruvate kinase and subsequently decreases glucose metabolism and GLUT-1
expression; mice treated with metastatic derived exosomes with high miR-122 content resulted
in rapid brain and lung metastasis [126]. Numerous studies portray the role of specific
microRNAs from cancer derived exosomes that are capable of conferring metastasis.
In a very interesting illustration of exosomal activity in establishment of the pre-metastatic
niche, Hood et al. demonstrated that melanoma exosomes rapidly stimulate the production of
endothelial spheroids and regulated inflammatory cytokines leading to promotion of endothelial
angiogenic responses that can contribute to the metastatic potential [127]. Similarly, Peinado et
al. demonstrated that exosomes from highly metastatic melanomas augmented the metastatic
potential of primary tumors by receptor-tyrosine kinase MET dependent alterations of bone
marrow progenitors [128]. Furthermore, it has been reported that melanoma excreted exosomes
exhibit a preference for sentinel lymph nodes, preparing the nodes for melanoma cell
recruitment and facilitating lymphatic metastasis [129]. Exosomes have apparent functions in
physical invasion and through content specific effects, mediate metastasis.
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5.2 Drug Resistance

Innate, acquired and de novo drug resistance remain a hallmark of cancer and drug
resistance is the major obstacle in devising therapies to achieve successful outcomes against
the disease. Innate multidrug resistance (MDR) occurs when cancer cells have drug resistant
phenotypes prior to any drug or treatment exposure. Innate drug resistance is often associated
with the over expression of ABC transporters; drug efflux pumps that include P-glyoprotein
(MDR1) and multidrug resistance protein 1 (MRP1). Acquired MDR is a gradual process where
cancer cells exposed to drug, radiation or targeted therapy educate themselves and alter their
genetic makeup to acquire drug resistance cancer phenotypes. De novo drug resistance on the
other hand is a transient strategy acquired by the cells to attain an environment-mediated drug
resistance, which involves signaling events in the tumor microenvironment that can lead to
soluble factor-mediated drug resistance (SFM-DR) or cell adhesion-mediated drug resistance
(CAM-DR) [130]. Cancer cells employ a combination of mechanisms to achieve multidrug
resistance such as decreased drug influx, increased drug metabolism and detoxification,
increased DNA repair mechanisms, increased drug efflux, and decreased apoptosis. Cancer
cells further mitigate radiation and chemotherapy via multiple different mechanisms such as
epigenetic modifications, poor drug penetration and through the presence of cancer stem cells
[131]. Given the central role of exosomes in cellular communication, it is undoubtable that
exosomes also contribute to MDR. One intuitive mechanism involving exosomes would be the
sequestration of cytotoxic drugs in the intracellular vesicles and subsequent expulsion, to
negate drug effect within the cells [132]. Luciani et al supported this by showing that melanoma,
adenocarcinoma and lymphoma cells confiscate drugs such as cisplatin, 5-flurouracil and
vinblastin in their endosomal compartment [133].
This was further confirmed when
melanosomes secreted from melanoma cells that were exposed to chemotherapy, were laden
with cytotoxic drugs [134]. The platinum drugs internalized by cancer cells are often segregated
into endosomal compartments such as lysosomes or vesicles that are destined for secretion
and this is often mediated by lysosome-associated protein (LAMP) and copper export proteins
ATP7A and ATP7B [135]. Safaei et al showed that exosomes secreted by cisplatin resistant
ovarian cancer cells showed 2.6-fold higher drug concentration along with higher content of
LAMP and other cisplatin transporters such as MRP2, ATP7A and ATP7B [136]. Cancer cells
exposed to radiation therapy also show an altered composition and rate of exosome secretion,
which suggests their role in resistance to radiotherapy [67, 137].
The major contribution of exosomes in imparting MDR to cancer cells cements from their
ability to transport molecular information such as proteins, mRNAs and miRNAs from one cell to
another. Exosomes facilitate cell-cell crosstalk within the tumor environment, which plays a
crucial role in augmenting MDR pathways. Boelens et al studied the exosome-mediated
interaction of stromal and breast cancer cells through paracrine and juxtacrine signaling and
demonstrated that such crosstalk helps cancer cells to abate the chemo- and radio- therapeutic
insults [138]. The noncoding RNA content from the stromal exosomes could activate a STAT1dependent response and subsequent NOTCH3 signaling pathway in the breast cancer cells,
which leads to decreased cell apoptosis and an increase in the tumor growth. A mechanistic
analysis reveals that the cancer cells induce secretion of exosomes rich in RNA polymerase III
transcripts, which contains 5-triphosphate, from the stromal cells to engage RIG-I receptors on
cancer cells to activate the STAT1 pathway [139, 140]. Similarly, transfer of drug efflux pump
proteins such as P-gp from docetaxel-resistant cells via exosomes renders drug-sensitive cells
resistant to the drug [139, 140]. Another report shows exosome-mediated transfer of miRNA
from adriamycin and docetaxel resistant MCF-7 cells to drug sensitive cells, leading to a drug
resistant phenotype [141]. Microarray and qPCR analysis confirmed that miR-100, miR-17, miR222, miR-342p and miR-miR-451 were considerably elevated in the exosomes derived from
both drug resistance cell lines and incubation of these exosomes with drug-sensitive cells led to
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an increase in these miRNA levels in the acceptor cells as well [141]. A detailed analysis of the
miR-222 target gene PTEN in drug resistant and sensitive cells showed that while drug resistant
cells show a reduced expression, the gene was highly expressed in the sensitive cells and its
activity was severely hampered upon treatment with a miR-222 mimic or exosomes from
docetaxel resistant cells. Xiao et al described a similar observation with their study on non-small
cell lung cancer A549 cells, where treatment of resistant cells with cisplatin led to an increase in
miR-21 content of the exosomes, which conferred resistance when exposed to drug sensitive
cells [142]. All these studies suggest that exosome mediate drug resistance through both the
direct shuttling of drugs out of the cells and through the horizontal transfer of
molecules/molecular signals that bestow drug resistance to the otherwise sensitive cells.

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5.3 Immune System Modulation

The work of Raposo et al demonstrating that the presence of major histocompatibility
complex (MHC) Class II-peptide complexes in exosomes secreted by both human and murine B
lymphoblastoid cells, which could activate antigen-specific T cell responses, was truly seminal
work; the report cemented an immunomodulatory role of exosomes in vivo [143]. Exosomes,
along with other tumor derived extracellular vesicles (EVs), regulate the immune system through
four distinct pathways involving antigen presentation to prime CD4+ or CD8+ T cells, activation
or suppression of the immune system, and modulation of the complement system (Figure 4).
Exosomes involve in antigen presentation with or without the engagement of antigen presenting
cells (APCs) such as dendritic cells (DCs). The direct presentation (Figure 4A, Pathway a)
involves interaction of the MHC-peptide complex, costimulatory and adhesion molecules on the
exosome surface with the corresponding T cells receptor, resulting in priming/activation of the T
cells [143]. The direct T cell stimulation capability of exosomes however is poor compared to
the APCs, which could be attributed to small size and limited sites of interaction [144]. Indeed,
increasing the concentration of the MHC-peptide complex in exosomes or increasing the total
particle size by immobilizing them on latex beads leads to significant improvement in their ability
to stimulate T cells [145-147]. Indirect presentation on the other hand ensues with the
interaction of exosomes with the APCs (Figure 4A, Pathway b and c) through surface receptors
such as integrins or intercellular adhesion molecule 1 (ICAM1; CD54) [145, 148]. The exosome
bound to the APCs could either decorate the surface and subsequently interact with the T cell
(Figure 4A, Pathway b; also known as cross-dressing) or could be internalized by the APCs,
where the antigen is further processed for presentation (Figure 4A, Pathway c) [148]. The
internalized exosome is degraded within the APCs and the antigen peptide is processed for
presentation on the surface of the APCs (Figure 4A, Pathway d) or could be secreted as APCderived exosomes where the antigen is presented on the MHC from the parent APC (Figure
4A, Pathway e). The fate of exosomes interacting with DCs depends on the status of the parent
cell; immature DC show efficient exosome internalization capability following processing of
peptides for presentation on their surface or release as DC-derived exosomes whereas mature
DCs mostly present the bound exosomes on their surface [149]. The cross dressing of
exosomes was confirmed in a study where DCs lacking MHC class II molecule IAb were
incubated with exosomes containing an IAb-peptide complex, following incubation DCs could
stimulate CD4+ T cells, suggesting that exosomes served as a source of IAb while DCs provide
the costimulatory molecule [150].
There is mounting evidence that besides presenting antigens, exosomes also serve as a
source of native antigen including not only proteins/peptides but also functional mRNA and
miRNA to regulate the immune system and therefore have promising therapeutic potential [145].
Wolfers et al studied exosomes derived from different syngeneic and allogeneic tumor models
to show a strong T cell-mediated antitumor response and cross protection, suggesting that
exosomes could be a source of antigens to mount an immune response. Using tumor-derived
exosomes as a source of native antigen for antitumor protective therapy, they demonstrated a
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strong CD8+ T cell response leading to rejection of tumors not only by the autologous
exosomes but by allogeneic exosomes as well, confirming that the antitumor response is not
mediated by the MHC class I-peptide complex [151]. Similarly, microarray analysis of exosomes
from murine (MC/9) and human mast cells (HMC-1) as well as primary murine mast cells
contain an enormous

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Figure 4. Exosome Regulation of the Immune System in Cancer.

Schematic of the immune/exosome (tumor-derived extracellular vesicles; EVs) interface. As

depicted in each panel, there are 4 different scenarios for exosome/immune interfacing
involving antigen presentation to prime CD4+ or CD8+ T cells (A), activation (B) or
suppression (C) of the immune system, and modulation of the complement system (D).
Panel A: Exosomes involve in antigen presentation with or without engagement of antigen
presenting cells (APCs) such as dendritic cells (DCs). In the direct presentation pathway (a),
the MHC-peptide complex on the exosome surface interacts with the corresponding T cells
receptor, resulting in priming/activation of the T cells. Indirect presentation (b and c) involves
interaction of exosomes with the APCs. The exosome bound to the APCs could either
decorate the surface and subsequently interact with the T cell (b; also known as crossdressing) or could be internalized by the APCs, where the antigen is further processed for
presentation (c). The exosome is degraded within the APCs and the antigen peptide is
processed for presentation on the surface of the APCs (d) or could be secreted as APCderived exosomes where the antigen is presented on the MHC from the APC (e). Panel B:
Exosomes can directly activate macrophages, neutrophils, natural killer (NK) cells, and
APCs. APCs subsequently activate CD4+ or CD8+ T cells. Panel C: Exosomes also
function to suppress an immune response, protecting cancer cells from immune cell
recognition and destruction. Tumor derived exosomes can trigger apoptosis in T cells,
reduce activity of NK cells and macrophages, and increase the population of myeloidderived suppressor cells (MSDCs). Panel D: Exosomes avoid immune recognition through
the expression of surface molecules that prevent activation of the complement system,
resulting
in
a
subsequent
escape
from
opsonization.
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pool of functional mRNAs that are absent in the cytoplasm of parent cells, could be translated in
vitro to corresponding proteins and importantly, could be shuttled between transferring capability
of exosomes have been related to immune response against pathogens mast cells (therefore
termed exosomal shuttle RNA as previously mentioned) [62]. Exosomes can directly activate
immune effector cells including macrophages, neutrophils, natural killer cells, and APCs (Figure
4B). Proteomic analysis of exosomes derived from cancer cells demonstrate a repertoire of
cancer-specific antigens such as heat shock proteins (HSPs), melan A, mesothelin and
carcinoembryonic antigen (CEA), that can activate APCs to mount an anti-cancer immune
response, a phenomenon often referred to as immune surveillance [145, 151, 152]. These
immunogenic abilities of tumor-derived exosomes have galvanized the efforts to use them as
anti-cancer vaccines.
Exosomes interestingly perform an equally contrasting role of immunosuppression in
cancer, which helps cancer cells to escape from immune recognition and therefore promotes
tumorigenesis [153]. In this regard, exosomes play a multifaceted role in affecting the immune
system at different levels to obliterate the innate and adaptive immune response against cancer
cells. TEX induce T cell apoptosis, reduce NK cells activity, inhibit IFN- dependent class II
expression of macrophages and alter monocyte differentiation to increase the myeloid-derived
suppressor cell (MSDCs) population, which leads to a collective failure of the immune system in
containment of cancer growth (Figure 4C) [153-158].
The ability of tumor and APCs-derived exosomes to evade immune recognition (Figure
4D) is another intriguing aspect that has been a focus of research, especially to improve the
therapeutic use of exosomes in vaccines and drug delivery. Cells express surface molecules
such as CD46 (membrane cofactor protein), CD55 (decay accelerating factor) and CD59 that
prevent activation of the complement system and thus they escape opsonization [159]. Clayton
et al demonstrated that APC-derived exosomes express CD55 and CD59 but not CD46 on their
surface, which inhibits the formation of complement membrane attacking complex and helps
them avoid clearance. They further showed that blocking CD55 with an antibody increased the
surface binding of C3b upon incubation in serum and resulted in exosomal lysis. CD59 blocking
with an antibody gave a similar result and the exosomal lysis increased significantly when both
CD55 and CD59 were blocked, suggesting their preventive role [160]. More importantly, this
indicates that exosomes are not shed by cells as a means of a secretory pathway to remove
redundant molecules but are functional cellular nanocarriers. Collectively, these studies
illustrate the mechanism behind the stability of exosomes in the blood by avoiding opsonins,
complement systems and coagulation factors. Exosomes appear capable of delivering almost
any biological message, and in cancer, exosomal communication contributes to metastasis,
drug resistance, and cancer immunology. The question now is how to manipulate exosomes for
cancer therapy?
6. Therapeutic Manipulation of Exosomal Content
Due to their eloquent function as natural nanoparticles, the therapeutic potential of
exosomes is immense. As a starting point, studying exosomes as a model of an ideal drug
delivery system is aiding the development of more effective therapeutics. Beyond using
exosomes, natures nanoparticles, as a model for drug delivery, there is great interest in
applying exosomes as therapeutics and in manipulating exosomal content for therapeutic
outcomes [161]. Although exosomal content can significantly vary depending the cell of origin
[162], exosomes can be manipulated for drug delivery applications. We discuss the therapeutic
application of exosomes to control metastasis and for cancer immunotherapy (with illustrative
examples in Table 2) and discuss the current status of exosome based clinical trials.
6.1 Therapeutic Applications of Exosomes in Preventing Metastasis
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The main approaches for inhibiting exosome-mediated metastatic potential has been
focused on blocking the production of exosomes by tumor cells and blocking the communication
of tumor cells with the immune system and other cells. It has been shown that exosomes from
4T1 cancer cells are released in a Rab27a-dependent manner, and it has been suggested that
inhibiting Rab27a results in reduction of exosomal release and a decrease of primary tumor
growth and metastatic potential [163]. A study performed on HeLa cells by Ostrowski et al.
showed that knocking down Rab27a and b inhibited the production of exosomes. This was
achieved by silencing two Rab27 effectors, Slp4 and Slac2b by using shRNAs [164].
Alternatively, exosome production can be inhibited by blocking H+/Na+ and Na+/Ca2+ channels
[165, 166]. Chalmin et al. used the molecule dimethyl amiloride, which can block Ca2+
channels, in combination with cyclophosphamide treatment, an alkylating agent capable of
eliminating regulatory T-cells, to examine the effects on CT26 tumor bearing mice and the
potential inhibition of exosome secretion [167]. Dimethyl amiloride treatment resulted in
reduction of exosomes in vitro and in vivo, while also inhibiting Stat-3 activation and
demonstrated a synergistic effect of the combination treatment [167]. The combination
treatment was ineffective in nude mice, demonstrating a critical role of T cells and the regulatory
effect of exosomes [167]. Another therapeutic approach that can be used, although it is not
specific for only inhibiting metastasis, is selective loading of exosomes and the use of exosomes
as therapeutic carriers; surface modification and active targeting of tumor or immune cells can
also be used with this approach [168, 169].

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6.2 Therapeutic Applications of Exosomes for Immunotherapy

Cancer immunotherapy is the exploitation of the immune system to recognize and attack
tumor cells; it is based on the principle that the immune system can be boosted, guided or even
activated in order to attack tumor cells, resulting in inhibiting tumor growth, metastasis or further
more in tumor regression [170]. In a number of publications, it has been reported that exosomes
originating from dendritic cells can strongly stimulate the immune system [171-173]. This can be
achieved even without the presence of mature dendritic cells, through a CD8+T cell priming
pathway [173]. The presence of MHC proteins, class I and II, in exosomes originating from
dendritic cells allow exosomes to exhibit strong antigen presenting properties [169]. Segura et
al. [174] demonstrated that the ability of exosomes to stimulate strong immune responses
correlated with the maturity of the dendritic cells they originated from. Although the exosomes
secreted from immature and mature dendritic cells share similar morphological characteristics
and protein content, the exosomes originating from mature dendritic cells are enriched in MHC
class II, B7.2 and ICAM-1 proteins while demonstrating a 50 to 100 fold stronger T cell
stimulation [174].
In an interesting study, dendritic cells were cultured in vitro and were pulsed with L1210
lymphocytic leukemia cell antigen and lipopolysaccharide. It was demonstrated that the isolated
and purified exosomes derived from the pulsed dendritic cells were able to induce spleen cell
proliferation in vitro and activate them to kill L1210 cells. Furthermore, the dendritic cell derived
exosomes, in combination with cyclophosphamide and polyinosinic-polycytidylic acid sodium
salt, suppressed L1210 tumor growth in vivo and produced improved survival time in mice [175].
In another study, Zitvogel et al. used exosomes derived from dendritic cells which in turn were
pulsed with acid-eluted peptides (AEP-P815 or AEP-TS/A) to immunize mice in vivo carrying
P815 tumors. A week after a single intradermal administration of the exosomes, tumor growth
was halted and by day 60, 40 to 60% of the animals were tumor free, illustrating the applicability
of exosomes as a cell-free vaccine alternative to dendritic cell therapy for cancer
immunotherapy [176].

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Origin of Exosomes

Melanoma

TEX

Melanoma

TEX

Melanoma

TEX

Mouse Breast
Cancer

TEX

Cervical Cancer

TEX

Mouse colon cancer

TEX

Prostate Cancer

TEX

Breast Cancer

TEX

Leukemia

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DEX

Leukemia

TEX

Mouse Mastocytoma

DEX

Melanoma

DEX

Mouse Mastocytoma

TEX

Glioma

TEX

Lymphoma

TEX

Mouse
plasmacytoma

TEX

Conclusion
Exosomes promote metastatic niche at distant
sites [127]
Exosomes augmented metastatic potential of
primary tumors [128]
Exosomes accumulate at sentinel lymph nodes
for promoting metastatic niche [129]
Inhibition of Rab27a inhibited TEX secretion,
primary tumor growth and metastatic potential
[163]
Inhibition of Rab27a and b, inhibited exosome
production [164]
Inhibition of Na+/Ca2+ channels and exosome
secretion using dimethyl amiloride. Improved
anticancer efficacy for cyclophosphamide
treatment [167]
Exosomes transferred drug resistance from
resistant cells to non-resistant cells [140]
Curcumin modified the ubiquintinated protein
content of the TEX, inhibiting their
immunosuppressive properties [177]
Exosomes pulsed with tumor specific antigen
induced spleen cell proliferation and activated
them against tumor cells, inhibiting tumor
growth [175]
Exosome immunization caused partial tumor
growth inhibition [178]
Exosomes pulsed with acid-eluted peptide
immunized mice and caused tumor regression
[176]
Exosomes carried Mart-1 tumor antigen to
dendritic cells for specific lymphocyte
stimulation [179]
Exosomes carried native tumor specific
antigens to dendritic cells for potent T-celldependent antitumor effect [180]
Exosomes carried antigen presenting
molecules and tumor antigen, capable of
causing dendritic cells to activate T
lymphocytes against glioma cells [181]
Exosomes by heat-shocked tumor cells
contained higher number of HSP proteins and
were able to induce dendritic cell maturation
and potent T cell responses [182]
Exosomes carrying surface HSP caused more
efficient dendritic cell maturation [183]

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Table 2. Investigational Exosome Therapies

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Table 2: Illustrative studies involving exosomes from different cell origin, their action, and
possible therapeutic utilization
*DEX: Dendritic cell derived exosomes; TEX: Tumor cell derived exosomes

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6.3 Clinical Trials and the Future of Exosome Therapeutics

The clinical translation of exosome therapeutics is being tremendously aided by
improved methods for the isolation and purification of exosomes in a current good
manufacturing practice (cGMP) like environment. Anosys SA, Inc. (France) pioneered the
methodology for exosome purification and laid down the quality control criteria for their clinical
application [184]. Exosomes prove to be clinically significant in a wide area of anticancer
therapy since their role in cancer immune biology, angiogenesis, drug resistance, disease
progression and metastasis is well studied, characterized and outlined. However, their first
clinical implementation came as anticancer vaccines due to obvious advantages. As outlined
earlier, exosomes derived from tumors cells and components from immune system are involved
in antigen presentation, transport of native antigen and modulation of the immune response.
Exosomes carry the necessary repertoire of surface molecules to avoid immune recognition and
clearance from circulation, which make them an ideal candidate for therapeutic applications.
Institut Gustave Roussy and Institut Curie (France) conducted a Phase I clinical trial to
assess the safety and surrogate markers for efficacy of DC-derived exosomes (also known as
Dex) in melanoma patients [185]. A total of 15 patients suffering from metastatic melanoma
(Stage IIIB or IV) were recruited for the study and 1.5 ml of their blood was drawn for separation
of CD14+ monocyte cells by leukapheresis. The monocytes were differentiated into immature
DCs by treatment with GM-CSF and IL4; loaded with melanoma antigen gene (MAGE) peptide
(10 g/mL) and subsequently the exosomes were isolated and purified for vaccination. No
Grade 2 toxicity was observed in any of the patients and 5 out of 15 patients showed clinical
efficacy to the vaccination regimen. Concomitantly, another Phase I clinical trial was undertaken
at Duke University (Durham, USA) where 13 patients suffering from stage IIIb/IV NSCLC were
recruited for vaccination with an aim to ascertain the safety profile of an exosome-based
vaccine and its efficacy [186]. The patients were vaccinated four times subcutaneously or intradermally at a weekly interval and 9 out of the 13 patients completed the therapy. No acute
exosome-related toxicity was observed in any patient while only grade 1-2 toxicity was observed
in a subset of patients, which included reaction at site of injection, flu-like illness and peripheral
arm pain. 52 665+ days post-vaccination survival was recorded in patients; MAGE-specific T
cell response was observed in 1 out of 3 patients and lytic activity of NK cells was reinstated in
2 out of 4 patients. Another Phase I clinical trial in 40 patients with advanced colorectal cancer
(stage III/IV) was initiated using ascites-derived exosome (Aex) with or without granulocytemacrophage colony-stimulating factor (GM-CSF) [187]. Both vaccination regimens were welltolerated by patients with some counts of grade 1-2 adverse effects were noted including
reaction at injection site, nausea, pain, fever and fatigue. Importantly, a significant
carcinoembryonic antigen (CEA)-specific antitumor cytotoxic T lymphocytes (CTSs) response
was observed in patients vaccinated with the Aex and GM-CSF combination, suggesting their
clinical benefits. The promising safety profile from the initial clinical studies has led to initiation of
several clinical trials based on exosome-mediated intervention (Table 3).
The favorable outcome of past clinical trials and the future pipeline suggests that
exosome-mediated intervention is aggressively moving towards clinical translation. It is not
surprising since exosomes present several liposome-like desirable attributes such as the ability
to load hydrophobic as well as hydrophilic drugs, improved residence time and minimal inherent
toxicity. However, like any other delivery systems, exosome-mediated therapeutic approaches
have several uphill challenges. One of the major obstacles remains the successful loading of
the active pharmaceutical ingredient into the luminal space without disrupting their biological
properties. Source of exosomes and scalability is another challenge that limits their applicability.
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Exosomes carry the signature proteins from the host and therefore their application in a nonhost patient could have immunogenic implications that have to be considered. The concluded
clinical trials have all used the host blood as the source monocyte-derived dendritic cells (MDDCs), which have been utilized for exosome production and isolation. However, during the
clinical trials, a large variability was observed in the quantity of exosomes that could be isolated
from an individual patients blood, which would depend on the number of isolated monocytes,
their successful differentiation to DCs and finally the exosome producing capability for
subsequent application. Such variability in a clinical environment is impractical for successful
translation of the technology and a more robust approach has to be devised to overcome them.
Successful answers to these questions would certainly boost the status of exosomes as a front
line therapeutic approach against cancer.
Table 3: Exosome based clinical trials
Source

Active Ingredient

Trial

Status

DCs

MAGE A3 peptide

Phase I

MAGE A3
MAGE A4
MAGE A10
MAGE-3DPO4
GM-CSF

Phase I

Completed
[185]
Completed
[186]

Phase I

Completed
[187]
Completed

Lortab

Phase I

Recruiting

Curcumin

Phase I

Recruiting

Phase I
Phase II
Phase I

Phase I

Recruiting
Not Known
Recruiting by
invitation
Recruiting

NU

Role
Vaccine
Vaccine

DCs

Colorectal

Vaccine

Malignant Glioma

Vaccine

Oral mucositis in Head

& Neck chemotherapy
Colon
Pancreatic
NSCLC
Type I Diabetes

Drug
Delivery
Drug
Delivery
Vaccine
Vaccine
-

Ascitesderived
Tumorderived
Grapederived
Grapederived
DCs
-

Parkinsons

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Melanoma

Phase I

Table 3: A list of completed or ongoing clinical trials based on exosome-mediated therapy.

(Abbreviation- DC: Dendritic cells; NSCLC: non-small cell lung cancer, MAGE: melanoma
antigen gene)
7. Conclusions
In the mere 30 years since their discovery, exosomes have surged to the forefront of cell
biology, cell signaling, cancer biology, immunology, and drug delivery, and are paving their way
into clinical diagnosis and clinical therapy trials. The progression of exosomal science and
therapeutics has been truly rapid and remarkable. The clinical success of exosomal therapies
will assuredly set a precedent for fast-tracking from discovery to application. Exosomal
biology is governed by the endocytotic, endosomal, and exocytotic pathways. The first step in
exosome formation is endocytosis, followed by the signaling endosome, which dictates
exosomal content through cytoplasmic interactions. Exosomal content is determined by the (1)
surface receptors, pores, and channels of the endosome that function, as in the plasma
membrane, to internalize specific constituents, (2) the lipid composition of the endosome and
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the lipid facilitated loading of contents such as ceramide mediated internalization of microRNA,
and (3) sorting machinery such as ESCRT. As the exosomal process is aberrant in cancer, we
conceptualize that this can be attributed to both, the function of the signaling endosome in
selecting content, and to the cancer cell-specific distinctions in receptor endocytosis and plasma
membrane remodeling. This concept explains exosomal variations from cell to cell as a
reflection of the signaling pathways that dominate a cells phenotype (i.e. quiescent verses
proliferative); in this way, exosomes can be released in response to a microenvironmental
selection pressure or to achieve a particular effect/result from a signaling pathway (i.e.
movement or invasion).
One of the most distinguishing hallmarks of cancer derived exosomes are the high level of
microRNA content. This miRNA varies from cell type to cell type but has proved to be an
important bioactive component of exosomes and is useful in biomarker diagnostics. Cancer
exosomes contain functionally active transcription factors, delivered in complexed and activated
forms. This depicts the dysregulation of cancer. Exosomes clearly function in metastatic
invasion, metastatic transformation, and establishment of a pre-metastatic niche. Exosomes
also promote a drug resistant microenvironment, can sequester and extrude drugs, and can
transform a non-resistant cell into a resistant cell through the direct transfer of functional drug
efflux pumps (P-gp). Exosomes regulate the immune system through antigen presentation,
immune activation, immune suppression, and modulating the complement system. Due to their
phenomenal ability to effectively deliver contents and their central role in cell communication,
exploratory exosomal therapies are being rampantly developed and exosomal therapies are fast
tracking to the clinic. The timeline of progress in exosome applications is almost as amazing as
the function of exosomes in cellular communication and invasion. Before long, exosomes will
surely reach the clinic as natures nanoparticles for cancer therapy.

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