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Exosomes as divine messengers are they the Hermes of modern molecular oncology

Review

Exosomes as divine messengers:are they the Hermes of modern molecular oncology?

C Braicu 1,9,C Tomuleasa 1,2,9,P Monroig 3,4,A Cucuianu 2,5,I Berindan-Neagoe*,1,6,7and GA Calin*,3,8

Exosomes are cell-derived vesicles that convey key elements with the potential to modulate intercellular communication.They are known to be secreted from all types of cells,and are crucial messengers that can regulate cellular processes by ‘traf?cking’molecules from cells of one tissue to another.The exosomal content has been shown to be broad,composed of different types of cytokines,growth factors,proteins,or nucleic acids.Besides messenger RNA (mRNA)they can also contain noncoding transcripts such as microRNAs (miRNAs),which are small endogenous cellular regulators of protein expression.In diseases such as cancer,exosomes can facilitate tumor progression by altering their vesicular content and supplying the tumor niche with molecules that favor the progression of oncogenic processes such as proliferation,invasion and metastasis,or even drug resistance.The packaging of their molecular content is known to be tissue speci?c,a fact that makes them interesting tools in clinical diagnostics and ideal candidates for biomarkers.In the current report,we describe the main properties of exosomes and explain their involvement in processes such as cell differentiation and cell death.Furthermore,we emphasize the need of developing patient-targeted treatments by applying the conceptualization of exosomal-derived miRNA-based therapeutics.Cell Death and Differentiation (2015)22,34–45;doi:10.1038/cdd.2014.130;published online 19September 2014

Facts

Exosomes are key elements that facilitate intercellular communication;depending on their vesicular content (‘cargo’),they can modulate tumor cells by in?uencing major cellular pathways such as apoptosis,cell differentia-tion,angiogenesis and metastasis.

This communication can involve the exchange of mole-cules such as small noncoding RNAs (e.g.miRNAs)between malignant,nontransformed and stromal cells (in all directions).

Exosomal miRNAs represent ideal candidates for biomar-kers,with multiple applications in the management of an array of pathologies such as cancer.

Manipulating exosomal miRNAs suggests new alternatives for patient-tailored individualized therapies.Open Questions

What are the mechanisms through which exosomal contents (e.g.,miRNA)are selected to be further secreted

from tumor cells?Are these mechanisms similar/different when the secretion is from nontransformed or stromal cells?Are the miRNAs conveyed in exosomes a re?ection of the cellular miRNA composition?

How are the molecules sequestered in exosomes in?uen-cing the cancer hallmarks (e.g.,mediating immune evasion or establishing metastatic niches)?In ancient Greek mythology,Hermes was the wing-shod messenger of the Olympians,the beloved son of Zeus and of the nymph,Maia.He was committed to numerous responsi-bilities given by Zeus,and the most important one was to serve as a link between two worlds,taking messages from the gods to mankind.1By applying the wisdom of ancient philosophy to modern biomedical research,there is clear resemblance between the way the two worlds –mankind and gods –co-evolved,with the ways ontogenesis and oncogen-esis are thought to develop:by communicating through messengers that for years were unknown to scientists.

The release of membrane-bound vesicles is a highly conserved biological event in prokaryotes and eukaryotes,a

1

Research Center for Functional Genomics and Translational Medicine,‘Iuliu Hatieganu’University of Medicine and Pharmacy,Cluj-Napoca,Romania;2Department of Hematology,‘Ion Chiricuta’Oncology Institute,Cluj-Napoca,Romania;3Department of Experimental Therapeutics,The University of Texas MD Anderson Cancer Center,Houston,TX,USA;4University of Puerto Rico School of Medicine,San Juan,Puerto Rico;5Department of Hematology,‘Iuliu Hatieganu’University of Medicine and Pharmacy,Cluj-Napoca,Romania;6Department of Immunology,‘Iuliu Hatieganu’University of Medicine and Pharmacy,Cluj-Napoca,Romania;7Department of Functional Genomics and Experimental Pathology,‘Ion Chiricuta’Oncology Institute,Cluj-Napoca,Romania and 8Center for RNA Interference and Non-Coding RNAs,The University of Texas MD Anderson Cancer Center,Houston,TX,USA

*Corresponding authors:I Berindan-Neagoe,Department of Functional Genomics,The Oncology Institute,Research Center for Functional Genomics,Biomedicine and Translational Medicine;Department of Immunology,‘Iuliu Hatieganu’University of Medicine and Pharmacy,Cluj-Napoca 400349,Romania.Tel:+40264598361;Fax:+40264598885;E-mail:ioana.neagoe@umfcluj.ro

or GA Calin,Department of Experimental Therapeutics,University of Texas MD Anderson Cancer Center,South Campus Research Building 3(3SCR4.3424)1881East Road,Unit 1950,Houston,TX 77030,USA.Tel:+17137925461;Fax:+17137454528(Fax assistant t17137456339);E-mail:gcalin@https://www.wendangku.net/doc/0c10014782.html, 9

These authors contributed equally to this work.

Received 02.3.14;revised 24.6.14;accepted 07.7.14;Edited by RA Knight;published online 19.9.14

Abbreviations:CLL,chronic lymphocytic leukemia;CML,chronic myeloid leukemia;ESCC,esophageal squamous cell carcinoma;miRNA,microRNA;mRNA,messenger RNA;ncRNA,noncoding RNA;MBL,monoclonal B-cell lymphocytosis

Cell Death and Differentiation (2015)22,34–45

&2015Macmillan Publishers Limited All rights reserved 1350-9047/15

https://www.wendangku.net/doc/0c10014782.html,/cdd

fact that attributes these vesicles an important role in regulating physiological cellular processes.2Interestingly, recent studies have discovered that transformed-tumor cells can take advantage of these endogenous‘traf?cking systems’by transferring molecules that activate cancer-related path-ways such as anti-apoptotic,proliferative or other tumorigenic ones.Initially,malignant tumor cells develop and proliferate in their local niche through the activation of endogenous oncogenic proteins and pathways.However,after some time, these cells recruit endogenous systems such as vesicle secretion,to broaden communication within the local tumor microenvironment and beyond.For example,at the vascular interface they orchestrate the enrollment of endothelial, perivascular or in?ammatory cells,as well as platelets and clotting factors to supply tumor requirements.Actions such as these lead to the disruption of the local vascular homeostasis and also to the alteration of vital pathways that can favor the development of a tumor microenvironment with metastatic potential.3,4Through their‘traf?cking’,membrane-bound vesicles transport‘molecular machinery’with the potential of causing physiological effects that can very well favor tumorigenesis.Several key elements have been shown to be sequestered and transported through these vesicles: cytokines,growth factors,proteins,lipids,messenger RNAs (mRNAs)or noncoding transcripts,including microRNAs (miRNAs).2–5

MiRNAs are short single-stranded(19–25nucleotides in length)nonprotein-coding RNA transcripts(ncRNA)that are initially produced in the nucleus and then transported into the cytoplasm,where they undergo a series of steps to acquire maturation.Mature miRNAs regulate gene expression by binding(through watsonian complementarity)to the sequence of a target mRNA.This interaction results in translational repression and/or mRNA cleavage,which consequently decreases the levels of the mRNA coding protein.6,7MiRNAs have been found to be aberrantly expressed in many diseases.8–12For example,in cancer,the tumor microenvir-onment contains deregulated miRNA levels,and a reason for their altered levels is because they are being actively secreted as membrane-bound vesicular content.

Cells can secrete‘molecular machinery’through several types of vesicular carriers that are composed of both membrane and cytosolic constituents.These carriers have been shown to contain nuclear/cytoplasmic proteins,but also nucleic acids13as well as other molecules.The biogenesis and main characteristics of these vesicular systems are presented in Table1and Figure1a.Vesicular carriers can be classi?ed based on their tissue of origin,their size and targeted function.In this manner,they can be categorized as either exosomes,microvesicles or apoptotic bodies.13–15Out of the three,exosomes are currently studied the most.They are depicted as vesicular carriers involved in mediating both cell-to-cell communication16and the traf?cking of several molecules that are regarded as insoluble or classi?ed as endogenous cellular components.Exosomes are usually transported through biological?uids such as plasma or serum.Thus,body?uids comprise a complex variety of extracellular vesicles that are being secreted from different tissues and therefore different cell types.The mechanisms regarding exosomal formation from their tissue of origin,their traveling through body?uids and their evolutionary signi?-

cance has been studied in the past.7,8It is known that exosomes display a set of proteins that re?ect their tissue of

origin and more speci?cally their target cells.17Furthermore,

their traf?cking is cell speci?c,18,19and the exosomal content

of the vesicles is highly regulated by a group of proteins that together form the endosomal sorting complex required for transport(ESCRT).2,3This complex determines the relative abundance of proteins,mRNA,20miRNAs(...and so on),that

will be segregated into a vesicle to be further transported elsewhere.

In this review,we aim to emphasize the roles that exosomal miRNAs have as essential communicators between the cancer cells and all other cell types.We also describe

the consequences of their‘molecular traf?cking’in cancer development,progression and metastasis.

Exosomes,Their Synthesis and Internal Content

Exosomes are naturally produced,membrane vesicle-like structures that range in size between40and100nm.15They

have a very unique cup-shaped morphology when they are observed through transmission electron microscopy.16,21

After being secreted,these structures have been described

to have a buoyant density of1.10to1.21g/ml in sucrose.

Aside from lipids such as cholesterol,ceramide or sphingo-lipids,their composition includes proteins like Rab GTPases, annexins or Tsg101,as well as integrins or tetraspanins(such

as CD63,CD9or CD82)8,15,22,23(Figure1b).

Several experimental procedures have served as assess-

ment tools to detect the presence,size,purity and content of exosomal structures.In general,exosomal content can be assessed by a wide range of approaches such as western

blot,mass spectrometry,?uorescence-activated cell sorting (FACS)or immunoelectronic microscopy.These techniques

have individually proven that exosomes contain a variety of lipids,hundreds of proteins,thousands of RNA species21 including over500different miRNAs.24,25

Immediately after their synthesis,15exosomes are released

and can remain in the extracellular space near the cell they originated from.Alternatively,they can also travel through body

?uids such as blood,urine,amniotic?uid,saliva,lung surfactant,malignant effusions or breast milk.The end result

of this dynamic process is a variety of regulative molecules

being transported to different tissues in different places,and

in?uencing cellular processes.Exosomes have been shown to

carry proteins,26many of which have the potential to in?uence multiple regulatory mechanisms.For example,exosomes can transport annexins that have the ability of altering the dynamics

of the cytoskeleton.They can also transport Rab GTPases that promote a wide range of cellular events regarding membrane fusions or endosomal sorting complexes desired for trans-

port.26–28In hematopoietic tissues,exosomes express high

levels of MHC class I or II molecules on their surfaces as well as

other types of adhesion molecules such as CD146,CD9, EGFRvIII,CD18,CD11a or LFA-3/CD58.29–31Speci?c surface markers such as these allow exosomes to be uptaken even by distant cells with a high level of speci?city.This mechanism facilitates the transfer of proteins,mRNAs,miRNAs,lipids or

other types of molecules to different parts of the body,and thus Exosomes as divine messengers

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in this way‘exosome-traf?cking’becomes a type of particle-based endocrine system.

Exosomes as Key Determinants of Intercellular Communication

The release of cellular invaginations from the membrane into the extracellular space has been known and described by Black32over three decades ago.Initially,exosomes were described as key players with important roles in cell-to-cell communication as well as key physiological and pathological processes.33However,it was recently proven that vesicles released by cells could be indicative of the cell type and/or its function.2,14With the progression of exosomal-related studies,these vesicles have now become a useful tool with diagnostic utility,and a novel strategy to develop highly speci?c drug delivery systems.

Exosomes can contain a variety of differentiation factors that could be released into body?uids affecting key processes of cellular homeostasis and differentiation.18The disruption of this homeostasis has important repercussions that may result in very different outcomes such as programmed cell death or, the contrary,chaotic cell proliferation and growth.34 A deregulated homeostatic environment can eventually lead to the development of several pathological conditions, including malignancies such as cancer.Thus,the contribution of exosomes in the altering of normal intercellular as well as intracellular signaling has been proven to be as signi?cant,or even more so,as lysosomes or proteasomes.13

Adult tissues,more speci?cally the neoplastic ones,are heterogeneous complex systems composed of a variety of cell types that continuously interact with one another.Both normal and tumor cells typically secrete proteins,and thus different subtypes of exosomes can be responsible for carrying these proteins from cell to cell,resulting in a type of ‘message delivery’.In cancer,the‘messages’are highly dysregulated and therefore intracellular circuits are either disrupted or overwhelmed,resulting in tumor initiation and development.Exosomes are the link between disruption of homeostasis in normal tissues and the development of oncogenesis(because they in?uence crucial modulators of cell cycle,proliferation,apoptosis,etc).35–39

Exosomal miRNAs in Cell Death and Differentiation Exosomes are important for various aspects of tumor biology, such as cell de-differentiation and programmed cell death. Yang et al.40have proven that puri?ed exosomes from the supernatant of T24bladder cancer cells demonstrated to have overexpressed levels of Bcl-2and cyclin D1,but reduced levels of Bax and caspase-3proteins.This same research group also proved that there is a direct,proportional link between the amount of secreted exosomes and the expres-sion of phosphorylated Akt and extracellular signal-regulated protein kinases(ERKs),both of which are crucial elements regulating apoptosis and cell differentiation processes.‘Auto-crine’or‘paracrine’intercellular traf?cking of proteins/genetic material via exosomes has proven to be extremely ef?cient for intercellular communication(speci?cally in tumors).For example,the release of exosomes may protect tumors from cell death,and surge the transmission of signals that may lead to an increase in cell proliferation.41The fact that exosomes can tightly regulate cellular processes such as these,most certainly implies that these vesicles could be targeted therapeutically to induce apoptosis and cell differentiation or to inhibit proliferation.4

The physiological role of exosomes is still a controversial topic,although to this date increasing amounts of experi-mental data have implicated them in various key processes.28 Nevertheless,some studies are still considered speculative. For example,Turchinovich et al.42suggested that circulating miRNAs are components or remaining parts of exosomes from dying cells that persist in the extracellular environment. This group proposed that those same miRNAs could act in a paracrine manner because exosomes supply a stable intravesicular environment for them as well as for other proteins that they require for their biogenesis and maturation (e.g.Ago2).42Similarly,additional studies have demonstrated

Table1Extracellular vehicles and their characteristics

Extracellular

vehicles

Size Biogenesis Characteristics Content Role References

Apoptotic bodies 50–5000nm Broad plasma membrane blob-

bing and breach of cell

fragments

Heterogeneous group of vesicles;

contains surface markers for the

recognition by phagocytic cells

markers:Annexin-V

Fragmented

nuclei as

well as cyto-

plasmic

organelles,

histone,

DNA

fragments

Activated as

response to a

cellular stress or

injury

5,22,23,30

Microvesicles50–1000nm Bud directly from the plasma

membrane through unique cel-

lular mechanisms No characteristic markers;lack of

transferrin receptors;secreted

during normal cellular processes

Cellular pro-

teins,lipids

and RNA,

including

miRNAs

Intercellular

communication;

transfer of pro-

teins and

genetic material

5,22,23,30

Exosomes30–100nm Originate from the endosomal

network named multivesicular

bodies(MVBs)and released

upon MVB fusion with the

plasma membrane More homogenous in size than

other vesicles;highly enriched in

transferrin receptors;secreted

during normal cellular processes;

markers:CD9,CD63,Alix,?otillin-

1and Tsg101

Cellular pro-

teins,lipids

and RNA,

including

miRNAs

Intercellular

communication;

transfer of pro-

teins and

genetic material

5,22,30

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that molecules,but speci?cally miRNAs,embedded in exosomal vesicles are protected from RNAases in the blood because they are surrounded by a lipidic membrane.43Thus,by increasing their stabilization and protecting them from degradation,exosomes enable molecules such as miRNAs to evade immunosurveillance mechanisms and thereby prevent events such as apoptosis.In this manner,exosomes allow an increase in pathological cancer-related processes such as angiogenesis,and even more,they confer tumors the ability of resisting chemo-and radiotherapy.43

Recently,it has been demonstrated that exosomes are uptaken in a selective way by neighbor cells but also by distant ones (including immune cells,through microenvironment cooperation),and that their uptake can lead to the activation of cell proliferation and differentiation.43,44They can con-tribute to these processes by embedding (as part of

their

Figure 1Exosome structure and function.(a )Exosome biogenesis and function:once released,exosomes can travel through biological ?uids (e.g.serum,lymph)and furthermore,they can be internalized by cells from nearby tissues,or more so,reach body sites very far from their tissue of origin.(b )Main classes of molecules included in the exosomal cargo.MT,mitochondria;MVB,multivesicular bodies

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‘molecular cargo’)miRNAs that are able to reprogram multiple cellular mechanisms in recipient cells.44

Recent reports suggest that exosomes are implicated in facilitating tumors with immune-evasive strategies(a known cancer hallmark).45,46Previous literature has suggested that these processes may occur as an effect of the direct interactions of immune effector cells and cancer exosomes; however,several other molecular intermediates are thought to be contributing as well(although the exact detailed mechanisms are not completely understood).4,5,45,46Through predictions,it has been speculated that a group of exosomal derived-miRNAs(e.g.,let-7,miR-17-92cluster,miR-21,miR-25,miR-106,miR-155or miR-200family)participate as mediators that alter immune responses by regulating T cells.45–47Further research aims to focus on deciphering not only which miRNAs mediate immune evasion,but also the mechanisms through which they do so(Figure2).

The current direction regarding the study of exosomal miRNAs contributing to immune evasion,is to verify their vesicular content by recovering them from body?uids.With strategies such as reversing immune dysfunction clinicians could obtain an increase in the initial therapeutic response from patients,or alternatively,the reversal of therapeutic resistance,or relapse prevention.48Exosomes or more speci?cally their vesicular content have already shown to target immunosuppressive pathways.For example,exoso-mal-derived miR-155directly in?uences tissues and their microenvironments by causing an evasion of immune cell response.49Its inhibition actually proved to sensitize CD4tTh cells for TREG suppression.50Therefore,miR-155 represents a primeval modulator of the immune system with signi?cant clinical implications because it targets receptors/ ligands requested for anticancer immunity.Thus,the delivery of anti-miR-155through arti?cial exosomes could be con-sidered a potential novel therapy.

Exosomal miRNAs in Cancer

Generally,tumor cells secrete a large amount of exosomes. Exosomal release has been proven to have prognostic relevance,where a higher amount of serum exosomes typically predicts unfavorable outcomes.12,44,45In2007, Valadi et al.51made signi?cant progress by proving that genetic materials,mRNAs and miRNAs,are indeed transported through exosomal traf?cking of molecules. Further along,other studies con?rmed the presence of miRNAs in microvesicles or exosomes.52Several others have been published associating exosomal miRNAs and their in?uence in cancer.For example,glioblastoma-derived exosomes were proven to be able to transport actively translated mRNA to different target cells.Even more,these exosomes were capable of inducing proliferation and tumor growth.53Similarly,in metastatic gastric cancer,the let-7 miRNA family(known to have a tumor-suppressive role by targeting oncogenes such as RAS and HMGA2)is selectively released through exosomes and this allows the targeting of oncogenic signals during metastasis development in the recipient tissue.54For a detailed example/model of exosomal content contributing to metastasis in cancer cells,refer to Figure 3.In addition,tumor-derived exosomes may also transport proteins that inhibit normal cellular processes such as apoptosis.MiRNA transportation from tumor cells into the vascular system promotes a rapid method of exosome traf?cking,which can promote processes such as disease spread/metastasis(refer to Figure4).

Interestingly,when cells are subjected to stressful condi-tions,miRNAs can promote tumor survival through an exosomal-mediated pathway,increasing the levels of oncogenes involved in survival and evasion of apoptosis.4 For example,in a study regarding cervical tumor cells,the secretion of survivin through exosomes was noticed after cells were exposed to radiation treatment.These mechanisms may be considered important for cancer cells to develop strategies that facilitate drug resistance.55On a different study,proteins such as FasL or TRAIL were shown to be expressed on the surfaces of exosomes in melanoma and colorectal carcino-mas.37This has negative consequences on patient therapeu-tic responses,as they inhibit the ability of T cells to enable apoptosis.In this way exosomes can interfere with cellular pathways not only by traf?cking speci?c molecules,but also by interacting with immune cells through surface–surface interactions.

To some extent,exosomes have been thought to set up an environment that could favor tumor development.Peinado et al.56described exosomes as key elements in the assembly of the pre-metastatic niche.This last study presented for the ?rst time the role of exosomes as part of the‘seed and soil’theory.Exosomes isolated from highly metastatic melanomas have been shown to increase the metastatic potential of primary tumors by facilitating(with their vesicular content) bone marrow progenitor cells to acquire a pro-metastatic phenotype via tyrosine kinase receptor activation.57A more recent investigation reported that exosomal miR-105 enhances angiogenesis in a cancer context by targeting ZO-1(a protein that in humans is encoded by the TJP1 gene).58In this way exosomes target proteins related to cell junctions and increase epithelial cell permeability,thereby promoting tumor invasion.

Several studies have stated the importance of exosomal-derived miRNAs in determining the overall effect of(already known)chemotherapies in cancer patients.For example,in breast cancer,exosomes released by HER-2-positive cancer cells have their expression modulated by various growth factors that include EGF and heregulin,a pleiotropic molecule that binds to the ErbB receptor family.These are some of the most extensively studied HER-2cross-activating ligands and are secreted in large quantities in the surrounding neoplastic niche.Through the expression of these receptors,exosomes manage to escape/inhibit the activity of trastuzumab,a previously established therapeutic strategy for this type of cancer.59Furthermore,in this subtype of breast cancer,the HER-2display on the surface of exosomes has been correlated with an increase in the binding of trastuzumab to exosome vesicles and not the original target(which are the cancer cells). This most de?nitely hinders therapeutic inef?ciency.60In addition to exosome-mediated mechanisms,the clinical resistance to herceptin therapy is also related to the cancer cells protecting themselves from NK-cell mediated antibody-dependent cytotoxicity(ADCC),which minimizes the chances of reducing tumor burden.61,62Thus,in this way exosomes

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confer cancer cells additional strategies that can allow them to overcome the effects of pre-existing therapeutics.

Exosomal contents such as miRNAs typically relate to the ones being expressed in the tissues from which they are secreted.However,in some cases the exosomal miRNA content does not correlate to miRNA pro?les of a particular cell type.33On occasions,tumor-derived exosomes establish a pre-metastatic niche which can regulate ‘premetastatic behaviors’because of the miRNAs that have been transferred from other tissues of origin (Figure 4).For example,in 2008,Taylor and Gercel-Taylor 31observed circulating tumor micro-vesicles in the peripheral blood of patients diagnosed with ovarian cancer,and these vesicles contained speci?c ‘molecular cargo’when compared to vesicles from

healthy

Figure 2Immune surveillance of T cells and speci?c miRs of exosomes released from tumor

cells

Figure 3Exosome secretion by tumor cells.The released exosomes are uptaken by neighboring cells and are capable of inducing pathways involved in cancer initiation/progression (e.g.migration,invasion),or alternatively,they can modulate immunogenic responses.Their downstream effect is attributed to their molecular cargo,which can be rich in miRNAs

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patients.This suggests that vesicular cargo such as miRNAs are different in their content when they are secreted from a tumor microenvironment versus when they originate from the exact same tissue,but from a healthy patient.Similarly,studies done in melanoma also described signi?cant differ-ences in mRNA and miRNA signatures when comparing exosomes derived from melanoma with the ones arising from normal melanocytes:the proteins such as HAPLN1,GRP78,syntenin-1and annexin A1were present in melanoma-derived exosomes,but not in normal melanocytes.63

Many oncogenes and processes promoting carcinogenesis (as well as other diseases)have been related to speci?c miRNAs and exosomes,and herein we present some examples.Exosomal-derived let-7was proven to be involved in the modulation of Toll-like receptor 7.64In addition,exosomal miR-21and miR-29a were demonstrated to stimulate Toll-like receptor-7and -8,causing a prometastatic in?ammatory response and activating tumor growth and metastasis.65Furthermore,Ferrajoli et al.66described an overexpression of miR-155in free circulating and blood microvesicles for MBL (monoclonal B-cell lymphocytosis)and CLL (chronic lympho-cytic leukemia)patients.Separately,in another study,11exosomal miRNAs were observed overexpressed in prostate cancer patients and miR-141and miR-375were presented as potential markers of metastatic prostate cancer based on exosomal and microvesicle samples.67More so,exosomal miRNA expression pro?ling proved that AZ-P7a cells (a metastatic gastric cancer cell line)release let-7miRNAs via exosomes into the extracellular environment to maintain their oncogenesis.54In addition,exosomal miR-223was shown to be released by macrophages,and to be ef?ciently transferred

to breast cancer cells,thereby targeting cellular invasion.68Finally,it has already been reported that exosomes are able to transfer phenotypic characteristics from tumor cells to other cell types,conferring aggressive behaviors to receptor cells.69

Exosomes as Novel Cancer Biomarkers

Exosome-associated signatures from various malignancies are expected to predict the presence of early disease before a formal diagnosis is done through classic imaging techniques such as MRI,CT or a PET scan.70–72In addition,these signatures might contribute by con?rming or discarding the results of biomarkers that are nowadays considered standard of care,such as the prostate-speci?c antigen (PSA),73carbohydrate antigen CA-90or CA-125.74

Exosomes are intercellular carriers of many molecules and,thus,they may be considered as novel sources of unknown potential biomarkers.75It would be very useful to develop new mechanisms to monitor the transcriptome expression pattern based on the use of noninvasive biological ?uids.76–78As the composition of nucleic acids (speci?cally RNA)re?ect in most cases the nature and origin of the parental cell,exosomes may be considered valuable carriers of cell-speci?c informa-tion because their content provides an insight glance of the proteins being transcribed in the original tissue.79–81In this manner,exosomes may be used in the clinic to achieve early diagnostics and prognosis information,but also to stratify and separate patients into different clinical categories in order to determine speci?c targeted therapies.With the increase in studies aiming to ?nd selective methods to isolate and purify exosomes,these approaches seem hopeful and

Figure 4Tumor-derived exosomes are secreted via a constitutive/inducible pathway that can be activated by a p53-mediated response.Through this pathway (and possibly additional ones that remain unknown),exosomes can transport microRNAs into the vascular system and regulate important cellular processes such as immunosurveillance,apoptosis and angiogenesis.For example:(a )TP53can induce the activity of TSAP6proteins,thereby modulating key functions associated with tumor-derived exosome traf?cking.Exosomes are generated by reverse budding of the membrane of multivesicular bodies (MVBs)that fuse with the plasma membrane,causing the release of these particles outside the cell.(b )From this moment on,exosomes enter the vascular or lymphatic system and circulate freely until they bind to their speci?c target.(c )The release from the cell is possible after the membrane is stimulated by calcium ionophores or phorbol esters.Inositol 3-kinase inhibitors can inhibit this process

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promising as researchers might actually?nd relevant biomar-kers to diagnose speci?c diseases or to better de?ne disease stages.16,26

Various functional studies have proven that exosomes display a delimited cell tropism because they target certain cells or tissues by displaying receptors in the membranes (including malignant ones).82,83For example,urine-derived exosomes from prostate cancer patients display(continu-ously)speci?c molecular markers such as PCA-3or TMPRSS2which are tissue speci?c.84In addition,exosomes are present in all bodily?uids,minimizing the need of performing invasive or potentially harmful diagnostic proce-dures(such as continuous rectal examinations for prostate cancer screening).These facts make them strong candidates for biomarkers of disease presence,progression or even therapeutic response.85,86

Recent data promote the use of dendritic cell-derived or even tumor-derived exosomes to understand which therapies are more favorable to use in the clinic.87,88For more details regarding studies using exosomes for anticancer vaccines refer to Table 2.On a separate note,by analyzing the proteomic pro?le of exosomes isolated from neuroblastoma, Marimpietri et al.89discovered that neuroblastoma cells secrete extracellular multivesicular bodies that express the GD2disialoganglioside,a protein involved in cell immune response,cell differentiation,cell proliferation and progres-sion.Thus,in this manner,proteomic pro?les of exosomes could be used to improve prognosis predictions,and diagnostic accuracy but also to predict of potential disease relapse in patients receiving chemotherapy or radiation treatment.Interestingly,in chronic myeloid leukemia(CML), Mineo et al.90showed that a co-evolution between endothelial cells and CML cells are essential for leukemia progression and resistance to therapy.This is possible because of the fact that K562malignant cells secret growth factors and various miRNAs and transport these‘endothelial inducing factors’via exosomes.As a result,tube formation is stimulated,even when treating with imatinib,a tyrosine kinase inhibitor that targets the Philadelphia chromosome-positive(Pht)myeloid leukemia cells.In this case,the development of angiogenesis was reported to regulate the progression and dissemination of this hematological malignancy.In cases such as these, exosomes provide an alternative strategy to target angiogen-esis in order to potentially achieve an even better therapeutic response in these patients.

Several studies have already proven successful in identify-ing exosomes as biomarkers.66For example,increased levels of exosomal miR-141and miR-375were capable of predicting metastasis or disease recurrence in prostate cancer.91 Exosomal miRNAs have shown distinct pro?les in ovarian cancer versus benign ovarian tumors.Moreover,the lack of exosomal miRNA in normal controls has also been observed and proven useful to distinguish healthy patients from others who have the disease.92Similarly,in a lung cancer study, signi?cant differences in total exosomal RNA levels in patients versus controls was found;93and a similarity between the circulating exosomal miRNA and the tumor-derived miRNA patterns for lung cancer patients was identi?ed.94Finally, miR-1246was considerably elevated in exosomes from serum samples of esophageal squamous cell carcinoma (ESCC)patients,and this was determined to be a powerful independent risk factor for poor survival.95All together,these data sets suggest that circulating exosomal miRNA would be extremely useful tools to develop novel cancer biomarkers (Table3).

Exosome-Based Therapy

Exosomes may have signi?cant contribution in drug delivery, based on the facts that they are considered natural carriers of multiple types of molecules,including nucleic acids.They have an ideal prototype to?t the role of‘physiologic liposomes’because of their nano-scaled size.96,97One potential applica-tion could be stimulating the body’s natural immune system to recognize and eliminate the neoplastic cells through exoso-mal-mediated recognition.These endogenous delivery sys-tems can overcome the dif?culties confronted when trying to use arti?cially created vesicles.20,21Based on this initial idea, several clinical trials have already started using exosomes with the aim of developing effective anticancer vaccines (Table4).One of the?rst steps in developing them has been reducing their immunogenicity98and increasing their half-life in the circulation,thereby assuring protection of their ‘therapeutic cargos’,particularly in the case of nucleic acids (miRNA or small interfering RNAs(siRNAs)).99,100Thus, exosomes are actively investigated as delivery systems of therapeutic nucleic acids structures,proteins or macromole-cular drugs in immunotherapy,101,102cancer and neurological diseases.103–106

Cho et al.75published one of the?rst reports using engineered exosomes to express a speci?c cancer antigen that could generate an immune antitumor response.In their study,they demonstrated that arti?cially introduced exo-somes have the ability to stimulate and shrink the tumor mass.To this date,liposomes are the predesigned carriers that have been proven to effectively deliver genetic material, drugs,cytokines,adjuvants or antigens into the body. However,nowadays,exosomes seem to be even better carriers because they can be introduced exogenously,but as they are‘naturally endogenous vesicles’,they possess a high biosafety pro?le.97

Table2Exosomes application in immune-driven vaccines

Cancer type Results Reference

Melanoma Exosomes from dendritic cells promote

the differentiation of melanoma-speci?c T

cells and thus produce IFN-g lymphocytes 57

Leukemia Inhibition of tumor growth by lysis of

cancer cells 86

Lymphoma Heat-shocked exosomes against

neoplastic cells 83

Leukemia Induction of spleen cell proliferation

and inhibition of cancer cells 87

Lung cancer Anticancer response of dendritic

cell-derived exosomes 94

Pancreatic cancer Induction of apoptosis via Notch signaling112

Pancreatic cancer Induction of apoptosis via PI3K/Akt/

GSK-3b pathway

113

Melanoma IL-I5R a-dependent cell proliferation81 Lymphoma Inhibition of tumor growth88Exosomes as divine messengers

C Braicu et al

41

Cell Death and Differentiation

Emerging progress in nanotechnology has allowed the development of novel exosome-based carriers that can be used in preclinical and clinical trials.In the past,nanoparticles that could mimic the physical characteristics of various viruses have shown to induce a very strong immune response when testing them as vaccines for similar clinical management.107 Exosomes are very similar to nanoparticles because they could express/present certain antigens on their surface; however,their toxicity is minimal or none.For example,Lee et al.108used extracted exosomes from autologous cancers and described them as nano-sized particles enriched with speci?c antigens.After combining them with dendritic cells in vitro,a very strong anticancer effect was noticed in both cell cultures and in vivo models of primary and advanced metastatic malignancies.

The highest level of applicable therapeutics expected to be achieved is the use of exosome-based treatments where their content involves miRNAs that can be traf?cked to speci?c tissue types to achieve the silencing of oncogenes(Table4). In a recent study,Wahlgren et al.109successfully delivered siRNAs using several types of human exosomes as delivery systems to mononuclear blood cells.Similarly,exosomes expressing let-7a were intravenously injected into a xenograft breast cancer model and the treatment resulted in signi?cant antitumoral activity caused by the targeting EGFR recep-tors.96Furthermore,some studies have suggested that exosomes themselves can become natural carriers of exogenous siRNA to human cells in vitro and in vivo.110An example of this was seen when trying to knock down RAD51, a potential therapeutic target for tumor cells.111 Conclusions and Perspectives

In order to achieve early cancer detection,we are in need of discovering and validating better standard-of-care assays that can identify the presence of malignancies through noninva-sive techniques.Discovering that exosomes mediate inter-cellular communication through the delivery of molecules (such as miRNAs and other nucleic acids)has underlined a particular interest for the use of these microvesicles as potential cancer biomarkers.As exosomes express speci?c markers on their surface,they are ideal candidates that could lead to more ef?cient management of diseases such as

Table3The miRNA exosomes in screening and diagnostic

Cancer type Results Observation Role Reference

MBL and CLL miR-155(m)Exosomal and whole blood of224samples from

2independent training and validation cohorts Diagnostic,

patient

strati?cation

66

Prostate cancer (PCa)miR-141(m),miR-181a-2*(k),miR-301a(m),miR-

326(m),miR-331-3p(m),miR-375(m),miR-432(m),

miR-574-3p(m),miR-625*(m),miR-210(m)

Plasma and serum microvesicles PCa versus

healthy controls,106patients for screening;

validation on119patients for metastatic versus

localized PCa(only for mir-141miR-375)

Diagnostic,

prognostic

67,73

Ovarian cancer miR-21(m),miR-141(m),miR-200a(m),miR-

200c(m),miR-203(m),miR-205(m)and miR-

214(m)Different stages of ovarian cancer(50cases)

versus benign ovarian adenoma(n?10)

Diagnostic92

Lung cancer miR-17-3p(m),miR-21(m),miR-106a(m),miR-

146(m),miR-155(m),miR-191(m),miR-192(m),

miR-203(m),miR-205(m),miR-210(m),miR-

212(m),miR-214(m)Exosomal and free miRNA in plasma for lung

cancer versus control group

Screening

and

diagnostic

94

Melanoma miR-31(m),miR-185(m),and miR-34b(m)Tumor cell-derived exosomes and normal

melanocyte-derived exosomes Biomarkers63

Esophageal squamous cell carcinoma (ESCC)miR-1246(m)Serum and exosomes Diagnostic,

prognostic

95

The symbol‘m’indicates overexpressed and‘k’indicates downregulated Table4Exosome-based miRNA,small interfering RNAs and protein delivery Therapeutic agent Therapeutic target Reference Nucleic acids delivery

miRNA

let-7a Targeting EGFR-expres-

sing breast cancer

(in vitro/in vivo)96

miR-29Tat and morphine-mediated

neuronal dysfunction in HIV 99

miR-223mimics and inhibitor Targeting b-catenin

pathway in breast

cancer cells

68

siRNA

Exosomal BACE siRNA formulation from dendritic cells expressing Lamp2b (assures and reduces immunogenicity)Knockdown of BACE1,

a therapeutic target in Alzheimer’s disease,in

wild-type mice.

114

Proteins

MelanA/Mart-1and

gp100(released by colon carcinoma cells express CEA and HER2)Stimulate CD4tand

CD8tT cells and exo-

somes(obtained in vitro)

leading to in vivo activation

of T-cell responses and

inhibition of tumor growth

98,101,

108,110

Dendritic cell-derived exosomes pulsed with tumor-derived antigens Antitumor T-cell responses

and tumor regression

(animal models)

109

Delta-like4(a trans-membrane ligand for Notch receptors)Inhibition of angiogenesis in preclinical models

104

Dexosomes Melanoma and non-small-

cell lung cancer phase I

clinical trial 105,106

Exosomes as divine messengers

C Braicu et al 42

Cell Death and Differentiation

cancer.Major regulatory pathways such as cell differentiation, proliferation,survival or apoptosis have been demonstrated to be in?uenced by the transfer of genetic material,such as noncoding RNAs through these secreted vesicles.MiRNAs are an exosomal component that has one of the most signi?cant functions in intercellular signaling and tumor progression. Through the dysregulation protein homeostasis miRNAs facil-itate tumorigenesis by reinforcing every single one of the cancer hallmarks.Thus,techniques such as molecular pro?ling of exosome miRNA signatures will not only add molecular knowl-edge to our understanding of communication between malignant cells and microenvironment,but will also facilitate the introduc-tion of novel cancer biomarkers and therapeutics.

Con?ict of Interest

The authors declare no con?ict of interest.

Acknowledgements.Dr.Berindan-Neagoe’s work was?nanced by POSCCE 709/2010grant with title:‘Clinical and economic impact of proteome and transcriptome molecular pro?ling in neoadjuvant therapy of triple negative breast cancer(BREASTIMPACT)’.Dr.Calin is The Alan M Gewirtz Leukemia&Lymphoma Society Scholar.Work in Dr.Calin’s laboratory is supported in part by the NIH/NCI grants1UH2TR00943-01and1R01CA182905-01,Developmental Research Awards in Prostate Cancer,Multiple Myeloma,Leukemia(P50CA100632)and Head and Neck(P50CA097007)SPOREs,a SINF MDACC_DKFZ grant in CLL, a SINF grant in colon cancer,a Kidney Cancer Pilot Project,the Duncan Family Institutional Seed Funds,The Blanton-Davis Ovarian Cancer-2013Sprint for Life Research Award,the Laura and John Arnold Foundation,the RGK Foundation,the Estate of CG Johnson,Jr.and by the CLL Global Research Foundation. The authors would like to thank Lucian Craciun for the assistance with the?gures.

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