Review pubs.acs.org/bc
Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
ExosomesSmall Players, Big Sound Diana Gulei,†,¶ Alexandra Iulia Irimie,‡,¶ Roxana Cojocneanu-Petric,# Joachim L. Schultze,§,∥ and Ioana Berindan-Neagoe*,†,#,⊥ †
MEDFUTURE-Research Center for Advanced Medicine, “Iuliu-Hatieganu” University of Medicine and Pharmacy, Marinescu 23 Street, 400337 Cluj-Napoca, Romania ‡ Department of Prosthetic Dentistry and Dental Materials, Division Dental Propaedeutics, Aesthetics, Faculty of Dentistry, “Iuliu-Hatieganu” University of Medicine and Pharmacy, Marinescu 23 Street, 400337 Cluj-Napoca, Romania # Research Center for Functional Genomics, Biomedicine and Translational Medicine, “Iuliu-Hatieganu” University of Medicine and Pharmacy, Marinescu 23 Street, 400337 Cluj-Napoca, Romania § Genomics and Immunoregulation, LIMES-Institute, University of Bonn, 53115 Bonn, Germany ∥ Platform for Single Cell Genomics and Epigenomics, German Center for Neurodegenerative Diseases, 53175 Bonn, Germany ⊥ Department of Functional Genomics and Experimental Pathology, The Oncology Institute “Prof. Dr. Ion Chiricuta”, Republicii 34-36 Street, 400015 Cluj-Napoca, Romania ABSTRACT: Incipiently named extracellular vesicles, exosomes are forming now a separate class of cellular mediators with important functions in physiological and pathological states. Their ability to transfer information between cells through encapsulation of proteins, nucleic acids and lipids for the preservation of the homeostatic equilibrium is translated also in pathological conditions. The recipient cells react to the reception of foreign molecules adjusting their molecular state according to the enclosed message. Cancer cells, in order to influence the microenvironment and facilitate the malignant expanding, exploit this intercellular trafficking. Immune cells are also producing exosomes that ensure the transportation of immune mediators and signaling molecules between cells. Current experimental attempts are concentrated on the adjustment of exosomes level for therapeutic purposes, enrolment of these vesicles as diagnosis or prognosis tools and also exosomes’ use as drug delivery vehicles or immune stimulatory agents.
1. INTRODUCTION The role of the immune surveillance in eliminating cancer cells has been strongly studied in the oncology area, where numerous immune-related therapies are now developed or tested in order to abolish the tumor development and to control the immune response.1,2 This natural capacity of the immune cells to eliminate the malignant ones from the organism is sometimes bypassed by the tumor formation through several mechanisms: (1) expression of lower levels of surface antigens; (2) secretion of surface proteins able to inhibit the activity of the immune system; (3) release of immune inhibitory microvesicles/substances in the surrounding microenvironment. Every type of these mechanisms and also combined ones are able to tilt the balance in favor of the malignant cells, allowing the development of carcinogenesis and metastasis.3−5 Exosomes secreted by the tumor cells can influence the immune cells encountered in circulation or in the malignant environment through delivery of inhibitory signals that impair their function against the cancer mass.6−8 Nowadays, immunotherapies counteract this unbalanced regulation between cancer and immune cells in order to increase the potential of the immune system or to decrease the © XXXX American Chemical Society
capacity of the cancer cells to escape from the immune surveillance. Recently, the role of exosomes has also been highlighted in the scientific area with the potential of therapeutic molecules as they can mediate communication between cells and also immune related mechanisms. More precisely, these nanovehicles can intervene in the opsonization process, antigen presentation, and also inhibition or activation of the immune response.8−12 Exosomes could be explored in novel therapeutic strategies, where their aberrant function in cancer could be experimentally modulated in order to impair the malignant phenotype through activation of the immune system or suppression of the malignant inhibitory immune mechanisms. Despite the fact that exosomes were initially considered the “garbage bins” of cells, advances in the field of cellular Special Issue: Bioconjugate Materials in Vaccines and Immunotherapies Received: January 3, 2018 Revised: January 23, 2018 Published: January 25, 2018 A
DOI: 10.1021/acs.bioconjchem.8b00003 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 1. Exosome biogenesis. Next to endocytosis, early endosomes are formed followed by maturation, which results in their budding into MVBs (Multivesicular Bodies) as ILVs (Intraluminal Vesicles). The MVBs that are not subjected to lysosomal degradation are merging with the plasma membrane allowing the exosomes to enter into the extracellular space. The interaction between exosomes and target cells can be mediated by different mechanisms including (1) receptor binding, (2) phagocytosis, or (3) membrane fusion as shown on the recipient cell. Once entered into the target cells, exosomes can modulate their state in a context dependent manner, depending on the state of the donor cell. The modulation is mediated by the exosomal cargo, mainly composed of DNA and RNA sequencesmRNA, miRNAlipids, and proteins, including those involved in antigen presentation (integrins, tetraspaninsCD9, CD63, CD81, and CD82 and MHC I and II). ER − Endoplasmic Reticulum; MVB − Multivesicular Body; ILV − Intraluminal Vesicles; MHC − Major Histocompatibility Complex; miRNA − microRNAs; mRNA − Messenger RNA.
stimulated potential therapeutic applications of these small vehicles in the context of cancer management. At the moment, there are several ongoing clinical trials comprising the use of exosomes for anticancer therapy with an increased interest in lung malignancies, melanoma, colon, and prostate cancer.22 The main approaches are (1) depletion of exosomes from biological fluids, (2) targeting of key molecules involved in the biogenesis of these small microvesicles, or even (3) conversion of the secreted vesicles into delivery vehicles for anticancer therapeutics. The therapeutic direction as delivery vehicles seems to be the most promising one due to the “self” profile of the exosomes and implicit lack of immune recognition against exosomes by the host.23−25 Despite all these advances there are still numerous unclarified aspects that need to be revealed in order to efficiently target cancer cells and immune cells serving tumor microenvironment via exosomes.
communication demonstrated that these small vesicles (40− 100 nm) are actually crucial players in the process of molecular trafficking, being able to perform intercellular transport and information exchange with different regulatory cargo molecules.12−15 The double lipid membrane structures are small vesicle-like bodies secreted by the cells and are ubiquitously present in all body fluids.16 The secretion of these transfer vehicles is a process which is naturally present in physiological conditions, facilitating the communication between cells in order to preserve homeostasis. The same process is replicated under pathological contexts, including cancer, promoting the establishment of a malignant environment with effects on tumor cell growth, proliferation, invasion, and metastasis, and immune system aberrant control.13,17 The content of exosomes, consisting primarily of various proteins and nucleic acids DNA, mRNAs, but also short and long noncoding RNAs provides information about the state of the cell of origin, and, at the same time, offers clues concerning their possible effects on the recipient cells.18,19 Studies have shown that exosomes are involved in many cellular activities, from physiological ones such as carrying immunogenic molecules (e.g., CD86, CD40, MHC-I, MHC-II, Hsp70, and galectin-5) or conveying biomessages to neighboring or distant cellsthus mimicking paracrine and endocrine communicationto pathological ones, for instance, cancer growth and priming of the pre-metastatic site in various tumors.18,20,21 The continuous expanding record of exosomes implications in malignant scenarios has also
2. EXOSOME BIOGENESISFROM DONOR TO TARGET CELL The beginnings of the 1980s marked an important milestone in extracellular vesicles (EVs) research when the concept that EVs are simply formed and released through outside budding of the plasma membrane was replaced by a more refined and realistic theory. Two independent studies described the formation and release pathways of these types of communication vesicles that actually implies an intercellular formation mediated by multivesicular bodies (MVBs) that is only afterward followed B
DOI: 10.1021/acs.bioconjchem.8b00003 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry by the discharge into the extracellular medium.26,27 Several years later, Rose Johnstone suggested the term “exosomes” for the vehicles of endosomal origins to the scientific community.28 Now, exosomes and microvesicles (MVs) are clearly distinguished in the scientific nomenclature and their biogenesis is clearly separated in terms of mechanisms.29 Meanwhile, exosomes became increasingly popular being now at the center of interest for cancer diagnosis, prognosis, and even therapy. The formation and release of exosomes is a finely tuned process that starts with the formation of an early endosome that will mature into late endosomes under the control of endocytosis-associated proteins and lipid complexes.30 The transformed vesicles are encapsulated into multivesicular bodies (MVBs), which also harbor specific proteins and nucleic acids originating from processing steps within the donor cell. At the final stage, the MVBs that do not undergo lysosomal degradation are expelled from the cell via RabGTPases, followed by the release of exosomes into the extracellular milieu (Figure 1).31 By these mechanisms, exosomes are able to target recipient cells by their engulfment and further modulation of the recipient cell’s activity in a paracrine or endocrine manner, depending on their location.30 As mentioned earlier, the biogenesis of exosomes is a strictly regulated process, with every step within the formation of the EVs being controlled and directed by specific pathway components. MVBs appear inside the cells as units that hold vesicles in their lumen (ILVs). These vesicles are actually the future exosomes that will be released outside of the cell or will go for degradation inside of the lysosome.32 ILVs and exosome biogenesis represents a well-documented research niche with two proposed controlling mechanisms that revolve around the Endosomal Sorting Complex Required for Transport (ESCRT) machinery.13,29,30 ESCRT is divided into four main groups (ESCRT-0, -I, -II, and -III) that associate with different proteins in order to accomplish their functions (VPS4, VTA1, ALIX named also PDCD6IP).33 Every type of complex has a designated role, where ESCRT-0 is responsible for the recognition and retreatment of transmembrane proteins that have been previously marked through ubiquitination; ESCRT-I and -II are involved in the deformation and invagination of the membrane that holds specific molecules (cargo) and ESCRTIII has been assigned a function as a vesicle scissor.33 The associated proteins, mainly VPS4 ATPase, have the task of detaching and recycling the entire machinery. The flow of vesicle formation via the ESCRT machinery consists of a continuous interaction between specific molecules that ends up with the emergence of vesicles with cell-derived cargo inside MVBs. Even if the recirculation via exosome release or degradation through the lysosome pathway of the cytosolic proteins is still an incompletely deciphered mechanism, it is assumed that these molecules are recognized by the HSC70 chaperone that forms a bridge between MVBs and target proteins toward their integration into the ILVs.34 Studies involving the experimental depletion of cells from key ESCRT members revealed that ILVs and exosome biogenesis could take place even in the absence of these complexes, their role being replaced by heat shock proteins, lipids, or tetraspanins.35 ESCRT-dependent and ESCRTindependent pathways are reviewed by Colombo and colleagues together with the implications for exosomes formation.34 Once exosomes are formed inside of MVBs, these final vehicles fuse with the cell membrane in order to release their
content into the extracellular space. The secretion of the nanovesicles is strictly controlled by Rab guanosine triphosphatases (GTPases) together with other parameters.13 High concentration of Ca2+ inside the cell elevates the secretion of exosomes.36 pH levels outside and within the cells are also critical factors for the expulsion of the vesicles, where low pH status determines the acceleration of exosome release and also uptake by target cells.37 Experimental evidences show that certain genes are also involved in the process of exosomes trafficking, the attention being now shifted toward oncogenes that are able to raise the level of exosomes from malignant cells targeted to healthy cells with the purpose of malignant transformations.13 Concerning the mechanism by which recipient cells collect the released nanovesicles, it has been proposed that there are three types of interactions: (I) receptor−ligand cooperation; (II) direct fusion with the membrane of the target cell; (III) endocytosis in case of phagocytosis related pathways.38
3. EXOSOME CONTENT Exosome content is usually specific to the cell of origin, and may vary according to the physiological and pathological condition. In this regard, the exosomal cargo can reveal the state of the donor cell and can also influence in a pathological manner the fate of the recipient cell.13 Moreover, the specificity of the molecular content can be exploited for the development of noninvasive diagnosis and prognosis tools, as well as targeted therapeutic strategies.39,40 The reliability of intercellular communication is maintained and translated by specific components within the exosomes. These components are generally made of proteins, lipids, DNA fragments, mRNA, and small RNA species. However, the cargo is not randomly distributed into exosomes, where the incorporation of these molecules is based on strictly regulated mechanisms referred as the term of “sorting”. Even if the pathways responsible for the sorting of exosomes content are not completely elucidated, the fact that some molecules are constantly incorporated and others are absent and also the fluctuating content that mirrors the state of the cell (homeostatic, pathological, stress conditions) are standing as proof of the controlled loading of exosomes.41 Regarding the protein composition, exosomes share a common panel of molecules, but also contain other differential proteins that vary according to the type and state of the donor cell. The common list includes proteins from the endosome, plasma membrane of the cell, and also from the cytosol. Another unifying aspect consists of the lack of proteins derived from mitochondria, Golgi complex, nucleus, and also endoplasmic reticulum, revealing the specificity of exosomes that are not just disposal units for random cell debris.34 The incorporation of proteins is thought to be controlled by lipiddependent mechanisms, ESCRT, and tetraspanins. As shown in the previous chapter, ESCRT mechanism is deeply involved in the biogenesis of exosomes.34 This complex is responsible for the identification and selection of ubiquitinated proteins, molecules that will be further processed into ILVs and then degraded upon the fusion of MVBs with the lysosome.42 In the case of exosomes, where the proteins escape from the degradation step encountered in the lysosomal media and are shipped extracellularly via these nanovehicles, the role of ESCRT is still debated. Experimental inhibition of different elements within the ESCRT complex showed that the protein content of exosomes is affected in different manners depending C
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Bioconjugate Chemistry Table 1. Commonly Used Procedures for Exosome Isolation: General Characteristicsa yield
purity
time
cost
Ultracentrifugation Ultrafiltration Density gradient centrifugation Precipitation polymerization Magnetic-activated cell sorting Immunoaffinity capture-based techniques Mass spectrometric immunoassay Acoustic nanofilter
principle
Centrifugation followed by ultracentrifugation Separation based on selective filters; Pressure or centrifugation Centrifugation; Supernatant + sucrose solution; Ultracentrifugation
general workflow
++ + +++
++ + +++
++ + +++
+ ++ ++
Incubation with solution for exosome precipitation; Centrifugation; Resuspension Incubation with magnetic beads; Centrifugation; Elution Low-speed centrifugation; Microplate-based immunoaffinity approach; Binding of specific membrane biomarkers Monolithic silica micropipette tips with specific antibodies
+ + +
+ ++ +++
+ ++ ++
++ ++ ++
+
+++
++
Separation based on size and density with ultrasound standing waves
+
++
+
Ciliated nanowire-onmicropillar ExoChip
Sequestration of exosomes based on the size: 40 and 100 nm; Elimination of bigger molecules and debris Separation based on immunoaffinity; Staining with membrane specific dye; Analysis of exosomes content through Western Blot and RT-qPCR
+
++
+
++ + ++ + +
+
+++
+
+
“+”, low; “++”, medium; “+++”, increased; these marks do not necessarily mean that “+” represents a disadvantage and “+++” an advantage, with this being context dependent, e.g., low cost = positive aspect, low purity = negative aspect.
a
on the targeted ESCRT member.43 Impairment of tsg101 (ESCRT-0/I), Hrs, and STAM1 affected the level of exosomes with CD63, CD81, and MHC-II.43 Hrs expression was also associated with a vital role in exosomes biogenesis in dendritic cells.44 In the case of Alix or Vps4B (ESCRT-III) inhibition, the situation was reversed, where the amount of exosomes increased (measured by exosomal MHC II).43 Besides ESCRT mechanisms, lipids also play a role in exosomal sorting, where the secretion of exosomes that are proteolipid (PLP)positive is not modified upon the inhibition of Hrs, Alix, or Tsg101.45 Lipids are also included in the exosomal cargo, although the studies regarding these types of molecules are more limited.34,45−47 The main lipid structures that were observed as being enriched in exosomes are cholesterol, saturated fatty acids, and sphingomyelin. More importantly, researchers reveled that exosomes contain reminiscent structures from the lipid rafts, but also lipid raft-associated proteins, GPI-anchored proteins, and flotillins, present on the surface of the plasma membrane of the cells.48,49 In light of these findings, it is still unclear if the ILVs form inside of the MVBs following mechanisms known for lipid raft formation, or whether domains present naturally in the membrane of the cell are enclosed together with the vesicles from the MVBs. The third mechanisms for protein sorting consist of the action of tetraspanins, membrane proteins found in high levels in exosomes.50 CD63, among others, is one of the tetraspanins that regulates the biogenesis but also the loading process of exosomes, where LMP1 protein integration into the nanovehicles is dependent on CD63.51 A similar dependence mechanism takes place in the case of PMEL incorporation into ILVs within melanogenesis.41 The presence of nucleic acids inside the exosomes had the biggest impact, especially in oncology, due to the specificity of the genetic material in accordance with the physiopathological state of the donor cell. Within the nucleic acids spectrum, miRNA sequences are the most studied ones, once it was discovered that the secreted noncoding cargo could affect the expression of genes within the recipient cell and even induce pathological modifications in order to augment the process of malignant development.52−55 The fact that some miRNAs are abundant in exosomes, and some, even if present in the donor cells, are barely distinguished in the secreted vesicles, is a standing proof for a specific sorting mechanisms.56−59
HnRNPA2B1 is a wide encounter protein and has been associated with trafficking functions for mRNA in distal sites of neurons.60 This protein contains a binding motif in the 3′UTR for an RNA transport signal (RTS or A2RE),61 motif that is also overlapping with EXOmotifs (sequences that mediate the loading of miRNAs into exosomes59). Therefore, it is supposed that HnRNPA2B1 is actually playing a crucial role regarding the sorting process of miRNAs in exosomes. Moreover, the same protein is frequently encountered as sumoylated in the secreted vesicles, where this modification is mandatory for the uptake of miRNAs.59 Next generation sequencing experiments showed that actually the most abundant RNA structures from exosomes are represented by the class of structural RNAs and also small rRNA.56 Intriguingly, Wild and colleagues showed that the binding motif that mediates the forming of SRP-RNA−SRP protein complex is actually the EXOmotif: GGAG.62
4. EXOSOME ISOLATION METHODS Exosomes have been isolated from different cell types with ultracentrifugation being the most common method for separation of these vesicles. Most isolation methods are based on exosome size, which in general results in very good homogeneity of the obtained particles. Nevertheless, all methods still are not very reproducible and it is still difficult to obtain reliable results in validation studies or when comparing data derived from different groups. An important task for the field will be to standardize the technologies and make them more comparable. The most commonly used procedures for exosomes isolation are presented in Table 1. 5. MECHANISMS OF EXOSOMES IMPLICATION IN BOTH IMMUNE ACTIVATION AND INHIBITION Exosomes are important immune-modulators, able to trigger the activation of a diverse set of immune cells (Figure 2). In the late 1990s, Rapaso et al. discovered that B cells are able to secrete and deliver exosomes containing MHC (major histocompatibility complex) class II that induces a response by T cells.63 Now, it is generally accepted that dendritic cells (DCs) release exosomes bearing MHC I and MHC II class molecules, an action that can trigger an immune response against cancer cells when the exosomes are derived from mature DCs loaded with tumor antigens. These types of D
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Figure 2. Exosome mediated immune response in cancer versus pathogen−host interaction. Exosomes are crucial players in the activation of an immune response but at the same time can support tumor growth by suppressing immune cell activation. In the case of pathogen−host interactions, exosomes secreted by dendritic cells can activate effector CD4+ T and effector CD8+ T cells through direct antigen presentation or by cross presentation that requires the involvement of another set of mature DCs. Moreover, the same exosomes released by DCs can activate endothelial cells, promote the cytotoxic activity of NKs, and also trigger inflammation. In the case of malignant tumors, exosomes secreted by cancer cells can act in favor of the donor cells thereby suppressing differentiation and activation of DCs. Also, the vesicles can induce apoptosis of CD8+ T cells, inhibit the activity of NKs through downregulation of NKG2D receptors, and also induce the differentiation of CD4+ T cells into T regulatory cells. In this way, cancer derived exosomes can maintain a “light” immune environment that favors the proliferation and invasiveness of cancer cells within the body. NK − Natural killer; DC − Dendritic Cell; Treg cell − Regulatory T cell; TNF − Tumor Necrosis Factor; IL-15R − Interleukin 15 Receptor; BAT-3 − HLA-B-Associated Transcript 3; IL-1β − Interleukin 1 beta.
exosomes are able to directly activate CD8+ T cells and naive CD4+ T cells through an antigen-specific manner.64 Immature DCs are also able to discharge exosomes in the microenvironment, but in this case the direct activation of T cells is hampered and can only be induced following additional processing steps by the antigen presenting cells.65 Based on these findings, it has been suggested to use DC derived exosomes (DC-Exo) as a therapeutic option in cancer. For example, the administration of such vesicles and the subsequent incorporation of the immunogenic cargo into cancer cells increased the number of IFN-γ-secreting cells in the context of human breast adenocarcinoma cells cocultured with sensitized T-cells.65 Inversely, tumor derived exosomes hold immunosuppressive characteristics generated by their content (Figure 2). This is the case for Fas Ligand (FasL), a type II protein that has its place in the TNF family, and is often found in tumor-derived exosomes. Indeed, exosome-derived FasL has been shown to promote apoptosis of T lymphocytes.66 Another central inhibitory mechanism associated with tumor-derived exosome biology is
NKG2D (natural killer group 2, member D), a receptor that normally activates a number of immune cells (NK, gammadelta (+) T cells, CD8 (+), and NKT); however, it is frequently deregulated in cancer being one of the triggers for immune evasion. Cancers compromise immune activation by secreting growth factors (e.g., TGF-beta) and soluble NKG2D ligands thereby reducing the expression of NKG2D. Exosomes secreted by cancer cells are taking advantage of this mechanism and impair the activity of NK cells and CD8 (+) T cells by hijacking the expression of NKG2D present on the surface of the respective immune cells. Clayton et al. showed that this ability of cancer-derived exosomes is mediated by the presence of TGF-beta1 and NKG2D ligands and takes place in the moment of direct interactions between the vesicles and NK cells or CD8 (+) T cells.67 On the other hand, exosomes secreted by cancer cells can also act against the tumor mass, being a source of antigens for DCs inducing pathways by which an immune response can be triggered against malignant cells.68−70 Besides these immunomodulatory roles, exosomes are also involved in other processes like retroviral spreading within cell E
DOI: 10.1021/acs.bioconjchem.8b00003 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry Table 2. Immuno-Derived Exosomes and Their Content immune cell
function
Dendritic cells
Antigen presentation, Activation T cells, Immune tolerance, Immune memory Antibody production, Antigen presenting cells for T helper cells, memory B cells CD8+: Cytotoxicity, MHC-I antigen binding CD4+: MHC-II antigen binding, helper for cytotoxic T cells, helper for macrophage activation Cytotoxicity
B cells T cells
Natural killer cells Macrophages Mast cells
Inflammation, Phagocytosis, Phenotypic plasticity Immunomodulation, Allergy, Antigen presentation, Inflammation
exosome content
ref
MHC complexes, B71/CD80, B7-2/CD86, ICAM-1
76−79
CD19, CD37, MHC class II Treg: CD73, CD25, CTLA-4; CD4+: CD4, HLA Class I, TfR, TCR, Integrin β2, Fas Ligand; CD8+: Fas Ligand CD8, TCR Perforin, CD56, Granzyme B, Fas Ligand Bacterial antigens, Cofilin-1, GNB1, Actin, Cyclophilin A MHC class II, c-Kit, esRNA, LFA1, ICAM
cell line (THP-1), where miR-484 was found as significantly downregulated in LPS stimulated cells and miR-26a as upregulated with targets related to the inflammatory pathways. MiRNAs secreted by cancer cells through exosomes can modulate immune cells by binding to Toll-like receptors and mediate a pro-metastatic microenvironment: Fabbri et al. showed that miR-21 and miR-29a can target immune cells via exosomes and further bind and activate TLR8 that leads to stimulation of NFκB pathway and increased secretion of cytokines with inflammatory/prometastatic abilities.83 The role of exosomal miRNAs in activating/suppressing the immune system in malignant pathologies is still incompletely elucidated, hampering the smooth translation of exosome vaccines toward the clinical area. After showing that miR-203 can prompt the immune tolerance by decreasing the expression of TLR4 in dendritic cells in prostate cancer through exosomal trafficking,84 Que et al. implemented the possibility of depleting exosomes from miRNA and administered them as fortified therapeutic agents. Stimulation of dendritic cell/cytokineinduced killer cells (DC/CIKs) with modified exosomes lysate (without miRNAs) showed increased inhibitory activity on pancreatic cancer cells, demonstrating that the miRNA content can significantly influence the ability of the immune system to counteract the malignant development.85 miRNAs from exosomes can also travel between immune cells; miR-146a and miR-155 are released and subsequently target dendritic cells (regulation between dendritic cells) in order to manage the cell’s response to endotoxin, and implicitly regulate inflammatory related pathways. Moreover, these two miRNAs are acting in an antagonist manner, where miR-146a reduces the expression of inflammation genes and miR-155 promotes it.86 Challenging the cells with exosomes loaded with the specific noncoding sequence revealed the same pattern of inhibition/enforcement, underlining important therapeutic related options in terms of exosome administration in cancer and also inflammatory states. Even so, the data related to miRNAs enclosed in exosomes and their role in modulating the immune surveillance against or in favor of the cancer cell is still unsecured from a therapeutic point of view. The heterogeneity of the exosomal noncoding content is destabilizing the exact effect of naive exosome administration. However, it has been previously shown that these vehicles can be experimentally loaded with “custom made” cargos in order to actually take advantage of their “self” attributes and not necessarily of their content for the in vivo delivery of immune modulatory agents (miRNAs, but also other noncoding RNAs).
populations, where particles like HIV-1 take advantage of the biogenesis pathway of exosomes, escape from the lysosomal degradation and infect de novo additional cells.71,72 Exosomes are also present at the interphase between neurons and serve as “bridge” in terms of exchange of molecular information. This bridging function is apparently not present only under physiological conditions, but also in neurodegenerative diseases. For example, molecules like PrPsc (scrapie form of the prion protein), alpha-synuclein, or fragments of amyloid precursor protein (APP) can be transferred between cells via exosomes.38 These structures are key elements in neurodegenerative diseases, where PrPsc is responsible for or associated with Creutzfeldt-Jacob disease, alpha-synuclein for Parkinson and APP for Alzheimer, all of them being found inside secreted exosomes.73−75 Another important cellular system where exosomes seem to play an important role for information exchange between cells is the stem cell compartment. Stem cells also secret exosomes, and in light of this property, stem cell-derived exosomes are increasingly considered for therapeutic options in the area of regenerative medicine.38 The association of exosomes and immune cells together with their content is briefly described in Table 2.
6. IMMUNOMODULATORY ROLES OF EXOSOMAL MIRNAS One of the main cargos that can be found in exosomes consists of RNA sequences, transcripts that hold or not the ability to further codify functional proteins. The latter ones, noncoding RNAs, are especially composed of miRNAs and lncRNAs that are actively expelled from the donor cell in order to modulate the target cell or, even more, to free the donor cell of supplementary noncoding RNAs in order to conserve the homeostatic balance between mRNAs and miRNAs (or other noncoding sequences). In the latest years of research it has been shown that the noncoding exosomal profile is actual dependent on the cellular state of the donor cell and also on the general state of the organisms as a whole. In this sense, chronic inflammation, that represents an important risk factor for several types of cancers (e.g., colon cancer), was shown to be an active modulator of the transcriptomic landscape of exosomes, including modifications in miRNAs content.80 The RNA content analysis between RAW 264.7 cells stimulated or not with LPS detected 433 miRNAs with exosomal provenance. Within these noncoding sequences, miR-146a, miR-146b, and miR-21-3p were found to be dysregulated between the two experimental scenarios, miRNAs that have been previously shown to be involved in inflammatory pathways.81,82 The same approach was also conducted on a human derived monocytic F
DOI: 10.1021/acs.bioconjchem.8b00003 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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7. EXOSOMES VACCINESDEXS Dendritic cell-derived exosomes (DEXs), as the name suggests, are exosomes secreted by dendritic cells and are able to increase the fitness of primary and also secondary immune response. Their role was established in 1998 by Zitvogel et al. where they showed that DEXs present MHC class I and class II and also additional stimulatory molecules that can trigger a tumor inhibitory effect dependent on T cells.87 This distinct cargo endow exosomes with immunostimulatory properties, functions that are also potentiated through the presence of surface molecules that facilitate the fusion with the target cells in order to further produce an immune response. Among these proteins, α- and β-chains (αMβ2), milk fat globule EGF factor 8, and the Ig superfamily member ICAM-1 are within the ones that stimulate the fusion with the receiving cell. More specifically, milk fat globule EGF factor 8 is actually mediating the formation of a bridge between exosomes and αvβ3 or αvβ5 integrins from the target cell.88 Other facilitators of the exosomes uptake within the population of the targeted cells could be also the proteins from the tetraspanin family like CD81, CD9, and CD63 that are present on the outer membrane of the vehicles.88,89 Thery et al. particularly studied CD9 where they proposed that this member is the most abundant one within exosomes derived from dendritic cells and also a mediator for APC or T cell interaction.88 Even so, the most investigated aspects regarding the proteomic profile of exosomes consists of the presence of peptide−MHC class II complex that could function as stimulators of a T cell specific response via the Ag presentation model.88,90 DEXs previously stimulated with tumor peptides showed inhibitory effects on malignant development after exogenous administration in mice (injection). This action was rapidly translated into potential clinical use, where DEXs were proposed as cell-free tumor vaccine. Additionally to MHC class II complex, exosomes also present MHC class I proteins that are associated with activation of CD8+ T cells once are delivered into the recipient dendritic cell within the microenvironment.91 Hsc73 (heat shock protein) represents a common member between the endocytic space of dendritic cells and exosomes, being associated in the same time with potent inhibitory effect upon tumor cells in vivo.88 The accumulation of hsc73 in DEXs could represent another immune layer of regulation involving exosomes, where these proteins were characterized as modulators of tumor rejection and cytotoxic T lymphocyte activation in mice injected with heat shock proteins (hsp) derived from the tumor microenvironment.92 It was also suggested that dendritic cells and macrophages express receptors for hsp, as it was shown that these shock proteins could be actually transferred to APCs for Ag presentation.93,94 The presence of Mac-1 on the surface of exosomes could also represent an immune mediator mechanism for the interaction with cells that express the ligands for Mac-1. These ligands, ICAM-1 or ICAM-2, are usually present in endothelial cells, but also present in dendritic cells and lymphocytes, a fact that underlines a possible mechanism of communication between secreted DEXs and target cells.88 DEXs were also studied in the context of NK cells, as they were shown to present NKG2D ligands upon secretion by immature DCs that further can activate in a direct manner these cells (NK). The ex vivo observations are sustained also by in vivo studies where DEXs were able to promote the activation and proliferation of NK cells in a NKG2D dependent manner; the mechanism was encountered also in a previous clinical
study where 50% of the patients responded to DEX vaccination through activation of NKs.95 Another NK dependent tumor inhibition that renders the activity of exosomes could be through BAT3 (NKp30 ligand) that is actively expressed on the surface of the nanovesicles.96 Additional surface molecules like FasL and TRAIL mediate the stimulation of NK through DEXs, but also can potentiate the induction of apoptosis in cancer cells through activation of caspases.97 The potential of exosomes derived from dendritic cells was further explored in a clinical scenario in order to assess the feasibility of this immunotherapy. Two clinical trials marked an important point for DEXs, where MAGE3+ patients with small cell lung cancers and advanced melanoma received weekly doses of DEXs enclosing MAGE malignant antigens. Even if the response from T cells was not as high as expected, part of the patients were correlated with stable disease and also some of them presented increased NK functions.98,99 These initial data paved the way toward more advanced investigation regarding the potential therapeutic value of DEXs: mDEXs derived from mature DCs can be used with a superior effect in terms of T cell activation due to the fact that they express a higher amount of stimulatory molecules than those obtained from immature DCs (e.g., MHC class II, ICAM1).100−102
8. THERAPEUTIC POTENTIAL OF EXOSOMES IN CANCERBEYOND IMMUNOTHERAPY The discovery of the heterogeneous exosomes involvement in malignant progression has led to the idea of targeting or using these vesicles in the context of cancer therapy (Figure 3).14 One approach is focusing on the removal of exosomes from the circulation in order to inhibit the pro-carcinogenic actions of these agents. The microvesicular extinction could be achieved through biogenesis inhibition by prevention of microtubule formation and stability, modulation of endosomal distribution pathways, or through the administration of proton pump inhibitors.37,103−105 Another form of action related to exosomemediated therapy consist of the usage of these molecules as nanovehicles for the targeted delivery of drugs to malignant sites, avoiding secondary cytotoxicity and immune eradication of the vehicle. Moreover, exosomes can be loaded with different nucleic acid sequences such as miRNA inhibitors or mimics, or siRNAs (small interfering RNAs) for the modulation of gene expression, but also with specific antigens in order to trigger an immune response.14,103,106−108 In a recent study, Mathiyalagan and Sahoo revealed a protocol for the isolation of exosomes and their potential use as carriers of miRNA for gene therapy in cardiovascular disease. Even if the targeted pathology was not cancer, this protocol where they generated exosomes from human CD34+ stem cells containing exogenous miRNA sequences for targeted delivery can be easily translated into other health sectors.109 In another research study, Ohno et al. delivered engineered exosomes containing let-7 to EGFR expressing breast cancer cells.110 Let-7 was associated with tumor suppressor capacities in cancer and was also observed as downregulated in multiple malignant pathologies like colon, ovary, breast, and lung cancer.111 The targeting mechanisms was constructed based on the idea that numerous epithelial tumors express EGFR, therefore functioning as an intermediary factor for specific delivery. The downside consists of the fact that EGF, the ligand for this receptor, is extremely mitogenic, decreasing the reliability of this duo as mediators of targeted delivery strategies. However, GE11 peptide is efficiently recognized by EGFR and also is less mitogenic that EGF, G
DOI: 10.1021/acs.bioconjchem.8b00003 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
Figure 3. Diagnosis and therapeutic value of exosomes. Exosomes can be used as early and noninvasive diagnostic and prognostic tools (A) due to their ubiquitous distribution in body fluids and also due to their specific cargo that can reveal the state of the donor cell. The same vesicles can also be loaded with anticancer drugs (B), compromising a more efficient delivery vehicle due to the “self” characteristics and persistence within the circulation. Another therapeutic alternative related to exosomes consist of the inhibition of the biogenesis process or even more in the depletion of the nanovesicles with different compounds (C). In this way, the pro-tumoral activity of exosomes can be significantly reduced concomitant with a decrease in cancer development or progression.
depletion of exosomes to reduce the pathological transformation of surrounding cells. This type of strategy was successfully applied in mouse models of colon carcinoma where administration of DMA (dimethyl amiloride) promoted the effect of CTX (cyclophosphamide) by cancer derived exosome depletion. Moreover, a synonym compound, amiloride, capable to reduce the exosomes levels, is currently being used for the treatment of high blood pressure. Therefore, by analyzing the blood profile of metastatic colon cancer patients that are treated with amiloride to reduce blood pressure, an inhibition of exosome formation together with inhibition of the suppressive function of MDSC-like (Myeloid-derived suppressor-like) cells was observed.119 MDSC-like cells can inhibit T cell activation in cancer contributing in this way to the development of the disease.120 In this sense, exosomes secreted by colon cancer cells were associated with an increased activity of MDSC like cells, an effect that was attenuated in patients treated with amiloride. The KRAS gene that is mutated in approximately 30% to 40% of colorectal cancers represents another important therapeutic pathway related to exosomes in this pathology. Cancer cells that are holding a mutant gene secret exosome rich in proteins with tumor promoting roles like EGFR, KRAS, integrins, and SRC family kinases. The surrounding wild-type cells that are a target of these exosomes rapidly acquire malignant-like phenotypes and grow at a faster rate.121 In this sense, mutant KRAS or exosomes bearing mutant KRAS proteins are a target for cancer therapy, where the silencing of the oncogene or depletion of the tumor favoring vehicles could stand as a potent anticancer strategy.
being counted as the ideal peptide for this type of therapeutic based interaction.112 Therefore, Ohno et al. modified the exosomes in order to express the GE11 peptide and successfully delivered let-7 to EGFR-expressing cancer cells both in vitro and in vivo (RAG2−/− mice with breast cancer cells xenografts).110 The significance of miRNA replacement or inhibition therapy has already been established with numerous studies conducted on this strategy for cancer impairment; however, the constant issue remains the delivery method for these small sequences, where exosomes are gaining increased attention due to their advantageous characteristics (nonimunogenic, decreased toxicity, ability to survive in circulation and target cancer cells).106,108,113−115 Recently, Lugini et al. discovered that exosomes derived from colorectal cancer cells can promote the malignant development by influencing the phenotype of cancer mesenchymal stromal cells (cMSCs).116 The modifications to the level of cMSCs are associated with tumor-like changes, including increased proliferation and vesicle secretion, actions that favor the tumor microenvironment. Moreover, two tumor markers were found to be up-regulated in cMSCs following exosome uptake: vacuolar H+-ATPase (V-ATPase) and carcinoembryonic antigen (CEA). These two molecules are potent candidates for colon cancer therapy, where V-ATPase is associated with an advanced malignant state (metastatic cancer) and CEA is an established biomarker used for screening of the evolution, progression, or regression during followup of colorectal malignancies.117,118 Considering the unfavorable consequences of exosomes released by colon cancer cells on the surrounding microenvironment, it would be an attractive strategy to apply an anticancer therapy compromising the H
DOI: 10.1021/acs.bioconjchem.8b00003 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Control of exosomes level
Cell activation
Delivery vehicle
type of therapy involving exosomes
Modification of exosomes targeting specificity Modification of exosomes cargo through activation of donor cell Elimination of exosomes from circulation Inhibition of exosome release/biogenesis
Modification of exosomes donor cell
Direct modification of secreted exosomes
therapeutic strategy
Modification of exosomes release rate for a better response to treatment
Dialysis-like strategy for the depletion of specific vesicles (e.g., exosomes) from the blood of cancer patients
Stimulation of donor cell with exogenous substances in order to increase the expression of specific molecules into exosomes
Modification of donor cells in order to produce exosomes containing specific sequences for targeted delivery
Transfection of donor cells with the gene of interest that will be further packed into exosomes secreted by the transfected cells
Incubation of donor cells with the specific drug/molecule in order to further isolate the secreted exosomes that will contain the administrated therapeutic
Repeated freeze and thaw cycles of exosomes and drug mix for mechanical disruption of the exosome membrane and encapsulation of the drug (cycle: room temperature followed by liquid nitrogen or −80 °C freezer; protocol: minimum 3 cycles) Chemically based methods for attachment of drugs and molecules to purified exosomes
Extrusion of exosomes and drug mixture with a syringe-based lipid extruder equipped with a 100−400 nm porous membrane
Administration of Ketotifen (10 μmol L−1) in three cancer cells decreased the amount of secreted exosomes together with the promotion of cell response to doxorubicin (increased anticancer effect)
Aethlon ADAPT (adaptive dialysis-like affinity platform technology) system for the hemofiltration of exosomes based on the specific size (
