Exosomes are potential biomarkers for cancer progression and neurodegenerative disease

Electron microscope image showing a maternal exosome from human endometrial fluid binding to the mouse trophoectoderm. The embryo receives the contents of the maternal exosome to establish the first communication between the mother and the embryo in the period prior to implantation.

There is a growing demand for diagnostic markers for early disease detection and prognosis.

Exosomes are potential biomarkers for cancer progression and neurodegenerative disease, but it can be difficult to identify what tissue a specific exosome comes from.

Researchers at Uppsala University and spin-off company Vesicode AB have solved this problem by developing a method that maps surface protein complements on large numbers of individual exosomes.

Exosomes are released from all cells in the body.

They convey protein and nucleic acid cargos between the cells as a form of intercellular communication, and they represent potential circulating biomarkers for tumor progression and metastasis, as well as for early detection of neurodegenerative disease.

In order to use exosomes as biomarkers of diseases in different tissues it is vital to distinguish them according to their surface protein complements.

Researchers at Uppsala University and Vesicode AB, along with collaborators, have developed a method that can map surface protein complements on large numbers of individual exosomes.

The novel proximity-dependent barcoding assay (PBA) reveals the surface protein composition of individual exosomes using antibody-DNA conjugates and next-generation sequencing.

The method identifies proteins on individual exosomes using micrometer-sized, uniquely tagged single-stranded DNA clusters generated by rolling circle amplification.

“This technology will not only benefit researchers studying exosomes, but also enable high-throughput biomarker discovery.

We will further develop and validate the PBA technology and provide service to researchers starting later this year.

We believe single exosome analysis will allow this exciting class of biomarkers to reach its full potential,” says Di Wu, researcher and inventor of the PBA technology and founder of Vesicode AB, commercializing the technique.

“This new technology will allow large-scale screens for biomarkers in disease, complementing a panel of methods for sensitive and specific detection of exosomes that we have previously established,” says Masood Kamali-Moghaddam one of the group leaders at the Molecular Tools unit at Uppsala University.

More information: Wu et al. (2019) Profiling surface proteins on individual exosomes using a proximity barcoding assay, Nature CommunicationsDOI: 10.1038/s41467-019-11486-1

Journal information: Nature Communications
Provided by Uppsala University

Exosomes are small endosomal derived membrane microvesicles that have observed increasing attentions over the past decade.

The presence of exosomes in extracellular space was identified as early as in late 1980s [1].

However, exosomes secreted from cells were initially proposed as cellular waste resulting from cell damage, or by-products of cell homeostasis, and have no significant impact on neighboring cells.

It is only recently that these extracellular vesicles are functional vehicles that carry a complex cargo of proteins [2], lipids [3], and nucleic aids [245], be capable of delivering these cargos to the target cells they encounter, which may ultimately reprogram the recipient cells distal from their release.

Thus, exosomes represent a novel mode of intercellular communication, which may play a major role in many cellular processes, such as immune response [6], signal transduction [7], antigen presentation [8].

As exosomes can be released by practically all eukaryotic cells, it is well considered that their cargos may greatly differ from each other for function of the originated cell types and their current state (e.g. transformed, differentiated, stimulated, and stressed).

Thus, exosomes and their biologically active cargos may offer prognostic information in a range of diseases, such as chronic inflammation [9], cardiovascular and renal diseases [1011], neurodegenerative diseases [12], lipid metabolic diseases [13] and tumors [14].

In this review, we endeavor to provide a brief description of exosome biogenesis, molecular properties, and exosomal functional activities in cell–cell communications, as established to date.

In addition, strategies for their isolation and characterization also be summarized. Finally, we discuss the feasibility of exosomes as clinical biomarkers and therapeutic potential of engineered exosomes as vehicles for targeted therapy.Go to:

Exosome biogenesis

Exosomes are constitutively generated from late endosomes, which are formed by inward budding of the limited multivesicular body (MVB) membrane.

Invagination of late endosomal membranes results in the formation of intraluminal vesicles (ILVs) within large MVBs [15].

During this process, certain proteins are incorporated into the invaginating membrane, while the cytosolic components are engulfed and enclosed within the ILVs.

Most ILVs are released into the extracellular space upon fusion with the plasma membrane, which are referred to as “exosomes” [1617].

Alternatively, these components are trafficked to lysosomes for degradation.

Canonical exosomes display a particular biconcave or cup-like shape when produced by artificially drying during preparation, while they appear spheroid in solution under transmission electron microscopy [18].

Typically, they have a density range from 1.13 g/mL (B cell-derived exosomes) [19] up to 1.19 g/mL (epithelial cell-derived exosomes) [20] on sucrose gradients.

Evidence has revealed that the formation of ILVs requires the endosomal sorting complex required for transport (ESCRT) function.

It is an intricate protein machinery composed of four separate protein ESCRTs (0 through III) that work cooperatively to facilitate MVB formation, vesicle budding, and protein cargo sorting [2122].

The ESCRT mechanism is initiated by recognition and sequestration of ubiquitinated proteins to specific domains of the endosomal membrane via ubiquitin-binding subunits of ESCRT-0.

After interaction with the ESCRT-I and -II complexes, the total complex will then combine with ESCRT-III, a protein complex that is involved in promoting the budding processes. Finally, following cleaving the buds to form ILVs, the ESCRT-III complex separates from the MVB membrane with energy supplied by the sorting protein Vps4 [21].

Despite the controversy of whether exosome release is an ESCRT-regulated mechanism, different ESCRT components and ubiquitinated proteins have already been identified in exosomes isolated from various cell types.

Additionally, the typical exosomal protein Alix, which is associated with several ESCRT (TSG101 and CHMP4) proteins, has been reported to participate in endosomal membrane budding and abscission, as well as exosomal cargo selection via interaction with syndecan [23]. These observations led to a hypothesis implicating ESCRT function in exosomal biogenesis.

Interestingly, recent evidence favors an alternative pathway for sorting exosomal cargo into MVBs in an ESCRT-independent manner, which seems to depend on raft-based microdomains for the lateral segregation of cargo within the endosomal membrane.

These microdomains are thought to be highly enriched in sphingomyelinases, from which ceramides can be formed by hydrolytic removal of the phosphocholine moiety [24].

Ceramides are known to induce lateral phase separation and coalescence of microdomains in model membranes.

Moreover, the cone-shaped structure of ceramide might cause spontaneous negative curvature of the endosomal membrane, thereby promoting domain-induced budding. Consequently, this ceramide-dependent mechanism emphasizes a key role of exosomal lipids in exosome biogenesis [25].

Proteins, such as tetraspanins, also participate in exosome biogenesis and protein loading. Tetraspanin-enriched microdomains (TEMs) are ubiquitous specialized membrane platforms for compartmentalization of receptors and signaling proteins in the plasma membrane [26].

It has been shown that TEMs together with tetraspanin CD81 play a key role in sorting target receptors and intracellular components toward exosomes [27].

Apparently, several specialized mechanisms exist to ensure the specific sorting of bioactive molecules into exosomes, either the ESCRT-dependent or -independent mechanism (involved tetraspanins and lipids), may act variously depending on the origin of the cell type.

In addition to exosomes, other types of membrane vesicles produced by cells include plasma membrane-budded microvesicles (MVs) and apoptotic bodies. MVs are heterogeneous populations of membrane vesicles generated by outward budding from the plasma membrane.

They are 100–1000 nm in size with variable shapes, and are predominately characterized as products of platelets, endothelial cells (ECs), and red blood cells.

The density of MVs has been reported to be between 1.25 and 1.30 g/mL [28]. Apoptotic bodies are exclusively released from the plasma membrane during the late stage of apoptosis, range in size from 1 to 5 mm, comparable to that of platelets, and contain several intracellular fragments, cellular organelles, membranes, and cytosolic contents. Apoptotic bodies are closed structures with a higher sucrose gradient density than MVs, ranging from 1.18 to 1.28 g/mL [29].

The features and characteristics of these cell-derived MV types are listed in Table 1. Finally, the comparatively smaller size and unified shape allow exosomes to successfully escape clearance by the mononuclear phagocyte system, not only prolonging their circulation time, but also implying their superiority in cell–cell communication.

The complex architecture of exosomes

Exosomes have been regarded as mini version of the parental cell, for the complex architecture of exosomes in terms of specially sorted proteins, lipids, nucleic acids, and respective content that highly dependent on the status quo of the cell type of origin.

A large variety of constitutive elements have been identified in exosomes from different cell types, including approximately 4400 proteins, 194 lipids, 1639 mRNAs, and 764 miRNAs, which illustrate their complexity and potential functional diversity [3031].

Typically, exosomes are highly enriched in proteins with various functions, such as tetraspanins (CD9, CD63, CD81, CD82), which take part in cell penetration, invasion, and fusion events; heat shock proteins (HSP70, HSP90), as part of the stress response that are involved in antigen binding and presentation; MVB formation proteins that are involved in exosome release (Alix, TSG101); as well as proteins responsible for membrane transport and fusion (annexins and Rab) [32].

Among these proteins, certain members participate in exosome biogenesis (Alix, flotillin, and TSG101), rendering exosomes distinct from the ectosomes released upon plasma membrane shedding, while others specifically enriched in exosomes are widely used as exosomal marker proteins (e.g. TSG101, HSP70, CD81, and CD63). A detailed summary of protein components found in exosomes is shown in Table 2.

Aside from selected proteins, exosomes also contain different patterns of RNAs that can be incorporated into recipient cells.

RNA sequencing analysis showed that microRNAs (miRs) were the most abundant in human plasma derived exosomal RNA species, making up over 42.32% of all raw reads and 76.20% of all mappable reads [33].

Other RNA species including ribosomal RNA (9.16% of all mappable counts), long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%).

Once miRs are packed into exosomes, they can undergo unidirectional transfer between cells, resulting in the establishment of an intercellular trafficking network, which, in turn, elicits transient or persistent phenotypic changes of recipient cells [8].

MiRs, such as miR-214, miR-29a, miR-1, miR-126, and miR-320, which participate in angiogenesis, hematopoiesis, exocytosis, and tumorigenesis, have already been reported in exosome-based cell to cell communication [34].

Interestingly, besides miRs, long RNA species, especially long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) have recently been reported to be existed in exosomes, and impact a variety of biological processes including the development of cancer [35].

They may function together to transduct cell signals so that local cellular microenvironments will be altered or maintained.

Early in 2013, Kogure and his group identified several dramatically altered lncRNAs in human hepatocellular cancer (HCC) cell-derived exosomes [36].

Among them, the novel lncRNA TUC339 was the most highly significantly expressed one, which was functionally implicated in modulating tumor cell growth and adhesion.

Thus, they prompted that exosomes-mediated transfer of intercellular functionally active lncRNA as a mechanism of intercellular signaling in HCC. Later, Alice et al. reported that lncRNA H19 could be packaged inside CD90+Huh7 cells-derived exosomes, and be delivered to endothelial cells (ECs), influencing ECs in a pro-metastatic way via the exosome-mediated vascular endothelial growth factor (VEGF) increase [37].

These studies indicate that exosome-mediated transfer of lncRNA is an important mechanism existed in tumor development, and play a crucial role in regulation of the tumor microenvironment via influencing major cellular pathways.

Other lncRNAs transmitted by exosomes include lncRNA CRNDE-h in colorectal cancer [38], lncARSR in sunitinib resistance of renal cancer [39], lncRNA Hotair in rheumatoid arthritis [40], lincRNA-p21 and ncRNA-CCND1 in bleomycin-induced DNA damage [41].

CircRNAs were demonstrated high stability, and were not susceptible to exonuclease cleavage, which proposed a possible tumor diagnostic marker. In 2015, Li et al. identified existence of circRNAs in exosomes through RNA sequencing analyses of hepatic MHCC-LM3 cancer cells and cell-derived exosomes [42].

In a comparison of healthy donors and colorectal cancer patients, 67 circRNAs were missing and 257 new circRNA species were detected in cancer patients. Further overexpression of miR-7 in HEK293T cells and MCF-7 cells showed significant downregulation of circRNA CDR1as in exosomes, suggesting that the process of circRNAs entering exosomes may be regulated by intracellular miRs. In a recent study, Exosomal circRNA_100284 derived from arsenite-transformed cells, has been reported to promote malignant transformation of human hepatic cells, via regulation of EZH2 by miR-217 [43].

The bioactivity of exosomes exists not only in their proteins and nucleic acids, but also in their lipid components. Generally, exosomes are enriched in phosphatidylserine (PS), phosphatidic acid, cholesterol, sphingomyelin (SM), arachidonic acid and other fatty acids, prostaglandins, and leukotrienes, which account for their stability and structural rigidity (listed in Table 3).

Moreover, exosomes also have some functional lipolytic enzymes, which can produce units of various bioactive lipids autonomously.

These exosomal bioactive lipids may be internalized into recipient cells, concentrating lipid mediators within the endosomes.

Evidence has revealed that accumulation of prostaglandins and fatty acids brought by exosomes during 1 h can result in micromolar concentrations for prostaglandins, and millimolar concentrations for fatty acids, which are enough to trigger prostaglandin-dependent biological responses [44].

Meanwhile, exosomal lipids may interact with lipid transfer proteins in recipient cytosolic, such as fatty acid binding proteins (FABPs), or receptors such as PPARg for the 15dPGJ2, for conformation of the cytosolic complex AA/FABP/PPARg [45], which will be further addressed to the nucleus.

As a result, the exosome derived 15d-PGJ2 and PGE2 may provide a natural way to supply intracellular PGE2. The endosomal AA brought by exosomes can be delivered to the PGE synthase and COXs present in endoplasmic reticulum for an additional PGE2 synthesis.

Similarly, the unsaturated 22:6 fatty acid DHA released from exosomes can potentially be addressed to the microsomal antioestrogen binding site in the recipient endoplasmic reticulum [46] and inhibit the activity of the cholesterol epoxide hydrolase which cleaves the cholesterol 5,6 epoxide into cholestanetriol [47].

Since exosomes can be released and taken up by target cells to modulate cell lipid metabolism, exosome-mediated intercellular lipid exchange should be taken into account in the pathogenesis of cholesterol-related storage disease, such as atherosclerosis.

Internalization of T cell-derived exosomes by monocytes via the PS receptor has been shown to effectively facilitate cholesterol accumulation into lipid droplets, implying a role of exosomes in atherosclerosis development [20].

Since exosomes have been shown to play a role in lipid-related pathologies, the lipid content of exosomes may act as another molecular signature for disease diagnosis and prognosis, in addition to protein and RNA biomarkers.

Exosome-mediated intercellular communication

Traditionally, cells communicate with neighboring cells through direct cell–cell contact including gap junctions, cell surface protein/protein interactions, while communicating with distant cells through secreted soluble factors, such as hormones and cytokines, to facilitate signal propagation [48].

Moreover, electrical and chemical signals (e.g. nucleotides, lipids, and short peptides) are also involved for communication [49].

Interestingly, it is now recognized that exosomes with a cell-specific cargo of proteins, lipids, and nucleic acids may act as a novel intercellular communication mechanism.

This concept is based on the observation that exosomes released from parental cells may interact with target cells, leading to the subsequent influence of target cell behavior and phenotype features [50].

The success of exosomal biological applications is highly dependent on effective delivery of genetic materials, which can be achieved via receptor-ligand interactions, direct fusion of membranes, or internalization via endocytosis [51].

Once internalized, exosomes may fuse with the limiting membrane of endosomes, resulting in the horizontal genetic transfer of their content to the cytoplasm of target cells.

The bioactive molecules contained in exosomes have been shown to impact target cells via the following mechanisms:

(1) direct stimulation of target cells via surface-bound ligands;

(2) transfer of activated receptors to recipient cells; and

(3) epigenetic reprogramming of recipient cells via delivery of functional proteins, lipids, and RNAs [52] (Fig. 1).

As a result, parental cells can communicate with specific proximal or distal target cells through exosome amplification.

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Fig. 1
The schematic diagram of pathways involved in exosome mediated cell-to-cell communication. (1) Exosomes signal recipient cells via direct surface-bound ligands. (2) Exosomes transfer activated receptors to recipient cells. (3) Exosomes may epigenetically reprogram recipient cells via delivery of functional proteins, lipids, and RNAs

In immune system, exosomes have an important function in immunoregulation, including antigen presentation, immune activation, immune suppression, and immune tolerance via exosome-mediated intercellular communication.

Exosomes derived from CD4+ T cells and CD8+ T cells can bind to dendritic cells (DCs) through peptide/major histocompatibility complex MHC/TCR and ICAM-1/LFA-1 interactions, which lead to the apoptosis of DCs and thus mediate the DC-mediated T cell silence in antigen-specific way [53].

Exosomes secreted by regulatory T cells contain Let-7b, Let-7d, and microRNA-155, which are able to inhibit Th1 immune response and mediate immune suppression [54]. In addition, CD73-expressing Treg-derived exosomes can produce adenosine which may further inhibit the activation and proliferation of CD4+ T cells [5556].

Meanwhile, B lymphoblast-derived exosomes have also been shown to induce human and mouse-antigen specific T cell activation, for the presence of MHC–peptide complexes, and even co-stimulatory molecules on them [57].

DCs are professional antigen presenting cells with the unique capacity to induce primary and secondary immune responses.

It has been reported that exosomes derived from DCs pulsed with tumor peptide can eradicate or suppress growth of established murine tumors via presentation of class II-peptide complexes to naive T lymphocytes and the priming of specific cytotoxic T lymphocytes in vivo [58].

Mesenchymal stem cells (MSCs) derived exosomes are prompted to be the main effects on wound healing.

The therapeutic capacity of MSC-‘exosomes’ derived from different organs, have been tested in various disease models, demonstrating a similar or even superior functional capacity to MSCs themselves, such as reducing myocardial infarction size [5960], preventing adverse remodeling after myocardial ischemia/reperfusion injury [61], providing therapeutic effects in cutaneous wound healing [62], acute kidney injury [63], hepatic injury [6465], neonatal lung injury [66], promoting survival of retinal ganglion cells in optical nerve crush [67], ameliorating retinal laser injury [68] and orchestrating neurological protection by the transfer of miRs [69].

Surprisingly, in contrast with wide spread cell origin, exosomes do not randomly interact with any recipient cell that happens to be in the vicinity.

They may display distinct tissue/cell homing, probably on account of their high expression levels of adhesion molecules, such as integrins and tetraspanins, and their potential target capability [70]. Hence, the selective transmission of exosomal genetic information makes them attractive candidates for the diagnosis and treatment of diseases.


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