Exosomes are membrane-enclosed vesicles that are derived from the endocytic compartment and released at the plasma membrane into the extracellular space. The plasma membrane is not the source of the lipid bilayer of exosomes; rather, exosomes originate from luminal pinocytosis of early endosomes. These vesicles range from 30 nm to 100 nm in diameter and traffic cargo in an autocrine, paracrine, and endocrine fashion. Exosomes contain a subset of biologically active macromolecules present in the cell: protein, lipids, and multiple RNA species. The composition of exosomal cargo is unique to the producing cells, giving healthy and diseased cells a specific exosomal signature. The trafficking of these molecules into neighboring cells alters their behavior, a process involved in neuronal signaling, fetal development, tissue homeostasis and repair, adaptive immunity, and cancer progression.
In contrast to the shedding of microvesicles which bud at the plasma membrane, exosomes are contained as vesicles within endosomal compartments, termed multivesicular bodies (MVB). Their unique biogen- esis is also reflected in their lipid composition resembling that of the early endosomes. Exosomes originate as inward buddings of the endosomal membrane to create intraluminal vesicles (ILVs). ILVs accrue during the transition from early to late endosomes, also known as multivesicular bodies. Move- ment toward the plasma membrane is controlled by the cytoskeleton and small GTPases. Endosomes move along microtubule tracks via molecular motors dictated by Rab GTPases and the phosphoinositide profile on the outer lipid leaflet of the vesicles. Secretion occurs when endosomes fuse with the plasma membrane and release their exosomes. This allows the cell to manage exosomal output in a temporally and spatially controlled fashion by using multiple cytoskeletal and membrane proteins to mediate fusion and secretion. Subject to an active process, cells change both the output of exosomes and the cargo within them under various stress-inducing conditions.
Exosomes contain internal maturation proteins and the proteins bound for recipient cells within the lumenal compartment. To date, close to 4,600 different proteins have been shown to associate with exosomes. The cellular origin of exosomes accounts for the fact that many exosomal proteins are involved in endosomal pathways. The most common proteins include the tetraspanins CD9, CD63, and CD81, which act as protein scaffolds; flotillin, which aids in vesicle formation; Alix, an adaptor protein required for endosomal trafficking; and TSG101, a regulator of vesicular trafficking. These proteins are frequently used as markers for the classification as exosomes. Exosomes also contain cytoplasmic proteins that act in recipient cells. These include metabolic enzymes, signal transducers, and transcription factors.
One mechanism by which proteins are sorted into exosomes relies upon ESCRT (endosomal sorting complexes required for transport), a complex of proteins that coordinates both budding and the sorting of proteins into ILVs. This complex was initially described as being required for the sorting of ubiquitinated proteins destined for the lysosomal degradation, but it has recently been shown to sort proteins into exosomes. The posttranslational modifications that ESCRT recognizes for sorting are still unclear, but seem to be primarily orchestrated by a combination of the ubiquitin profile and association of other “guide” proteins. This recent work on sorting was performed on viral proteins and may vary for endogenous protein exportation.
Exosomes are enriched in cholesterol, ceramides, sphingomyelin, and saturated species of phosphatidyl- choline and phosphatidylethanolamine. The lipid composition of exosomes contributes to both ILV formation and trafficking within the cell of origin. In studies where components of the ESCRT complex are knocked out, ILVs are still generated in a mechanism that seems to be aided by the increased incorporation of ceramides, which increase membrane curvature. Sphingosine-1-phosphate was also shown to contribute to ILV formation, as a reduction in flotillin+ exosomes was observed in sphingosine kinase 2 knockout cell lines. Importantly, in contrast to microvesicles whose bilayer reflects that of the plasma membrane, exosomes contain additional lipid moieties of endosomal origin.
Deep sequencing of exosomes has revealed the existence of a vast array of RNA species in exosomes. Importantly, the RNA content of exosomes is not a proportional reflection of the transcriptome of the cell, but exhibits enrichment and exclusion of specific transcripts. The mechanism by which selection is accomplished remains under investigation, but may utilize miRNA sequence motifs. This “EXOmotif,” in tandem with a sumoylated heterogenous ribonucleoprotein A2B1 (hnRNPA2B1), selectively sorts miRNA into exosomes. Similarly, cis elements on mRNA transcripts have also been identified to be correlated with exosomal sorting. One study demonstrated interplay between miRNA and mRNA in the sorting efficiency of transcripts, and another study showed the presence of miRNA/RISC complex within exosomes. Currently, more research needs to be performed to elucidate these sorting mechanisms, but it is clear that RNA sorting is dependent upon both cis- and trans-elements.
Exosomes are relatively resistant to strong shearing forces and enzymatic degradation, making them suitable candidates for the delivery of fragile or cell impermeable molecules. Alternatively, exosomes in the extracellular space can traffic back to the cell of origin, neighboring cells, or into the bodily fluids for transmission to other organs and tissues. Several mechanisms have been described for cellular entry of exosomes. Recipient cells take up exosomes in a fashion similar to cell-cell adhesion, through the use of ICAMs and integrins. Surface receptors on the recipient cells recognize exosome proteins, glycoproteins, and lipid moieties, leading to cell-type specific trafficking. This is of particular importance in signaling within the immune system. For example, CD169+ macrophages recognize B-cell-derived exosomes particularly by the decoration of 2–3 linked sialic acid and glycoproteins containing specific mannose residues. This is important for communication between cells within the spleen and lymph nodes. Lectins on the surface of recipient cells contribute to preferential exosome uptake, as do clathrins, dynamin, and caveolae. Expression of these surface receptors modulates the specificity and affinity for exosome reception. Further, exosomes can either fuse with the recipient cell’s plasma membrane, releasing their cargo into the cytosol, or become engulfed and enter the endocytic pathway. Release of cargo into the cellular cytosol affects recipient cell behavior. Of recent interest within the exosome field is the trafficking of functional RNA molecules between cells. Messenger RNA transcripts generated in a donor cell have been shown to be translated to functional protein in recipient cells. Furthermore, miRNA generated in a producer cell can exhibit suppression on recipient cell gene expression. This type of cell-cell communi- cation contributes to tissue development and homeostasis and is especially important in the cross talk between the stromal and parenchymal components within organs.
Exosomes in cancer
The development of cancer is a dynamic process, and exosomes contribute to a variety of events that enable cancer to progress. Almost all tumor types appear to exhibit an increase in exosomal output upon transformation. The cell-autonomous accumulation of mutations and the subsequent signaling dysregulation allow tumors to overcome cellular checkpoints and growth restrictions, whereas intercellular signaling shapes their microenvironment by manipulating neighboring cells and tissue. Exosomal-mediated cell signaling appears to contribute to a tumor-proliferative microenvironment through interactions with neighboring stroma and via immune evasion.
Tumors are capable of transforming their surroundings into a state that supports malignancy. This includes modulation of the extracellular matrix (ECM) and induction of angiogenesis. ECM is altered in part by the manipulation of fibroblasts near the tumor. These cells are referred to as cancer-associated fibroblasts (CAF) and remodel the architecture and composition of surrounding ECM to promote metastasis and vascularization. Exosome-mediated transport of the signaling molecules TGF-b1 and FGF-2 contributes to the altered fibroblast phenotype. Multiple studies have also shown that exosomal output of stromal cells is altered in a tumor environment, which reinforces tumor proliferation by providing growth factors and signals back to the tumor cells. Angiogenesis is the process by which vascularization is introduced to a tumor. This process is required for solid tumors, as their size and growth rate require the delivery of oxygen to counteract their hypoxic environment. Hypoxia and nutrient depletion provoke the release of exosomes containing angiogenic miRNA and stimulatory signaling molecules that induce the neovascular formation of blood vessels in the tumor.
While the tumor microenvironment contains cells from both the innate and adaptive arms of the immune system, most tumors are able to suppress the local immune response. The communication between immune cells, mediated by cellular contact or the release of chemokines and cytokines, establishes the balance between pro- or anti-inflammatory responses. Exosomes influence this equilibrium through several mechanisms. Exosome-mediated immune suppression was shown in experiments demonstrating increased tumor proliferation and decreased immune response when transplanted tumors were accompanied by injections of exosomes derived from the same tumor. Candidate mechanisms in support of this observation include delivery of proapoptotic ligands to reduce tumor infiltrating lymphocytes in the microenvironment, delivery of anti-inflammatory cytokines, and induction of regulatory T cells that increase the immune tolerance of the tumor.
The unique content of tumor-derived exosomes allows for the use of these vesicles as biomarkers for the detection cancer relapse. As they equilibrate with the bloodstream, circulating exosomes provide a minimally invasive source of substrate for cancer detection from solid tumors and hematologic malig- nancies. Sampling of body fluids (e.g., blood, urine, saliva, CSF) provides minimally invasive sources of exosomes, allowing more frequent screening and potentially earlier detection. Recently, the exosomal miRNA profile has provided a promising platform for disease surveillance, as this RNA population shows increased enrichment in cancer-specific transcripts. Accordingly, the utility as biomarkers is not limited to cancer, but includes infectious disease and degenerative neurological conditions.
Lцtvall J, Hill AF et al (2014) Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3. doi:10.3402/jev.v3.26913
Mittelbrunn M, Vicente Manzanares M, Sбnchez-Madrid F (2015) Organizing polarized delivery of exosomes at synapses. Traffic. doi:10.1111/tra.12258
Roma-Rodriguez C, Fernandes AR, Baptisa PV (2014) Exosome in tumour microenvironment: overview of the crosstalk between normal and cancer cells. Biomed Res Int. doi:10.1155/2014/179486. Epub 2014 May 21
Villarroya-Beltri C, Baixauli F et al (2014) Sorting it out: regulation of exosome loading. Semin Cancer Biol (28) doi:10.1016/j.semcancer.2014.04.009