The biology of extracellular microvesicles

Abstract

The study of extracellular vesicles (EVs) is a rapidly evolving field, owing in large part to recent advances in the realization of their significant contributions to normal physiology and disease. Once discredited as cell debris, these membrane vesicles have now emerged as mediators of intercellular communication by interaction with target cells, drug and gene delivery, and as potentially versatile platforms of clinical biomarkers as a result of their distinctive protein, nucleic acid and lipid cargoes. While there are multiple classes of EVs released from almost all cell types, here we focus primarily on the biogenesis, fate and functional cargoes of microvesicles (MVs). MVs regulate many important cellular processes including facilitating cell invasion, cell growth, evasion of immune response, stimulating angiogenesis, drug resistance and many others.

1 |. INTRODUCTION

Extracellular vesicles (EVs) comprise a varied and heterogeneous group of particles released from cells originating largely from endosomes and/or the plasma membrane.^1,2 Initially described as the disposal mechanisms used by cells to discard unwanted material, ensuing research has shown that EVs are important mediators of intercellular communication in normal physiology and pathophysiology.^3–7 Recent studies have shown that EVs contain various proteins, lipids, glycolipids, glycoproteins and nucleic acids including DNA, mRNA and noncoding RNAs.^8–18 Thus, EVs have the potential to deliver multiplexed information to surrounding tissues and systemically through the body.

The prevalence of EVs in disease has made them an attractive focus for the development of diagnostics and as a noninvasive or minimally invasive screening tool for detecting pathology in diseases such as cancer,^19,20 diabetes^21,22 and cardiovascular disease.^23,24 EVs have been detected in multiple body fluids including urine, saliva and blood, making them readily available for analysis.^22,25–27 Efforts have also been directed at investigating the therapeutic potential of EVs, both by preventing their formation,²⁸ and as tools to package and deliver disease modulating proteins or drugs, for enhanced uptake by target cells. This could be in the form of harvested vesicles with innate therapeutic properties,^29–31 vesicles which are harvested from cells which have been altered to enhance the presence of particular cargo,^32–34 or vesicles which are loaded with cargo artificially, after their isolation from cells.^33–35 Despite intense research in the biology and therapeutic implications of EVs in recent years, categorization of these vesicles into various subtypes is still rather tenuous. Here, we will describe this expanding and complex field of research by first outlining the subtypes of EVs with subsequent focus primarily on the biogenesis, cargo and fate of 1 EV subtype, namely, microvesicles (MVs).

2 |. TYPES OF EXTRACELLULAR VESICLES

Currently assigned into the broad categories of exosomes, MVs, or apoptotic bodies based upon their mechanism of formation, mode of release from cells, and size² (Table 1), these classifications somewhat belie the heterogeneity that may exist within vesicle classes in terms of both cargo and functionality.³⁶ Exosomes, the best characterized of the EV subtypes, are formed first as intraluminal vesicles within a multivesicular body (MVB), which are released into the extracellular space upon MVB fusion with the plasma membrane.³⁷ MVs, sometimes referred to as ectosomes or microparticles, are formed by outward blebbing of the plasma membrane and subsequent fission of plasma membrane blebs.² MVs derived from human cancer cells (Figure 1A) have received a good deal of attention because of their ability to participate in the horizontal transfer of signaling proteins between cancer cells and to contribute to their invasive activity. Apoptotic bodies are formed during cellular blebbing and fragmentation upon cell death² (Figure 1B), and their impact upon other cells is not well-studied. Vesicles may also be released from nanotubular structures radiating from the plasma membrane.³⁸ At present it appears that most, if not all, cell types form EVs,^25,39 however, in general, cell stress and disease frequently upregulate their formation and alter the cargo contained within.⁴⁰

TABLE 1.

Characteristics of natural and synthetic biological vesicles

Vesicle type	Size	Origin	Function
Nanovesicle	8–12 nm	• Unknown at present	• Intercellular communication • Promote cancer progression
Exosome	50–100 nm	• Intraluminal vesicles of multivesicular body • Released upon MVB fusion with plasma membrane	• Intercellular communication in healthy and diseased cells • Condition/educate the extracellular microenvironment • May promote disease pathogenesis
Microvesicle	200–1000 nm+	• Direct budding from plasma membrane • Released via acto-myosin-driven fission into extracellular space	• Intercellular communication in healthy and diseased cells • Condition/educate the extracellular microenvironment • May promote disease pathogenesis
Apoptotic body	500 nm+	• Random plasma membrane blebs of cytoplasm and organelle fragments	• Breakdown of apoptotic cells • Potential intercellular signal transduction
Large oncosome	1–10 μm	• Direct budding from plasma membrane	• Intercellular communication sent from cancer cells • Condition/educate the extracellular microenvironment • May promote disease pathogenesis
Giant plasma membrane vesicle	1 μm+	• Formed by artificially stimulating cells to shed blebs of lipid-rich plasma membrane	• Study plasma membrane protein behavior • Study plasma membrane lipid organization
Uni- and multilamellar vesicles	Unilamellar: Small: <100 nm; Large: 100–400 nm; Giant: 1–100 μm; Multilamellar: Small: 80–120 nm; Large: 200–3000 nm	• Synthesized in vitro as mixtures of cholesterol and phospholipids in an organic solvent. May require sonic or mechanical processing to form the single lipid bilayer (unilamellar) or multi-bilayered (multilamellar) vesicles	• Study properties of the plasma membrane such as lipid rafts and phase separation • Delivery of therapeutics

FIGURE 1.

Micrographs of EVs. A, Microvesicles (arrows) are shed by invasive LOX melanoma cells invading fibrillar collagen matrix (confocal reflection of matrix shown as red). Cells were labeled for β1 integrin (green). B, EVs (arrows) released by melanoma cells following apoptosis induction with hydrogen peroxide. Cells were labeled for β1 integrin in red, and cleaved caspase-3 is shown in green

Overall, EVs comprise a wide spectrum of vesicles ranging from 8 nm to several microns. MVs generally range from 200 nm to several microns in diameter, whereas exosomes are smaller, and range from 50 to 100 nm in diameter.^2,41 Nanovesicles ranging from 8 to 12 nm in peripheral blood have also been recently described,⁴² and these very small vesicles may be errantly isolated with exosomes using many of the currently utilized isolation protocols. Due to the considerable heterogeneity in the size of MVs, there are likely to be subpopulations within this category of vesicles. The term “oncosomes” has been used to describe MVs or even all EVs that have been released from cancer cells.⁴³ However, of note, large oncosomes (LO) refer to a subtype of plasma membrane-derived MVs, 1 to 10 μm in diameter, which are released from tumor cells.^44,45 Nomenclature, as well as sizes attributed to various EV subtypes, has not been without debate.^46,47 Complicating this matter is the fact that isolation methods for EV subtypes are still in development and consensus best-practices are not yet reached, leading to differences in the populations of vesicles being evaluated between studies.^36,48 As a result of the lack of clear distinction between EV subtypes, in many studies collective EV populations have been utilized. For the purposes of this review, we use the term “extracellular vesicles” for all studies that have not been attributed to a given population or when the isolation methods suggest that multiple subtypes were likely being investigated. We have attributed characteristics to “microvesicles” if the methodologies for vesicle isolation have enriched for the MV fraction.

It is likely that mode of biogenesis and EV cargo will play a critical role in defining criteria for classification. In this vein, recently, subpopulations of EVs have been described by characterization of cargo, size and density,^36,49,50 as well their effects upon recipient cells.⁴⁹ It is possible, if not likely, that as the field moves forward additional vesicle classes, or modifications to the current classification systems, will emerge.

Although not classified along with the above, additional vesicle types have been described primarily as research tools to study lipid and protein behavior. These may be formed upon artificial stimulation of cells, as in giant plasma membrane vesicles (GPMVs), which are formed upon the treatment of live cells with stimuli such as laser irradiation, salt treatment or the chemicals paraformaldehyde and dithiothreitol.^51–53 This results in the formation of large (micron sized) vesicles which are frequently used to study lipid and protein organization at the plasma membrane^52,54,55 (Figure 2). Additional vesicle types are prepared fully synthetically in vitro, as in small, large and giant uni- and multilamellar lipid vesicles,⁵⁶ including variants referred to as artificial plasma membrane vesicles.⁵⁷ These synthetic vesicles have also been used for therapeutic purposes, providing improved stability and uptake of drugs and nucleic acids,^58,59 likely exploiting the same mechanisms utilized by naturally-derived vesicles.

FIGURE 2.

Artificially-stimulated EVs. Phase contrast image of GPMVs (arrows) formed by live HeLa cells after stimulation with paraformaldehyde and dithiothrietol. Scale bar is 20 μm

3 |. MICROVESICLE BIOGENESIS

How cargo is trafficked to MVs is still an area of ongoing investigation, and is less well characterized relative to exosomes. However, several mechanisms of cargo trafficking have emerged from investigations in tumor cells. In general, MVs appear to form by the outward pinching of the plasma membrane. ARF6-positive recycling vesicles have been found to traffic cargo to the cell surface for incorporation into MVs.³ The v-SNARE VAMP3 has been shown to regulate the delivery of cargo such as MT1-MMP to facilitate matrix proteolysis during tumor cell invasion, in a process dependent on the association of VAMP3 with CD9.⁶⁰

The biogenesis and release of MVs from the cell may be regulated by multiple mechanisms. MVs are generated from sites of high membrane blebbing,^2,45 and their formation is stimulated in cells invading through compliant matrices.⁶¹ Interestingly in this regard, cells invading more rigid matrices frequently utilize a different invasive structure termed invadopodia at their ventral surface to mediate matrix proteolysis, and studies have indicated that exosomes are released at invadopodia to facilitate invasion,⁶² with the integral invadopodia protein cortactin regulating the trafficking and docking of MVBs.⁶³ Plasma membrane bleb formation is regulated by a host of factors which modulate deformability and bending of the membrane, including the lipid composition and organization of the peripheral cytoskeleton, to alter membrane fluidity and deformability. MVs display unique lipid characteristics, such as the externalization of the phospholipid phosphatidylserine (PS),⁶⁴ which has been demonstrated to promote uptake by recipient cells.⁶⁵ The phospholipid lysophosphatidylcholines, the sphingolipid sphingomyelins, and acylcarnitines, the fatty acyl esters of L-carnitine, have also been found to be enriched in MVs.⁶⁶ The prominent plasma membrane lipid cholesterol is thought to play an important role in MV formation, as its depletion reduces MV formation.⁶⁷ Ceramide, a cone-shaped lipid which promotes membrane bending, has also been shown to regulate MV formation.⁶⁸ Once loaded with cargo, the bleb must be pinched free from the cell in a process governed by acto-myosin contraction. This requires a tightly regulated balance of cytoskeletal elements which promote or suppress the required blebbing and pinching. Rho family GTPases have emerged as critical mediators of MV formation, which is not surprising, given their integral roles in regulating actin reorganization and cortical contractility.^61,69 RhoA activity has been demonstrated to promote MV formation, through the downstream kinases ROCK (Rho-associated coiled-coil containing kinases) and ERK (extracellular signal-regulated kinases).⁶¹ In tumor cells this is antagonized by an elevation in Rac1 activity when cells encounter firm extracellular matrix conditions that are not conducive to MV-mediated invasion. Activation of myosin light chain (MLC) at MV “necks” is required for MV fission into the extracellular space, and this is regulated by increased activity of MLC kinase and the suppression of MLC phosphatase,^3,61 mediated by RhoA activity, to promote enhanced myosin contractility. Cofilin phosphorylation downstream of Rho also promotes MV generation.⁶⁹

Several additional mechanisms have been shown to affect MV formation. The extracellular concentration of calcium can impact MV formation, with increased calcium eliciting increased vesiculation.⁷⁰ This is particularly interesting in light of the fact that calcium signaling is frequently dysregulated in cancer,^71,72 and multiple oncogenes and tumor suppressors impact calcium regulation.⁷² Increased calcium levels have been demonstrated to induce membrane phospholipid scrambling and increased MV formation in erythrocytes and platelets,⁷³ and a similar mechanism may be at work in cancer cells. G-protein coupled receptor 30 (GRP30) has been shown to stimulate the formation of EMMPRIN (extracellular matrix metalloproteinase inducer)-containing MVs from uterine cells,⁷⁴ which may contribute to tumorigenesis or cancer progression. Hypoxia, a common occurrence within solid tumors which is associated with tumor progression and therapy resistance,⁷⁵ has also been shown to promote MV release via a cellular process mediated by hypoxia-inducible factors (HIFs) and Rab22a.⁷⁶ Actin deimination, whereupon protein arginine deiminases (PADs) catalyze the hydrolysis of peptidyl-arginine to peptidyl-citrulline, has also been associated with facilitating MV biogenesis.⁷⁷ Additional work has shown a role for the ESCRT protein TSG101, in concert with arrestin domain-containing protein 1 - (ARRDC1), in regulating MV formation at the plasma membrane, in a process that also requires the VPS4 ATPase for vesicle release.⁷⁸

Suppression of the actin-nucleator diaphanous-related formin 3 - (DIAPH3) enhances formation of LO.⁷⁹ Interestingly in this regard, analysis of primary and metastatic human prostate tumors reveals a significantly higher frequency of deletion of the locus encoding DIAPH3 in metastatic tumors in comparison to organ-confined tumors. Additionally, the activation of EGFR (epidermal growth factor receptor),⁷⁹ overexpression of membrane-targeted Akt1,⁷⁹ caveolin-1 overexpression,⁴⁵ and stimulation with heparin-binding EGF (epidermal growth factor)-like growth factor combined with p38MAPK inhibition⁸⁰ have also been found to stimulate LO formation.

It is important to mention that there is overlap in the molecules which mediate the biogenesis of multiple vesicle types, best studied in exosomes and MVs. In these populations, proteins including ARF6 and ESCRT complex components such as TSG101, and lipids such as ceramide, have been shown to regulate the formation of both vesicle types,^{3,27,49,66,68,78,81} indicating that there may be coupling between the biogenesis of different vesicle families. Further work is required to determine if the aforementioned regulators work in concert or if the cellular machinery engaged is dependent on the content of the EVs being released.

4 |. UPTAKE OF EVS INTO CELLS

Modes of MV cargo uptake by recipient cells include simple plasma membrane-EV fusion with direct cargo deposition into the cytoplasm, and the uptake of intact vesicles via multiple mechanisms for trafficking to an endosomal or lysosomal compartment.^82–84 EV uptake by recipient cells is carried out by a variety of modes, including clathrin and caveolin-mediated endocytosis,⁸⁵ lipid raft endocytosis,^86,87 phagocytosis⁸³ and micropinocytosis,^85,88 likely guided by vesicle membrane composition and surface protein profile.⁸³ Further, low pH as provided by tumor microenvironments would be conducive to the fusion of MV membranes with recipient cells. Studies have shown that the cellular uptake of MVs is enhanced by the inclusion of a pH-sensitive peptide to promote membrane fusion of MV and endosomal membranes upon internalization.⁸⁹ Low pH has also been shown to promote exosome release and uptake,⁹⁰ indicating that this may be a conserved EV characteristic. Recent studies have indicated an importance for actin polymerization and dynamin, a key protein involved in membrane budding and fission, in macropinocytic-like MV uptake by alveolar epithelial cells.⁸⁸ An important regulator in EV internalization is heparan sulfate proteoglycans (HSPGs), and treating EVs with heparin reduces their uptake.^91,92 Blocking the scavenger receptor type B1 (SR-B1), a receptor for HDL (high-density lipoproteins) which mediates the uptake of extracellular material, with HDL nanoparticles also blocks exosome uptake by altering lipid raft composition and SR-B1 clustering,⁹³ and similar mechanisms may regulate MV uptake as well. It appears that the mode of uptake is likely dependent on the cell type and the ligands on the recipient cells, although much remains to be learned in this regard. Interactions of EVs with target cells have been shown to trigger a host of cellular signaling cascades, and their uptake in recipient cells may facilitate transfer of functional cargo or its degradation.⁸³

5 |. FUNCTIONAL CARGOES IN MICROVESICLES DERIVED FROM TUMOR CELLS

The cargoes contained within EVs reflect both the intracellular origin of the cargo as well as the cell type from which the vesicle was derived. We refer readers to the many profiling studies which have been published on this subject for further information.^{36,66,94–100} Prototypical exosome markers include tetraspannins such as CD9, CD63 and CD81, and ESCRT proteins Alix and TSG101.^37,101 Unique and universal MV markers are somewhat less well-defined. At present they are best studied in tumor cells, and frequently include selectins, flotillin-2, ARF6 and CD40.^3,102 As indicated above, an ever-evolving body of work reveals that many of these markers may not be exclusive to a particular class of vesicle,³⁶ underscoring the heterogeneity of EVs and the potential overlap in functions between vesicle classes.

In normal cells, EVs have been implicated in regulating many vital physiological activities, ranging from the maintenance of stem cell plasticity,¹⁰³ communication between mother and fetus during pregnancy,¹⁰⁴ and blood clotting¹⁰⁵; however, it is their role in disease which has garnered the most attention. MVs have been shown to play a role in a wide variety of pathologies, including cardiovascular disease,^24,106–111 diabetes,^{21,107,111–113} and neurodegeneration.^68,114–116 The functional roles of EV cargoes have been best characterized in cancer progression and the rest of this section will focus on tumor-derived EVs. EV cargo from invasive tumor cells contains cargoes such as matrix metalloproteases to enable extracellular matrix proteolysis and subsequent cell invasion.^2,62,64 Cancer cell-derived EVs have also been characterized to contain extracellular matrix proteins such as fibronectin, which may mediate interaction between the vesicles and target cells,¹¹⁷ as well as facilitate anchorage-independent growth in recipient cells,¹¹⁸ and promote the induction of pro-inflammatory cytokines in immune cells.¹¹⁹ Fibronectin in exosomes has been shown to be an important cargo to promote directional motility and chemotaxis in tumor cells,^120,121 and the abundance of fibronectin on EVs in the plasma of individuals with breast cancer has indicated its potential as a diagnostic biomarker.¹²²

The chemoattractant potential of EV cargoes provides the potential for invasive cells to deposit cues to facilitate the directed movement of subsequent cells, and the recruitment of other associated cell types such as stromal and endothelial cells.¹²³ Oncogenic cargo, such as the mutant EGFR variant EGFRvIII can stimulate transformation of naïve cells.¹²⁴ Notably absent from tumor cell MVs are cortactin and Tks5,² prominent markers of the aforementioned invadopodia invasive structures.

One of the key roles of MVs in tumor progression is the ability to alter the behavior of other cell types to facilitate tumor cell survival. In this regard, MVs may facilitate tumor progression by directing cytotoxic cargo toward circulating immune cells, as demonstrated by melanoma cells inciting apoptosis in lymphocytes via proapoptotic Fas ligand containing MVs.¹²⁵ A similar effect was induced by melanoma FasL and TRAIL-containing MVs on T-cell blasts,¹²⁶ again promoting tumor cell survival. MiR-214 has been shown to be released in tumor cell MVs, resulting in downregulation of PTEN in T regulatory cells while promoting Treg expansion and host immune tolerance.¹²⁷ MVs isolated from esophageal cancer cells have been shown to promote the differentiation of naïve B cells into TGFβ-producing regulatory B cells, which suppress immunity derived from CD8+ T cells.¹²⁸ MVs have also been shown to promote angiogenesis, carrying cargo such as TGFβ, stimulating migration, proliferation and tube formation in endothelial cells.¹²⁹ A recent study similarly found that MVs released from oral cancer cells stimulate angiogenesis via sonic hedgehog and RhoA.¹³⁰

Furthering the profound implications of MVs in tumor survival and progression are their roles in drug resistance. MVs have been noted to mediate the efflux of chemotherapeutics,^28,131 promoting cell survival, and the number of MVs formed upon drug treatment has been shown to correlate with their chemotherapeutic resistance.¹³¹ MVs have also been demonstrated to transfer the drug resistance molecule p-glycoprotein in ovarian cancer cells, mediating drug-resistance in previously drug-sensitive cells.¹³²

Additionally, MVs are thought to prepare a niche for metastatic tumor cells, allowing for the colonization of a secondary site. This can be accomplished utilizing a complex set of cargo, including proteases for matrix degradation and impairment of vascular integrity, molecules which alter stromal stiffness and organization, deposition of chemoattractant molecules, angiogenic stimulation and changes in immune cell behavior.^133–136

EVs, including MVs, have been characterized to contain a broad range of nucleic acids, including nuclear and mitochondrial DNA, mRNA and noncoding RNAs such as miRNA, circular RNA and long noncoding RNA.^8–17 This presents the potential for wide-ranging implications in cancer as a potential efflux mechanism for “unwanted” nucleic acids as well as to transfer DNA and RNA which directly support oncogenesis and tumor progression. Packaging within EVs likely provides protection from degradation as the cargo is shuttled from the cell of origin to the recipient, as well as enhanced uptake by targeted cells.¹³⁷ Potential diagnostic implications of this nucleic acid content are revealed in multiple cancers. miRNA expression has been noted to be altered in the EVs isolated from a host of cancer types, including lung, prostate, pancreatic, colorectal, melanoma, glioblastoma, hepatocellular, leukemic and ovarian.¹³⁸ For example, in chronic myelogenous leukemia, an upregulation of miRNAs associated with tumorigenesis was noted in blast-derived MVs.¹³⁹ The therapeutic use of nucleic acids in EVs is also an area of investigation, to evaluate their potential as a tool for the delivery of gene therapy.¹³⁷

A recent analysis of MVs in the blood of patients with locally advanced or metastatic lung, breast, colorectal or head & neck cancers indicated that the profile of the protein markers MUC1, EGFR and EpCAM correlated with tumor subtype, whereas the MMP-inducer EMMPRIN was increased regardless of tumor type and the increased abundance of EMMPRIN-positive MVs correlated strongly with poor overall survival.¹⁹ Another recent study demonstrated the differential phosphorylation of an array of proteins found in exosomes and MVs isolated from plasma in breast cancer patients as compared to healthy controls.¹⁴⁰ In plasma cell dyscrasias such as multiple myeloma, free antibody light chains in the blood are a prominent characteristic of disease, and these may be circulated through the bloodstream within MVs that also contain Hsp70, annexin V and c-src,¹⁴¹ presenting diagnostic possibilities and indicating the mode of cargo transfer between cells. Other studies have indicated that the overall prevalence of MVs in blood alone may be a diagnostic indicator, such as the increase in MVs that has been noted in multiple myeloma¹⁴² and in gastric cancer.¹⁴³

6 |. CONCLUDING STATEMENTS

Increasing evidence supports a role for MVs, as well as other types of EVs, in intercellular communication. This, coupled with the findings that EVs derived from many sites in the body can make their way into body fluids, have led to exciting possibilities of their use in diagnostic and therapeutic applications. However, there is need for general consensus on classification and content of these shed vesicles. Further research on biogenesis and cargoes of EVs will greatly facilitate more precise discrimination between EV populations and functional characterization of EV subtypes.