Current understanding of extracellular vesicle homing/tropism

Abstract

Extracellular vesicles (EVs) are membrane-enclosed packets released from cells that can transfer bioactive molecules from cell to cell without direct contact with the target cells. This transfer of molecules can activate consequential processes in the recipient cells, including cell differentiation and migration that maintain tissue homeostasis or promote tissue pathology. One controversial aspect of the EV’s biology that holds therapeutic promise is their capacity to engage defined cells at specific sites. On the one hand, persuasive studies have shown that EVs express surface molecules that ensure their tissue localization and enable cell-specific interactions, as demonstrated using in vitro and in vivo analyses. Therefore, this feature of EV biology is under investigation in translational studies to control malignancies and deliver chemicals and bioactive molecules to combat several diseases. On the other hand, some studies have shown that EVs fail to traffic in hosts in a targeted manner, which questions the potential role of EVs as vehicles for drug delivery and their capacity to serve as cell-free biomodulators. In this review, the biology of EV homing/tropism in mammalian hosts is discussed, and the biological characteristics that may result in their controversial characteristics are brought to the fore.

Introduction

The term extracellular vesicles (EVs) includes three major and distinct groups of small membrane-enclosed packets that contain bioactive molecules secreted by all cells. The different EVs are the products of distinct biogenesis schemes (1) (2),(3). Exosomes, or small EVs, range in size from 50 to 200 microns and are derived from intraluminal vesicles within multivesicular compartments that are in the endocytic pathway of cells; microvesicles range in size from 100 to 1,000 microns are formed by plasma membrane budding; apoptotic bodies range in size from 100 to 5,000 microns are formed by the disintegration of cells (4). Each subtype of EVs should be expected to be composed of unique molecular characteristics and functions; however, there is broad recognition that implementing a few standard protocols to isolate EVs based on their size and density is not adequate to yield homogenous populations of each sub-type. Recognition of this limitation in the isolation of EVs led the International Society for EVs (ISEV) to recomnd the use of “EV” as a broad classifier term for these types of vesicles (5, 6) Exosomes are often referred to as vesicles belonging to the group of small EVs(7), which is the term that will be used in this review. That said, there has been considerable interest in understanding the composition of EVs, the factors that influence the loading of EVs, and their functions (3, 8). Many studies on the composition of small EVs have shown an enrichment of defined membrane and cytosolic molecules from the endocytic pathway (5) (9). Membrane proteins in small EVs include MHC class II complexes (5), (9), members of the tetraspanin family (CD9, CD63, and CD81), endosomal sorting complex required for transport (ESCRT), and integrins (10), (11); membrane-associated molecules include Rab GTPases (Rab4, Rab11, and Rab27) (12), (13); cytoskeletal proteins including actin, tubulin, and cofilin; and cytosolic proteins including ALG-2-interacting protein X (Alix), tumor susceptibility gene 101 (TSG101) and heat shock proteins (e.g., Hsp70 and Hsp90). In addition, nucleic acids, including DNA, coding, and non-coding RNAs (mRNA, miRNA, circRNA, tRNA), and several categories of RNA binding proteins have been identified within the lumen of small EVs (14), (15), (16), (17). Together, these molecules can initiate, enhance, or inhibit cellular functions upon the delivery of EVs to recipient cells.

The functional role of EVs was discovered in several types of studies, including studies on the development of immune responses (18), (19) and studies that evaluated the therapeutic potential of adoptively transferring pluripotent cells to reconstitute impaired cellular functions (20), (21) or stromal cells (22). Examples include studies where the therapeutic effects of the transfer of mesenchymal stems cells (MSC) to mouse recipients were evaluated, and it was observed that factors derived from these cells, in the MSC-conditioned medium, had immunomodulatory, proangiogenic, and tissue-tropic activities that could substitute for the cells (23). Similar observations were made in studies on the potential of stromal cells to protect against tubular injury in a cisplatin-induced acute renal failure model (22). It was shown that conditioned media from cultured stromal cells induced migration and proliferation of kidney-derived epithelial cells. Moreover, intraperitoneal administration of this conditioned medium to mice injected with cisplatin diminished tubular cell apoptosis, increased survival, and limited renal injury. Together, such studies demonstrated that the cells’ secretome in the conditioned medium could serve as a cell-free therapeutic agent for treating inflammatory diseases (21).

Studies on the molecular composition of EV membranes

Like cells, EVs display molecules on their surface that can interact with cells and other surfaces in tissues. Molecules on the surface of EVs include lipids such as phosphatidylserine (PS), sphingomyelin, cholesterol and ceramides, carbohydrate moieties (24), (25) such as glycosphingolipid glycan groups (26), (27), and many types of membrane proteins. Some of these lipids are enriched in exosomes as compared to their parental cells, which was shown upon compilation of lipid data from several studies (24) Specifically, cholesterol, sphingomyelinases (SM), glycosphingolipids, and phosphatidylserine (PS) were found to be enriched by 2 to 3 times. These lipids can mediate binding of exosomes with recipient cells. For example, PS binds the immunomodulatory TIM-1 and TIM-4 (T-cell immunoglobulin- and mucin-domain-containing molecule) receptors on phagocytic cells(28), thus promoting selective interactions and uptake of EVs that express this molecule. It is noteworthy that the observed lipid content of EVs can inform on the purity of the preparation. Specifically, the presence of cardiolipin may suggest contamination of the preparation with lipids from internal organelles such as mitochondria (24). EVs are also enriched with a variety of membrane proteins. Some these proteins include selected cytokines and chemokines such as IL-8 and chemokine (C-X-C motif) ligand 1 (CXCL1) (29). Interestingly the cytokine incorporation in EVs including expression on the EV membrane is cell/tissue origin dependent(29). For example, most cytokines secreted by CD4+ T cells are within EVs as compared to placental villous tissue that preferentially secrete cytokines in free (soluble) form. Other surface molecules on EVs include the major histocompatibility antigens (MHC molecules) and accessory molecules necessary for antigen specific activation of both CD4+ and CD8+ T cells (30). The display of these molecules on EVs is also cell source dependent, which has been convincingly shown in studies of dendritic cells where EVs from mature DCs are relatively enriched for CD86 and intercellular adhesion molecule 1 (ICAM-1), whereas EVs from immature DCs are enriched with milk fat globule–epidermal growth factor–factor VIII (MFG-E8)(31).

Our understanding of how molecules are selected for packaging in EVs is incomplete. Insight on the selection mechanisms that dictate the presence of proteins in the delimiting membrane of EVs and the consequences of the differential expression of these proteins on EV functions has come from studies of tetraspanins and integrins. Tetraspanins are small transmembrane proteins that function in cell migration, signal transduction, and intracellular trafficking. Of the 33 tetraspanins in the human genome, more than a handful have been shown to localize to small EVs (32–34). These tetraspanins are often used as specific markers of exosomes (34), (35). Integrins are cell adhesion receptors that bind to the extracellular matrix, cell surface, and soluble ligands. They are transmembrane alpha-beta heterodimers, and at least 18 alpha and eight beta subunits are known in humans, generating 24 heterodimers (36). The functions of integrins are partly dictated by the fact that some integrins are limited to specific cell types or tissues and by the pairing of alpha and beta subunits that determines their ligand-binding specificity (reviewed in (36). Therefore, it stands to reason that the differential expression of tetraspanins and integrins on EVs would influence EV interactions and functions. The studies by Zöller and colleagues have provided great insight into the interactions of tetraspanins and integrins in EVs (37), (38). In one study, the authors focused on interactions between tetraspanin 8 and integrin beta 4 in the EV lysates from rat tumor cell lines (39). The levels of tetraspanin 8 in the tumor lines were modulated by introducing overexpression constructs and by knockdown of their protein levels. Moreover, the abundance of tetraspanins and integrins on EVs was affected by changes in the expression of these molecules in the parental cells from which these EVs are derived. In co-immunoprecipitation experiments, tetraspanins and integrins engaged in promiscuous interactions but also in few preferential interactions. For instance, Tetraspanin 8 interacts with alpha4 and beta4 integrin chains, which also affects the pairing of integrins in the resultant EVs. Notably, some changes in molecular interactions of tetraspanins and integrins that appeared to be subtle resulted in changes in the binding of EVs with cells and their tropism in vivo. Those observations complemented an earlier study that had shown that only small EVs expressing a defined set of tetraspanins, and associated molecules targeted endothelial cells (40). Moreover, interactions of EVs with hematopoietic cells, for example, that resulted in their binding and uptake, were favored by the expression of some adhesion molecules including CD54 (39). Regarding EV tropism, small EVs that expressed tetraspanin 8 were more readily taken-up by pancreas and lung and rarely by liver and gut. Taken together, those studies and others have well shown that alterations in abundance and identity of tetraspanin and integrins on small EVs affects molecular pairings on the small EVs surface and by extension, their functional interactions in vivo.

EV membrane composition is modulated by intracellular infection

Intracellular infection of cells can also modulate the composition of adhesion molecules expressed by EVs released from the infected cells. For example, in infections of cells by Epstein-Barr virus, EV content, including the expression of adhesion molecules, was modulated by Latent Membrane Protein 1 (LMP-1) (41). EVs isolated from the mouse macrophage cell line, RAW264.7, infected with either of the intracellular pathogens Leishmania donovani or Salmonella Typhimurium, were isolated and subjected to proteomic analysis (42), (43). Analysis of integrins in the EV preparations showed that S. Typhimurium infection resulted in altered levels of specific integrins (Tables 1 and 2). For example, S. Typhimurium infection of RAW264.7 cells leads to a decrease in integrin subunit alpha 4 (ITGA4), integrin subunit alpha M (ITGAM), integrin subunit beta 1 (ITGB1), integrin subunit beta 2 (ITGB2) in small EVs at 24 hpi (hours post-infection) (Table 1). However, at 48 hpi, the S. Typhimurium infection led to an increase in integrin subunit alpha M (ITGAM) and integrin subunit beta 2 (ITGB2) (Table 2). Tetraspanin expression EVs is also modulated by S. Typhimurium infection (Table 3).

Table 1.

Integrins in small EVs from Salmonella-infected RAW264.7 macrophages isolated at 24 hpi (MOI 5:1).

Symbol	Gene Name	Accession Number	Fisher’s Exact Test (p-value)	Expr Fold Change (24 hpi vs control)
ITGA4	integrin subunit alpha 4	Q792F9	0.0001	−5
ITGA5	integrin subunit alpha 5	P11688	0.78	1.7
ITGAM	integrin subunit alpha M	E9Q604	0.00055	−2.5
ITGAV	integrin subunit alpha V	P43406	0.38	−2.5
ITGB1	integrin subunit beta 1	P09055	0.0001	−3.333
ITGB2	integrin subunit beta 2	P11835	0.0001	−3.333
ITGB7	integrin subunit beta 7	P26011	0.38	−2.5

Table 2.

Integrins in small EVs from Salmonella-infected RAW264.7 macrophages isolated at 48 hpi (MOI 5:1).

Symbol	Gene Name	Accession Number	Fisher’s Exact Test (p-value)	Expr Fold Change (48 hpi vs control)
ITGAM	integrin subunit alpha M	E9Q604	0.025	1.8
ITGB2	integrin subunit beta 2	P11835	0.04	1.6
ITGA4	integrin subunit alpha 4	Q792F9	0.089	−1.429
ITGA5	integrin subunit alpha 5	P11688	0.13	4
ITGB1	integrin subunit beta 1	P09055	0.52	1
ITGAV	integrin subunit alpha V	P43406	0.74	1.4
ITGB7	integrin subunit beta 7	P26011	0.85	1

Table 3.

Tetraspanins in small EVs from Salmonella-infected RAW264.7 macrophages isolated at 24hpi (MOI 5:1).

Symbol	Gene Name	Accession Number	Fisher’s Exact Test (p-value)	Expr Fold Change (24 hpi vs control)
CD81	CD81 molecule	P35762	0.00029	−5
CD82	CD82 molecule	P40237	0.72	−1.111
CD9	CD9 molecule	P40240	0.084	−1.429
TSPAN14	tetraspanin 14	Q8QZY6	0.14	−5

The pattern of adhesion molecules expression on EVs dictates tissue tropism

As discussed earlier, EVs contain a range of molecules in their limiting membrane, including lipids, carbohydrates, and proteins that are likely to impact the cellular interactions of EVs and tropism. Although the molecules found in EVs are constrained by the molecular composition of their parental cells, EVs do not necessarily express the most abundant molecules of their parental cells (44), (45). This phenomenon suggests the existence of a selective mechanism to load specific proteins onto EVs that can be modulated for the expression of the desired molecular profile. It has been hypothesized that EV interactions with cells mirror the interactions of their parental cells and that EVs are preferentially homed to tissues that harbor their parental cells. Lyden and colleagues have contributed several studies on this topic (46), (47), (48). In one study, small EVs were recovered from tumor lines with known metastatic destinations. Small EVs from BxPC-3 and HPAF-II lines preferentially accumulated up to 4-fold in the liver as compared to other organs, which was consistent with the tissue tropism of those tumor lines. The small EVs from MDA-MB-231 exhibited a more than threefold accumulation in the lungs compared to small EVs from the tumor lines mentioned above. Moreover, 831-BrT EVs efficiently localized to the brain with a more than fourfold increase than small EVs from two other tumor lines that are not known to metastasize to the brain (46). The molecules that are likely most responsible for targeting the EVs to specific cells or organs are tetraspanins and integrins. Quantitative mass spectrometry of small EVs from these metastatic tumor lines identified six integrins among the top 40 most abundant adhesion molecules. For example, integrin alpha 6 (ITGα₆) and its partners ITGβ₄ and ITGβ₁ were present abundantly in lung-tropic EVs. By contrast, ITGβ₅, which associates only with ITGα_v was detected primarily in liver-tropic EVs. The critical requirement for EV-based ITGβ₄ in lung tropism was further confirmed by performing knock-down experiment of ITGβ₄. In the recipient tissues, EVs associated with molecules such as laminin and fibronectin were shown to activate Src phosphorylation and pro-inflammatory S100 gene expression (46). In another study, melanoma B16BL6 cell-derived small-EVs localized to the lungs within 10 minutes after injection of the EVs into animals. Treating the EVs with proteinase K that reduced vesicular integrin α6β1 displayed on the EVs diminished distribution to the lungs (49). Finally, the EVs with pronounced gut-homing properties express Integrin α4β7 (50). T cells induced by retinoic acid secrete the EVs that have increased integrin α4β7 and modify the expression of microenvironmental tissues to limit subsequent lymphocyte homing (50). Together, these studies support the hypothesis that integrin selection and its display on EVs plays a critical role in EV homing.

Studies of the small EVs transfer into hosts.

Studies seeking to determine the fate of EVs administered to recipient hosts have reported mixed results. Many studies have used various approaches to label EVs to track their entry and retention into tissues (51), (52). Some of the labeling approaches have included direct loading with dyes such as PKH26/27, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD)-lipophilic dye, or by genetic engineering of proteins in EVs with luminescent probes such as Renilla or Gaussia luciferases (53), (54). Most of those studies have evaluated the administration of heterologous EVs as compared to autologous EVs using varying routes of delivery that would influence the kinetics and biodistribution of small EVs. The picture that has emerged from analyses of those studies is quite mixed. Smyth and colleagues found rapid clearance and minimal tumor accumulation of intravenously-injected unmodified tumor derived small EVs, comparable to liposomes (55). This suggested to them that the unique protein and lipid composition of small EVs does not appreciably impact their bio-distribution. Nonetheless, their study found that innate immune mechanisms in a host impacted the biodistribution of small EVs. In a recent publication, Kang and colleagues compiled and analyzed over 29,000 reports on the biodistribution of small EVs published until June of 2019 (56). The outcomes of this analysis agreed with the observations of others that had shown that the site of EV retention after the transfer was dependent on several factors, including the route of administration [intravenous (IV) vs. intraperitoneal (IP) vs. subcutaneous (SC)], the number of particles transferred, the time at which transfers were evaluated and the recipient animals, just to name a few factors. Among the insightful observations from the analysis in (49) was that EVs are quickly cleared from the blood within 30 minutes of their administration. In most studies, the liver, followed by the lung, were the most reproducible accumulation sites of EVs. Other tissues at which EVs were retained were the spleen, GI tract, and kidney, in that order. The route of administration of EVs had some influence on the site of EV retention. A study by Wiklander and colleagues (57) included in the analysis by Kang et al. (49) evaluated the biodistribution of EVs prepared from three cell sources. Amongst the observations, there was an increased (56) trafficking of EVs to the pancreas and GI tract when EVs were administered SC or IP compared to preferential accumulation of EVs, from the same source, in the liver and spleen when the EVs were administered by IV. Specifically, EVs derived from C2C12, a muscle-derived cell line, showed more significant liver accumulation than EVs from B16F10-EVs, a melanoma-derived line, or EVs from bone marrow-derived dendritic cells. The EVs from the melanoma line preferentially accumulated in the GI tract. Other studies, including those in an acute kidney injury (AKI) model (58), showed EVs from mesenchymal stem cells to accumulate in injured kidneys. Hui and colleagues showed preferential accumulation of EVs prepared from Salmonella-infected macrophages in the GI tract and lungs, significantly more significant than the dye accumulation in these organs (59). The accumulation of EVs in the study by Hui and colleagues was also dependent on the route of EV administration (59). Together, it is evident that the route of administration of EVs affects the biodistribution in hosts. The analysis also underscored the impact of cell sources on the tissue destination of EVs.

Factors that have been shown to affect the homing of EVs

There are marked differences in the biodistribution of EVs from normal cells or tissues compared with EVs derived from immortalized cells. In the studies by Garofalo and colleagues, EVs were generated from murine lung and colon cancer lines; a human lung cancer cell line, and from human liver biopsy samples from healthy individuals, they showed that tumor-derived EVs, but not the EVs derived from healthy tissue, demonstrated a selective accumulation of the fluorescence at the tumor site 24 h after injection (51). Moreover, trafficking to and retention in tumors has been reproducibly observed by EVs derived from immortalized cells, even if their parental cells were not derived from that tumor. The tumor tropism of these EVs was further demonstrated when human EVs were also able to target the mouse mammary tumors (51), suggesting that damaged tissues (such as tumors) can recruit EVs derived from heterologous sources. Processes within the host appear to affect the distribution of small EVs. In a study by Smyth and colleagues, they showed that unmodified EVs administered by IV administration had a short half-life and were rapidly taken up by the mononuclear phagocyte system, particularly in the liver, lung, and spleen, leading to minimal accumulation in the intended tissues and undue delivery to unintended tissues (55). Their studies suggested that the innate immune system, with help from complement opsonization, contributes to removing tumor-derived EVs from circulation. Other observations by Smyth and colleagues (55) on the preferential trafficking of EVs to tumors complement the observations communicated in several other studies (60). Hypoxic conditions have been shown to stimulate the more significant release of small EVs by tumor cells (61), (62). Hypoxic conditions also increase the effects of EVs in recipient tissues by enhancing processes such as angiogenesis. However, the mechanism dictating the preferential recruitment of administered small EVs to tumors is not well understood. It has been shown that EVs derived from specific cells express surface antigens, including CD47, which limits their uptake by monocytes, therefore resulting in extended blood circulation times and increased opportunity to circulate widely (63), (64), (65). The absence of such molecules from EVs derived from normal cells or tissues may explain why evidence of directed homing of ‘normal EVs’ is challenging to find. It has been suggested that ‘damaged’ tissues, such as tumors, display greater expression of molecules such as CD54 (39) or S100, which are proteins in lymph nodes of melanoma patients and predictors of poor prognosis (66).

Strategies to reliably direct EV homing

The desire to exploit predictable EV distribution has been approached in several studies. In a study by Zhang and colleagues, monocyte membrane-decorated mesenchymal stem cell (MSC) small EVs were explored to improve the specific targeting capability of these EVs to injured hearts and promote heart repair in a myocardial ischemia-reperfusion injury model of mice (67). Monocyte membranes were isolated from the murine monocyte-macrophage cell line, RAW264.7 cells, while small EVs were derived from MSCs. The small EVs and macrophage-derived membranes were fused through a fusion and extrusion method. These monocyte membrane-decorated small EVs exhibited enhanced targeting efficiency to injured myocardium.

Peptides might also be important in the localization of EVs to specific compartments. As a proof of principle, small EVs can be engineered to increase their targeting capabilities by including targeting peptides fused to the extracellular region of proteins displayed on these EVs, such as an extracellular N-terminus is Lamp2b present on EVs (68). This principle has been used to target EVs to the central nervous system, where the rabies viral glycoprotein (RVG) peptide (YTIWMPENPRPGTPCDIFTNSRGKRASNG) fused to Lamp2b specifically binds to the acetylcholine receptor 3 and targets dendritic cell-derived EVs to the brain by using intravenous injections (68). T7 peptide can also be fused to Lamp2b to target EVs to the brain. In comparison, to control EVs, T7-containing EVs were shown to have increased delivery efficiency to the brain in the model of glioblastoma (69). Moreover, muscle-specific peptide (MSP) fused with Lamp2b was used to successfully deliver EVs to muscles (70).

Conclusions/Perspectives

A review of the biology of EVs regarding their homing/tropism has revealed the following observations: (1) More EVs are released from cells under stressful conditions that include such conditions as immortalization (malignancy), hypoxia, or pathogen infection than from normal cells from undamaged tissues. (2) EVs released under stressful conditions express higher levels of adhesion molecules, including tetraspanins and integrins (Figure 1). (3) The parental cell’s tissue origin predetermines the identities of tetraspanins and integrins expressed on EVs. (4) EVs released by cells under stressful conditions express molecules such as CD47 (do not eat me signals) that prevent their uptake by phagocytic cells, delaying their clearance from circulation. (5) Tissues undergoing stressful processes, including injury from malignant growth, express adhesion molecules that render them more receptive to EVs (Figure 1). Normal tissues are poor targets for administered EVs.

Figure 1.

Homing patterns of EVs is determined by their characteristics. Cells within damaged tissues such as damaged lung produce more EVs (including EVs with higher expression of adhesion molecules) and are more likely to uptake heterologous EVs than cells in normal, non-damaged tissues. The EVs that are preferentially retained by/targeted to lungs contain specific surface molecules, such as ITGα6, ITGβ4, or ITGβ1.

Efforts to modify EVs to improve their reproducible tropism have been promising. Those efforts will be supported by more studies on the molecular composition of homogenous preparations of EVs. Developing representative immortalized lines from each tissue should be a worthwhile undertaking. Experimental designs that are informed by a realistic understanding of EV biology are likely to deliver promising translatable findings.

Biographies

Biography

Dr. Peter Kima’s is on the Microbiology and Cell Science faculty in the College of Agriculture and Life Sciences (CALS) at the University of Florida. There are 3 major projects in Dr. Kima’s laboratory: 1) the evaluation of the role of PI3K signaling in Leishmania infections; 2) development of strategies to disrupt the formation of Leishmania parasitophorous vacuoles; 3) evaluation of the role of exosomes released from Leishmania-infected cells in infected tissue remodeling. Dr. Kima has been a member of several US and international grant review panels.

Dr. Mariola J Edelmann is a faculty in the Microbiology and Cell Science Department in the College of Agriculture and Life Sciences (CALS) at the University of Florida. Dr. Edelmannprimarily works on the role of host extracellular vesicles in Salmonella infection, including applying these vesicles as a cell-free vaccine and the function of extracellular vesicles in the immune response to bacterial infection. She is also exploring host-directed therapies for bacterial infections. Dr. Edelmann’s work in these areas has been funded by the National Institutes of Health and the Department of Defense.