Abstract
Extracellular vesicles (EVs) are tiny biological nanovesicles ranging from approximately 30–1000 nm in diameter that are released into the extracellular matrix of most cell types and in biofluids. The classification of EVs includes exosomes, microvesicles, and apoptotic bodies, dependent on various factors such as size, markers, and biogenesis pathways. The transition of EV relevance from that of being assumed as a trash bag to be a key player in critical physiological and pathological conditions has been revolutionary in many ways. EVs have been recently revealed to play a crucial role in stem cell biology and cancer progression via intercellular communication, contributing to organ development and the progression of cancer. This review focuses on the significant research progress made so far in the role of the crosstalk between EVs and stem cells and their niche, and cellular communication among different germ layers in developmental biology. In addition, it discusses the role of EVs in cancer progression and their application as therapeutic agents or drug delivery vehicles. All such discoveries have been facilitated by tremendous technological advancements in EV-associated research, especially the microfluidics systems. Their pros and cons in the context of characterization of EVs are also extensively discussed in this review. This review also deliberates the role of EVs in normal cell processes and disease conditions, and their application as a diagnostic and therapeutic tool. Finally, we propose future perspectives for EV-related research in stem cell and cancer biology.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
INTRODUCTION
Diverse communication systems mediate intercellular communication, both in physiological and pathological conditions, such as cellular junctions (tight junction, adherence junctions, gap junctions, and desmosomes), integrins, and selectins (Martin et al., 2013). Recently, a novel way of cell-cell communication mediated by extracellular vesicles (EVs) has gained lots of attention (Sverdlov 2012; Maia et al., 2018; Stahl and Raposo 2018). EVs are a heterogeneous group of cell-derived membranous nanovesicles with diameter around 30–1000 nm (sometimes as large as a few micrometers; Raposo et al., 1996). They are released from most cell types and biofluids such as blood, saliva, breast milk, semen, urine, cerebrospinal fluid (CSF), colostrum (de la Torre Gomez et al., 2018), tears (Grigoreva et al., 2017), bronchoalveolar fluid (Torregrosa Paredes et al., 2012), epididymal fluid (Gatti et al., 2005), amniotic fluid (Asea et al., 2008), bile (Masyuk et al., 2010), blastocoel fluid (Battaglia et al., 2019), middle ear effusion (Val et al., 2017), and ascites (Zhu et al., 2018). EVs can be broadly classified into exosomes, microvesicles (MVs), and apoptotic bodies (ABs), based on different biological properties such as biogenesis pathways, size and biomarker (Zhang et al., 2019).
Exosomes are a subset of EVs with a size of approximately 30–100 nm, which were found to be secreted upon the fusion of multivesicular bodies (MVBs) with the plasma membrane during the differentiation of reticulocyte for the first time (Harding et al., 1984; Pan et al., 1985). Further confirmation was provided by a similar pathway of exosomes in B-lymphocytes (Raposo et al., 1996), dendritic cells (Zitvogel et al., 1998), and many other cells including platelets, T-cells, mast cells, neurons, Schwann cells, oligodendrocytes, and epithelial cells (Simons and Raposo 2009; Théry et al., 2009; Becker 2014). In addition, an in vivo study demonstrated the release of exosome-like vesicles from prostate epithelial cells, referred to as prostasomes and thought to be essential for motility of spermatozoa (Ronquist and Brody 1985). Common biomarkers of exosomes include tetraspanins (CD9, CD63, and CD81), heat shock proteins (HSPs), membrane transporters and fusion proteins (e.g., GTPases, annexins, and flotillin), MVB biogenesis proteins (e.g., Alix and TSG101), phospholipases, and lipid-related proteins (Conde-Vancells et al., 2008; Subra et al., 2010). Exosomes are also enriched with lipids such as cholesterols, phospholipids, biphosphates, and sphingolipids (Kao et al., 2020). Although exosomes are heterogenous (Bernard et al., 2018; Kalluri and LeBleu, 2020) and their constituents resemble their cells of origin (Salomon et al., 2015), they are also shaped by the biogenesis pathway. The biogenesis of exosomes takes place via an endosomal pathway in which the introversion of early endosomes leads to the formation of MVBs carrying intraluminal vesicles, followed by the fusion of MVBs with the plasma membrane, resulting in the excretion of exosomes in the extracellular milieu (Johnstone et al., 1987). Schematic illustrations in Fig. 1A and 1B show a typical exosomal composition and different biogenesis pathways taken by different types of EVs, respectively.
MVs are generally about 2 µm in size and produced by budding and shedding of the cell membrane via a calcium-dependent pathway. Cells undergoing apoptosis release another set of EVs with size range of 1–5 µm, referred to as ABs (Dignat-George and Boulanger 2011; Colombo et al., 2014). There is no consensus about the nomenclature of exosomes and MVs because even nanovesicles that are similar in size to exosomes and released by budding off the plasma membrane are defined as exosomes (Booth et al., 2006). Therefore, we have used the more general term “EVs” in this review.
Scientists have recently discovered a wide range of biological or physiological activities of EVs in a diversity of fields including stem cell biology, cancer biology and therapy (Tai et al., 2019; Khawar et al., 2019). This review introduces the biology and historical perspective of EVs, the role of the crosstalk between EVs produced by stem cells and stem cells-niche, and EV-mediated communication between different germ layers in developmental biology. Further, the role of EVs in cancer progression will be elaborated, followed by a discussion of the application of EVs as therapeutic agents and delivery vehicles. The role of EVs in normal cell processes and disease conditions and their application as diagnostic and therapeutic tools will also be detailed. Finally, various methods for EV characterization will be discussed in the context of technological advancements in EV-associated research.
EVs AND THEIR CROSSTALK IN STEM CELL NICHE
Stem cells are a unique set of cells with the ability to self-renew. A single stem cell can produce clonal cell populations and further differentiate into several cell types (Tran and Damaser, 2015). Stem cells are broadly classified into embryonic stem cells (ESCs) and somatic stem cells (SSCs; also referred to as adult stem cells). ESCs are pluripotent and can differentiate into any type of cell including neural stem cells (NSCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), and endothelial progenitor cells (EPCs). SSCs were recently reported to exist in most adult tissues, maintaining tissue regeneration, turnover, and repair during injury, and are restricted to a specific lineage.
The fate of embryonic and adult stem cells is rigorously controlled by their microenvironment, known as stem cells-niche, through either inter- or intra- cellular communication (Pennings et al., 2018). Anatomically, stem cell niche refers to the location where the basic unit of the physiology of tissue is formed by combining signals for a balanced response from stem cells depending on the requirements of the organism (Ohlstein et al., 2004; Li and Xie, 2005). Extensive work has been carried out in various organisms to characterize the stem cell-niche (Kirkeby et al., 2016; Hsu et al., 2019). The hypothetical components of the stem cell niche include stem cells, stromal cells, extracellular matrix (ECM), and blood vessels (Pennings et al., 2018; Fig. 2A). The presence of each is not mandatory in a stem cell niche, although its structural organization and constituent elements provide a dynamic environment in which to control the cell number (stem cell pool) and the function of stem cells (Jones and Wagers, 2008). Interestingly, EVs produced from stem cells have recently attracted great attention owing to their essential roles in stem cell biology, such as regulation of self-renewal capacity (Ratajczak et al., 2006), mediating crosstalk (Deregibus et al., 2007; Camussi et al., 2010) between stem cells and niche (Hur et al., 2020), germ layer communication in organogenesis and development, and functions in stem cell differentiation and determination of cell fate (Aliotta et al., 2010; Quesenberry et al., 2010; Fig. 2B).
Regulation of self-renewal capacity of stem cells
EVs play a crucial role in regulating the self-renewal capacity of stem cells. To maintain proper functionality as well as self-renewal capacity, the crosstalk within the stem cells-niche is of prime importance. For example, mouse embryonic stem cell EVs (mESCEVs) facilitate the sustenance of self-renewal and expansion of adult stem cells; hematopoietic progenitor cells (HPCs) via delivery of specific mRNAs (such as octamer-binding transcription factor 4 (Oct-4), Nanog, Rex-1, stem cell leukaemia (SCL), and GATA binding protein 2 (GATA-2)) and proteins (such as Wnt family member 3 (WNT3) and OCT4) induce the overexpression of genes responsible for pluripotency. The mESCEVs increase survival and expansion of HPCs by inducing phosphorylation of mitogen-activated protein kinases (MAPK) p42/44 and serine-threonine kinase AKT. Further, the self-renewal phenotype of HPCs diminishes after inhibition of the function of mESCEVs by heat inactivation or pre-treatment with RNase, suggesting the critical effect of the protein or mRNA cargo within the mESCEVs on maintaining stemness of HPCs (Ratajczak et al., 2006).
Crosstalk between donor and recipient cells via EVs
The secreted factors in the stem cell-niche guide the fate of stem cells by facilitating communication, locally or remotely, by diffusing through the niche (Jones and Wagers, 2008). EVs have emerged as another way of communication (Camussi et al., 2010) between stem cells and the niche (Hur et al., 2020). Emerging evidence shows that EVs transport and deliver mRNAs that can reprogram target cells and modulate the phenotype of recipient cells (Ratajczak et al., 2006). Delivery of mRNA by EVs induces transcription in the recipient cells, and causes tissue-specific alterations (Aliotta et al., 2010), suggesting that mRNA in EVs is indeed functional. For example, EPC-derived EVs have been found to induce angiogenesis in terminally differentiated endothelial cells and proangiogenic character in dormant endothelial cells (Deregibus et al., 2007). Stem cells can also donate organelles including mitochondria (500–1000 nm) to neighbour cells to regenerate damaged tissues and cells (Zhang et al., 2016b; Jiang et al., 2019, 2016).
In addition, fibroblast-derived EVs can transport epidermal growth factor activity in the intestinal stem cell niche by carrying amphiregulin (Oszvald et al., 2019). Prostate cancer cell (PCa)-derived EVs carry cancer associated fibroblasts (CAF) that play a crucial role in communication between PCa and the tumor microenvironment (TME) to support pathways associated with stemness (Kato et al., 2017). In fact, EVs from PCa with AKT kinase activity have been reported to be taken up by normal prostate fibroblasts and induce their reprogramming via activation of stromal Myc pro-oncogene (Minciacchi et al., 2017).
Stem cell differentiation and cell fate determination via EVs
EVs produced from stem cells also play a significant role in the differentiation of stem cells and the determination of cell fate. The constituents of EVs are capable of affecting and determining the cell fate of impending populations of recipient cells via a stable alteration in genetic makeup (Quesenberry et al., 2010; Aliotta et al., 2010). Stem cells from embryos or adult stem cells originating from diverse adult tissues, including bone marrow, liver, and adipose tissue, and induced pluripotent stem cells (iPSCs), secrete EVs (Chen et al., 2017b). Quesenberry et al., proposed that the transfer of genetic information via EVs could be a key factor in the stem cell continuum model, where the decision of stem cells to differentiate was facilitated by the transit of cell cycle and environmental stimuli (Quesenberry et al., 2010). Jung et al., showed that EVs released during the differentiation of stem cells into white or beige adipocytes could promote cell reprogramming via miRNAs (Jung et al., 2020). Stronati et al., demonstrated that EVs could induce differentiation of neural stem progenitor cells (NSPCs). The NSPCs secreted EVs during proliferation as well as after differentiation, and treatment of proliferating NSPCs with EVs derived from differentiated NSPCs induced cell differentiation in a concentrated dependent manner (Stronati et al., 2019).
Therapeutic potential of EVs as a delivery carrier
The role of EVs as a therapeutic agent and a potential delivery vehicle has also been explored extensively. Like other EVs, stem cells EVs also recapitulate their parent cells, but are relatively less immunogenic and capable of crossing biological barriers (Bellavia et al., 2018), suggesting their therapeutic potential (Arslan et al., 2013; Ibrahim et al., 2014). Importantly, several clinical studies of EVs have been conducted, for example allogenic MSC-derived EVs were used in patients with acute ischemic stroke. ESCs-EVs carry various cargoes including miRNAs (miR-291, -294 and -295) and proteins (heat shock protein 90 (HSP90), delta like canonical Notch ligand 4 (DLL4), stress inducible protein (STI1), and Wnt-10b), and these cargoes cause cardiac regeneration, inhibition of glioblastoma growth, and retinal regeneration (Kanellopoulou et al., 2015; Khan et al., 2015; Cruz et al., 2018a; Peng et al., 2018; Wiklander et al., 2019; Zhu et al., 2019). Adipose tissue derived MSC-EVs also carry cargoes including miRNAs (miR-223, -146b, -126, -199a, -let7b, and -let7c) and proteins (CD9, CD63, HSP70, and CD81) that cause skeletal muscle regeneration, neovascularization, fibrosis inhibition, wound healing, and anti-inflammation. Umbilical cord-derived MSC-derived EVs carry miRNAs (miR-21, -146a, -181, -302a and -410) and proteins (CD63 and CD90) that cause anti-tumor and anti-apoptotic activity, neovascularization, and regulation of insulin resistance (Yao et al., 2019).
ROLE OF EVs IN INVERTEBRATES: A MIRROR TO THE POTENTIAL ROLE IN ANIMAL MODELS
Several scientists have developed and explored the possibility of utilizing a compliant invertebrate model to study the composition of EVs (Russell et al., 2020). Compared with complex mammalian models, Caenorhabditis elegans (C. elegans) and Drosophila (Beer and Wehman, 2017) can serve as a platform for isolation of EVs actively secreted into their extracellular space. Studies in classical genetic model organisms like C. elegans and Drosophila have begun to reveal the developmental and behavioural roles of EVs. These organisms help decipher the signalling function of EVs in multicellular organisms. Transgenic technologies and live imaging in C. elegans and Drosophila facilitate the in vivo monitoring of EVs via reporter genes such as fluorescence or luciferase genes (Panáková et al., 2005). These in vivo systems can be applied to explore the potential of EV-based signalling in the developmental process, behaviour change, or disease progression in metazoans. Moreover, the molecular mechanisms that underlie the release of EVs can be studied in-depth with these models. Samuel Liégeois et al., demonstrated the role of EVs in the normal development of the C. elegans cuticle. The release of lapidated morphogens in EVs was essential to the development of the cuticle (Liégeois et al., 2006). Moreover, EVs contain lipid-modified Hh-related proteins and the transmembrane protein CHE-14/Dispatched, which is involved in Hh secretion. Interestingly, Hh-related proteins are entrapped in MVBs aggregated in the cytoplasm when the release of EVs is inhibited via depletion of membrane-bound V0 sector of the vacuolar H+-ATPase (V-ATPase). Cuticle development is blocked upon depletion of V-ATPase, suggesting that EV-mediated release of Hh-related proteins in C. elegans provides a unique way to transport lipid-modified or transmembrane proteins during development (Liégeois et al., 2006).
Using Drosophila as a model, Koles et al., demonstrated the in vivo mechanisms of Evenness Interrupted (Evi)-EVs release (Koles et al., 2012). In addition, Korkut et al., showed a cellular mechanism through which a secreted WNT was carried across synapses by Wnt-binding protein and Evi-containing vesicles, trafficking between both the WNT-producing and the WNT-receiving cells (Korkut et al., 2009). The formation of EVs was also found to be essential for developing wings in Drosophila via Wingless (Wg) and Hh signalling (Gross et al., 2012; Gradilla et al., 2014; Matusek et al., 2014). Further, it has been found that filopodia in Xenopus embryos can act as super-highways for EVs in vivo, indicating a potential association between cellular extensions and EVs (Danilchik et al., 2013). Overall, EVs are involved in various signalling pathways by transporting morphogens across numerous cells as demonstrated in Drosophila.
On the contrary, abnormal secretion of EVs can impede the development of metazoans (Wehman et al., 2011). During embryogenesis, cell division occurs followed by a change in shape and cells migrating to a suitable location in the body. Interestingly, the loss of phosphatidylethanolamine (PE) flippase TAT-5 enhances the release of EVs, leading to disruption of cell adhesion and morphogenesis of C. elegans through lipid asymmetry (Wehman et al., 2011). EVs also play a vital role in the physiology of single-cell organisms (e.g., Chlamydomonas), multicellular organisms (e.g., C. elegans), and mammals (Wood and Rosenbaum, 2015). For example, in Dictyostelium, EVs released from cilia carry transmembrane protein crucial for sexual reproduction (Cao et al., 2015). The EVs derived from ciliated neurons in C. elegans carry proteins that enable mating behaviour of the male (Wang et al., 2014a; Maguire et al., 2015). EVs also play a crucial role in the mating behaviour of Drosophila. EVs released from large accessory gland cells in male Drosophila fuse with sperm after mating and interact with female reproductive tract epithelial cells. This inhibits female remating behaviour and contributes to the reproductive advantage of the first male to mate (Corrigan et al., 2014).
In the context of neuroscience, glial cells employ EVs to regulate various processes including formation of the myelin sheath and post-injury repair. EVs also facilitate the propagation of inflammatory signals induced by disease and tissue damage. They have also been reported to be associated with communication in the nervous system of intact organisms. For example, at the neuromuscular junction (NMJ) of Drosophila, Wnt signals are carried by EVs from neurons to muscles (Budnik et al., 2016). At the sensory neurons of C. elegans, EVs also conduct behaviourally relevant signals, for example the cilia of sensory neurons release EVs carrying the polycystin receptors, and the addition of these EVs to the rearing medium of naive animals enhances the frequency of locomotor reversal and tail chasing behaviour; a strategy employed during mating (Wang et al., 2014a; Budnik et al., 2016). Collectively, the numerous roles of EVs in classic genetic model organisms such as C. elegans and Drosophila may be a potential area of research to further determine their role in animal models.
EV-MEDIATED COMMUNICATION IN EMBRYONIC DEVELOPMENT
During embryonic development, various cell types are generated to form tissues and organ systems in an organism. The inner cell mass (ICM) of the blastocyst, which produces the entire embryo (in vivo condition), can be isolated and cultured as ESCs with the capacity of complete development. The ICM plays a crucial role by receiving various autocrine- or paracrine-(from neighbouring cells, including trophoblast or primitive endoderm) signals to facilitate the processes that lead to embryogenesis (Plusa and Hadjantonakis, 2014). Interestingly, intercellular communication in the ICM is via EVs. Recent evidence suggests that EVs transport morphogens like Sonic Hedgehog (SHh), and miRNAs like miR-21, and causes alteration in the phenotype of recipient cells like dendritic cells (Song et al., 2018; Sun et al., 2018). EVs also facilitate communication between the trophectoderm (TE) and ICM, traversing along filopodia that spread across the blastocoel associated with mural TE to the ICM in the mouse blastocyst (Alberti and Cochella, 2017; Gross et al., 2017). Several reports have shown that the filopodia-like protrusion in the cell membrane acts as a conduit during embryogenesis (Fairchild and Barna, 2014; Sagar et al., 2015), through signalling pathways such as Notch, SHh, Decapentaplegic (Dpp), EGF, FGF, Wnt, and bone morphogenetic proteins (BMPs; Heckman and Plummer, 2013; McMahon and Hasso, 2013), and controls the release and delivery of EVs (Cruz et al., 2018b). Video microscopy analysis has demonstrated that EVs move along filopodia and are rearranged in the dynamic cytoskeleton via signal transduction activity by the receptors fibroblast growth factor receptor 2 (Fgfr2) and Erb-B2 receptor tyrosine kinase 3 (ErbB3; Salas-Vidal and Lomelı́, 2004). Mouse ESC-secreted EVs were shown to be internalized by trophoblasts, leading to stimulation of trophoblast migration via the instigation of c-Jun N-terminal kinase (JNK) and JNK pathways. Furthermore, when injected into the blastocysts, the ESC-derived EVs enhanced the implantation ability of blastocysts in the uterus. This indicated that intercellular communication through EVs plays a crucial role to facilitate the interaction between ESCs and trophoblast during pregnancy (Desrochers et al., 2016).
EVs in Hh signalling
The core components of the hedgehog (Hh) signalling pathway are Hh ligand, transmembrane receptor Patched 1, intracellular G-protein Smoothened and transcription factors GLI1-3. Tanaka et al. showed that FGF signalling promotes secretion of EVs containing SHh and retinoic acid at the node of the gastrula’s primitive streak. They also revealed that fluid flow at the node promoted EV movement towards the left in embryos. The asymmetric accumulation of EVs specifically activated the non-canonical Hh signalling pathway in the left of the embryo, resulting in differential changes to intracellular calcium levels and gene expression, leading to a left-right gradient of morphogens (Tanaka et al., 2005).
EVs in Wnt signalling
Wnt signalling is involved in biological processes throughout embryonic development, such as proliferation, renewal, migration, differentiation, and polarity of stem cells (Loh et al., 2016). EVs are involved in Wnt signalling during extracellular transport in Drosophila through argosomes, where Wnt is released via EVs (Greco et al., 2001). Several reports indicate that EVs carry released Wingless (Wg) in neuromuscular junction and fly larval imaginal disc. The release of Wg has been found to be associated with Wg transmembrane binding protein Evi/Wntless (Wls)/Sprinter (Srt) and Wg is carried by Evi-containing EVs via synapses at the larval neuromuscular junction (KKorkut et al., 2009; oles et al., 2012). This entire process takes place during synaptic development (Koles et al., 2012).
EVs in Notch signalling
Notch is a transmembrane protein and its signalling is involved in various key developmental processes such as cell differentiation, decision, patterning, and polarity (Mašek and Andersson, 2017). The intracellular domain of Notch is cleaved after binding of the Notch ligand to its receptor and is translocated to the nucleus, so regulating the downstream processes. The involvement of EVs in Notch signalling was speculated when Delta, one of the Notch ligands, was identified in EVs (Le Borgne and Schweisguth, 2003; Kopan and Ilagan, 2009). Delta-like 4 (DLL4) was found in EVs derived from human endothelial cells. The EVs carrying DLL4 were taken up by other endothelial cells with subsequent increased angiogenesis upon inhibition of Notch signalling. Nevertheless the underlying mechanism responsible for inhibition of Notch signalling by EVs carrying DLL4 is unknown (Sheldon et al., 2010).
EVs in BMP signalling
Bone morphogenetic proteins (BMPs) belong to the superfamily of transforming growth factor-beta (TGF-β), responsible for morphogen-regulating organization of the body axis, tissue patterning, and development and preservation of stem cell niches at the time of embryonic development in diverse species. On the surface of the cell, BMPs bind to the receptors; bone morphogenetic protein receptors (BMRPs), by promoting the phosphorylation of members of the SMAD family that are translocated into the nucleus and regulate the expression of downstream genes (Wang et al., 2014b; Bier and De Robertis, 2015). The EVs derived from embryos of zebrafish were found to carry BMP2/4 that could activate gene expression of recipient cells. In addition, upon inhibiting the release of EVs, phosphorylation of SMAD1/5/9 and further activity of transcription were reduced, resulting in severe dorsalization phenotypes, reminiscent of disrupted BMP-signalling. This showed the crucial role of EVs in establishing the BMP morphogen gradient throughout the development of zebrafish (Draening et al., 2018).
EVs IN NORMAL CELL PROCESSES AND DISEASE CONDITIONS
Evidence has shown that EVs are crucial for physiological and pathological functions via cell-cell communication (De Toro et al., 2015; Thakur et al., 2017). In the normal physiological system, EVs play an essential role in the immune system (Raposo et al., 1996) and in intercellular communication between various immune cells (Corrado et al., 2013). The EVs in the immune system are capable of eliciting both immune activation and immunosuppression effects. For example, EVs released from mature dendritic cells (DCs) carry major histocompatibility complex (MHC) membrane molecules, causing an adaptive immune response via activation of T cell receptors. Especially during infection, the antigens are taken up by DCs, followed by discharge of MHC complexes, and this phenotype activates helper T cells and B cells, leading to the enhanced release of EVs containing MHC complexes in B cells. In addition, the DC-derived EVs also act as a carrier for antigens between neighboring DCs. Moreover, the EVs released from B cells can stimulate CD4+ T cells, implying that B cell-derived EVs are also crucial to facilitate modulation of the immune response. EVs released from immature DCs are involved in suppression of the adaptive immune response by causing apoptosis of T cells and a tolerogenic immune response. By inducing differentiation of T helper cells into regulatory T cells, EVs can also mediate the balance of pro- and anti-inflammatory effector T cells (Corrado et al., 2013).
EVs have been reported to play a crucial in the central nervous system (CNS), including the brain. The intercellular communication via neuronal EVs in neurons is vital in supporting various functions of the nervous system including myelination and maintenance of axonal integrity. The release of EVs is promoted by glutamate neurotransmitter via stimulation of ionotropic glutamate receptors, followed by the uptake of these EVs that transport their constituents to neuronal cells. Further, the released EVs from oligodendrocytes can protect neurons by elevating their tolerance to stress (Frühbeis et al., 2013; Fröhlich et al., 2014).
In the cardiovascular system (CVS), cardiac muscle cells release EVs, especially under hypoxic conditions (Gupta and Knowlton, 2007). The stressed cells under hypoxic conditions release EVs containing specific constituents, including TNF-α (Yu et al., 2012), that promulgate an inflammatory response. These EVs also carry genetic material and transfer between adjacent cells, leading to alteration of gene expression in recipient cells. This explains why EV-mediated intercellular communication is not specific to a cell type in the heart (Waldenström et al., 2012; M. and Das, 2014).
EVs are crucial to sustain both physiological and pathological functions through cell-cell communication (De Toro et al., 2015; Thakur et al., 2017). As well as regulation of normal cellular processes, EVs facilitate the progression of various pathological conditions such as cancer, neurodegenerative diseases, cardiovascular diseases, and infectious diseases (De Toro et al., 2015). In order to accomplish their physiological or pathological functions, EVs must be released from one cell type (donor cell) and be taken up by another (recipient cell); depending on whether this uptake is by donor cells or by different cells in the extracellular space, the uptake can be categorized as autologous or heterologous (2018; Menck et al., 2015). Menck et al., showed that tumor-derived EVs are crucial for the invasion of tumor cells via both autologous- and heterologous-dependent communication (Menck et al., 2015). Although much research has focused on EV biogenesis, it is quite fascinating and well-understood how EVs are selected and taken up by recipient cells.
APPLICATION OF EVs AS A DIAGNOSTIC AND THERAPEUTIC TOOL
The ability of EVs to carry endogenous proteins and nucleic acids and conduct their biological functions in recipient cells makes EVs an ideal candidate for diagnosis, therapeutics, and drug delivery.
EVs as a diagnostic tool
Several reports indicate that EVs represent the content of their parent cells, and thus serve as a fingerprint or image of their originating cells (Salomon et al., 2015; Sharma et al., 2017). Based on this feature, EVs have been widely used as a diagnostic tool for various diseases, including glioma (Basu and Ghosh 2019). Glioma is a type of brain cancer that occurs in the CNS (Thakur et al., 2018) although the presence of the blood-brain barrier (BBB) limits the diagnosis and prognosis of glioma. With the benefits of their small size and ability to the cross the BBB, EVs are an important tool for early detection of glioma. As noted previously, several research groups isolated EVs from various biofluids, and blood serum- or CSF-derived EVs have shown promising results in tracking the malignant progression of parent glioma cells (Whitehead et al., 2017; Thakur 2019). Similarly, the constituents of EVs have been used as a diagnostic tool to track the progression of other types of cancer (Soung et al., 2017), such as exosomal-NY-ESO-1 in lung cancer (Sandfeld-Paulsen et al., 2016), exosomal-PKG1, Ral GTPase activating protein catalytic subunit alpha 2 (RALGAPA2), nuclear transcription factor, X-box binding 1 (NFX1), tight junction protein 2 (TJP2), human epidermal growth factor receptor 2 (Her2), Glypican-1 in breast cancer (Melo et al., 2015; Chen et al., 2017a; Fang et al., 2017), exosomal-Glypican-1 in pancreatic cancer (Melo et al., 2015), exosomal-Glypican-1, carcinoembryonic antigen (CEA) in colorectal cancer (Li et al., 2017; Yokoyama et al., 2017), exosomal-prostate specific antigen (PSA), glycoprotein galactosyltransferase alpha (GGTA) in prostate cancer (Kawakami et al., 2017; Logozzi et al., 2017), and exosomal-CD24, claudin-4, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA-125) in ovarian cancer (Li et al., 2009; Zhao et al., 2016). Other research has demonstrated that exosomal monocarboxylate transporter 1 (MCT1) and CD147 can be used as biomarkers to track metabolic reprogramming and progression of malignant glioma. The authors utilized different methods including localized surface plasmon resonance (LSPR) and atomic force microscopy (AFM) to detect the presence of MCT1 and CD147 in exosomes isolated from normoxic and hypoxic glioma cells. The study revealed the correlation between the level of MCT1 or CD147 in exosomes, which represented the gain or loss of function of MCT1 or CD147 in glioma cells, and the malignant phenotypes of glioma cells, such as migration and proliferation (Thakur et al., 2020c). Use of EVs as a diagnostic indication has gained much attention because they can be detected in liquid biopsy samples such as blood, urine, and CSF (Pang et al., 2020). Furthermore, their biophysical properties have been utilized for development of an additional diagnostic biomarker (Paolini et al., 2018), as seen in the change of roughness, stiffness, and adhesion force of EVs in hypoxic vs. normoxic conditions. Interestingly, the utilization of EV-based non-invasive liquid biopsy as a diagnostic method has recently gained much attention due to the presence of EVs in various biofluids including malignant glioma (Saenz-Antoñanzas et al., 2019).
EVs as a therapeutic tool
The property of EVs to carry protein and nucleic acids that biologically affect recipient cells makes EVs an attractive therapeutic agent. EVs derived from some cell sources including mesenchymal stromal cells, DCs, and iPSCs, have shown therapeutic activities (Liu et al., 2017b; Yamashita et al., 2018). For example, mesenchymal stromal cell-derived EVs carry and deliver miRNAs or proteins such as interleukin-10 (IL-10) and TGF-β, resulting in anti-inflammatory effects by inhibiting cytokine expression, and tissue regeneration via increasing extracellular matrix modelling. Therefore, mesenchymal stromal cell-derived EVs hold therapeutic potential for several diseases such as cancer (Teng et al., 2015; Wang et al., 2017). In addition, they are relatively safer as they have a lower risk of forming teratoma and embolization (Sun et al., 2016; Liu et al., 2017b).
Several reports have explored the application of EVs as a vaccine. DC-derived EVs can be used as a vaccine against cancer, owing to their character as antigen-presenting cells (APCs). In the main, DC-derived EVs express major histocompatibility complex (MHC)-I, MHC-II, and co-stimulating factor (such as CD86; Théry et al., 1999, 2001). EVs isolated from mature DCs can induce a high T-cell response compared with those isolated from immature DCs (Segura et al., 2005). In addition, EVs isolated from DCs can activate natural killer cells via NKG2D ligands and IL-15Rɑlpha (Viaud et al., 2009). André et al. showed that DCs release profound MHC class I/peptide complexes in EVs to other naive DCs for adequate priming of CD8+ T cells in vitro. Moreover, mature DCs are required by EVs to facilitate the differentiation of melanoma-specific effector T lymphocytes, which could produce Interferon gamma (IFN-γ; Tc1) effector lymphocytes in human leukocyte antigen A2 (HLA-A2) transgenic mice (HHD2). Therefore, EVs could be associated with the transfer mechanism of functional MHC class I/peptide complexes to DCs for effective activation of cytotoxic T lymphocytes (CTL) in vivo (André et al., 2004).
Blocking pathological intercellular communication via EVs also holds therapeutic value. For example, an enhanced intracellular calcium level augments the release of EVs from K562 leukemia cells (Savina et al., 2003). Notably, blocking of H+/NA+ and Na+/Ca+ channels using dimethyl amiloride (DMA) dramatically reduces release of EVs in a mouse model of colon carcinoma, because these two channels control intracellular calcium levels (Chalmin et al., 2010). This suggests that developing techniques or drugs to obstruct the release of EVs has remarkable potential to inhibit cancer progression. Another study revealed that the enhanced level of MCT1 and CD147 in glioma cells could increase the release of EVs in an intracellular calcium-dependent manner. The enhanced level of MCT1 and CD147 in glioma cells is involved in the progression of glioma and their knock down reduces the migration and proliferation of glioma cells. Glioma cells exposed to hypoxia or lactate release a tremendously high level of EVs due to an enhanced intracellular calcium level. This was blocked by treating glioma cells with BAPTA-AM (a calcium channel blocker) as well as via genetic knockdown of MCT1 or CD147. This shows that targeting MCT1 and CD147 in glioma cells to reduce the release of EVs could be a crucial therapeutic target to inhibit glioma progression (Thakur et al., 2020c). Nonetheless in order to use EVs therapeutically, their large-scale production needs to be improved, as well as the ability to isolate EVs with high purity as well as yield, and optimization of storage conditions (Yamashita et al., 2018).
EVs as a drug delivery carrier
Numerous reports have shown that EVs can be an efficient drug delivery vehicle, delivering various cargoes to the target cells. These EVs can be either naturally obtained or synthesized with modification to enhance their drug delivery efficiency (Bunggulawa et al., 2018), and enable delivery of active pharmaceutical ingredients or siRNA (Aryani and Denecke, 2016). Interestingly, the hydrophilic core of EVs is an optimum environment for loading hydrophilic drugs (Jiang and Gao, 2017). Owing to their homing property, EVs can deliver the cargoes to distant target sites.
The EV-based delivery system has several advantages in terms of safety, specificity, and stability. EVs can evade phagocytosis and destruction by lysosomes and induce minimal immune response, owing to their small size and native formation (Ha et al., 2016). In general, stability is an important parameter in the evaluation of any delivery system, especially in the human circulation; the EV-based delivery system is stable in blood, facilitating long-distance transportation (Jiang and Gao, 2017). Osteikoetxea et al. showed that among three different types of EVs, exosomes were the least sensitive to detergents (Osteikoetxea et al., 2015), suggesting their suitability as a delivery carrier. Loading of cargoes in EVs can be before or after isolation from donor cells (van der Meel et al., 2014), and various methods have been used such as passive loading methods (incubation of cargoes with EVs, or donor cells) and active loading methods (sonication, extrusion, freeze-thaw, electroporation, incubation with membrane permeabilizing agent, click chemistry, antibody-mediated conjugation; Luan et al., 2017). Thakur A et al. showed that anti-cancer drugs such as doxorubicin and paclitaxel could be loaded into SF7761 glioma stem-like cells in the presence of saponin (acting as a permeabilizing agent) via a microfluidic Exo-Load device (Thakur et al., 2018, 2020d).
Surface modification of EVs has also been carried out to accomplish various goals such as enhancing target capacity (Tian et al., 2018) and site-specific delivery (Nakase and Futaki, 2015). EVs combined with pH-sensitive fusogenic peptide and cationic lipid have been explored for cytosolic delivery (Nakase and Futaki, 2015). EVs have been engineered with polyethylene glycol (PEG) and aminoethylanisamide (AA) to target malignant pulmonary cancer, such as lung adenocarcinoma (Kim et al., 2018; Liu et al., 2018). Fusion of EVs with liposome increased therapeutic outcome due to the enhanced targeting ability of modified EVs (Sato et al., 2016; Dimov et al., 2017).
ROLE OF EVs IN CANCER PROGRESSION
Cancer is the second leading cause of death in the United States, affecting approximately 1.7 million people with more than 600,000 associated deaths every year (Ma and Yu, 2006). Although the combination of chemotherapy, radiotherapy, and surgery is vital for cancer treatment (Ringborg et al., 2003), it is not effective in many cases (Vasan et al., 2019). The hallmarks of cancer include indefinite proliferation, evasion of apoptosis, angiogenesis, invasion, and metastasis (Chaffer and Weinberg, 2011; Baskar et al., 2012). One of the major reasons for treatment failure and cancer-related death is metastasis (Steeg, 2016). Cancer metastasis entails a series of stages controlled by cellular or molecular events. These include remodeling of the ECM, epithelial-mesenchymal transition (EMT), immune response, reprogramming of the tumor microenvironment, and involvement of bone marrow-derived cells such as MSCs in some cancers (Peinado et al., 2017). Notably, cancer cells employ angiogenesis, the growth of blood vessels from existing vasculature, to acquire nutrition and eliminate waste products from the tumor, facilitate survival and proliferation of cancer cells, and connect to the whole body circulation for distance trafficking and metastasis (Tonini et al., 2003).
Cancer cell-derived EVs and communication play an important role (He et al., 2019) in angiogenesis, migration, invasion, proliferation (Tian et al., 2019), immunomodulation (Baquir and Hancock 2017; Othman et al., 2019), and drug resistance (Zhang et al., 2018) in cancers. Mao et al. showed that EVs isolated from hypoxic esophageal squamous cell carcinoma (ESCC) cells promoted metastatic phenotypes by increasing tube formation (a measure of angiogenesis) of human umbilical vein endothelial cells (HUVECs; Mao et al., 2019). Ko et al. demonstrated that cancer cell (ovarian, colorectal, and renal cancer cell lines)-derived EVs could increase angiogenesis of endothelial cells (ECs) independent of EV uptake, indicating that EV uptake is not necessary to produce a functional change in the recipient cells (Ko et al., 2019). The phenotypic response was induced by a 189 amino acid-isomer of vascular endothelial growth factor (VEGF189), favorably localized on the membrane of EVs via its affinity to heparin. The interaction between VEGF189 and EVs significantly augmented the half-life of the ligand and decreased its recognition by bevacizumab (a VEGF antibody). Therefore, tumor angiogenesis stimulated by EV-associated VEGF (VEGF-EVs) could not be neutralized by bevacizumab. Moreover, the augmented VEGF-EVs were linked to lethality of disease when patients were treated with bevacizumab, suggesting that resistance to bevacizumab was potentially due to raised amounts of VEGF-EVs (Ko et al., 2019). Another study by Deng et al. showed that miR-155 in exosomes isolated from gastric carcinoma cells caused angiogenesis via targeting of the c-MTB-VEGF pathway in ECs, indicating that miR-155 in EVs can be a potential therapeutic target for gastric carcinoma (Deng et al., 2020). In addition, recent research has demonstrated the functionality of ovarian cancer-derived EVs carrying miR-205 that induce angiogenesis via regulation of the PTEN-AKT pathway in ECs in vitro and in vivo (He et al., 2019). Conclusively, the cancer-derived EVs can promote angiogenesis by carrying various functional cargoes and delivering them to recipient cells. Therefore, potential approaches for cancer treatment should target the release of EVs from donor cells, the pathways they regulate, or their uptake by recipient cells.
Several studies have suggested the role of remnant CSCs in the relapse or recurrence of cancer, tumor growth, metastasis, and drug resistance (Chang 2016), including the role of EVs in the progression of cancer (Wu et al., 2017). Certain stem cells including MSCs release EVs that facilitates communication in the tumor niche and plays a diverse role in metastasis, angiogenesis, and tumor formation. Nonetheless other studies have described the tumor-suppressing capacity of EVs derived from MSCs (Vakhshiteh et al., 2019). Therefore, the role of MSC-derived EVs is dependent on cell context and tissue microenvironment. It is important to understand the exact crosstalk among niche cells in the tumor microenvironment and the functional constituents of EVs during cancer progression. Several studies have suggested a potential role of EVs in assisting the formation of a pre-metastatic niche in the tumor microenvironment during cancer metastasis (Peinado et al., 2012; Costa-Silva et al., 2015; Steeg, 2016; Anderson et al., 2019). Costa-Silva et al., demonstrated that EVs from pancreatic ductal adenocarcinoma (PDAC) could initiate the generation of a pre-metastatic niche in the liver of naive mice and thereupon enhance the liver metastatic burden. The PDAC-derived EVs were engulfed by Kupffer cells causing secretion of TGF-β and enhancing the production of fibronectin by hepatic stellate cells. The fibrotic microenvironment promoted the use of macrophages derived from bone marrow. Interestingly, the PDAC-derived EVs contained a high level of macrophage migration inhibitory factor (MIF), and its impediment could obstruct the formation of a liver pre-metastatic niche (Costa-Silva et al., 2015).
TECHNOLOGICAL ADVANCEMENTS IN EV RESEARCH
Recent advances in technology have made it possible to effectively and efficiently isolate and analyze EVs (Ludwig et al., 2019). In general, the exosomes are isolated from cell culture conditioned medium (CCM) or biofluids using differential ultra-centrifugation, density gradient zone centrifugation, immunocapture by magnetic beads, exosome precipitation, or chromatography (Witwer et al., 2013; Yu et al., 2018).
Differential ultra-centrifugation
Differential ultra-centrifugation is a simple and widely used method that involves the centrifugation of CCM at different speeds to remove cell debris and obtain exosome pellets (Théry et al., 2006). Despite its widespread use, it is labor-intensive, tedious, reliant on a heavy centrifuge machine, and reduces yield because of damage to exosomes due to repeated centrifugation (Merchant et al., 2017). The method is not suitable for the isolation of exosomes from a small volume sample such as biofluids (blood serum; Witwer et al., 2013) or CSF. Another major hurdle of this method is the purification of exosomes from MVs or ABs, as the size of exosomes overlaps that of MVs and ABs (Zhang et al., 2017). There is a continuous need for better alternatives to isolate different types of EVs.
Density gradient zone centrifugation
Density gradient zone centrifugation is another means to isolate exosomes where the sample is subjected to centrifugal sedimentation after adding an inert gradient-based medium. Sucrose-based density gradient zone centrifugation is the most commonly employed method, where different components of the samples, including exosomes, are separated based on their density in the various iso-density zones (Kamerkar et al., 2017; Momen-Heravi, 2017). Compared with differential ultracentrifugation, this method separates exosomes with better purity and higher efficiency. Additionally, the separated components will not re-mix and the probability of damage to the exosomes is negligible. Nonetheless the method has limitations. The process is time-consuming, the yield of exosomes is relatively low, the intricate process requires preparation of the inert gradient medium in advance, and instruments required to perform the experiments are costly. These factors prohibit widespread use of this method (Zhang et al., 2017).
Precipitation method
Precipitation method is another widely employed method for isolating EVs. It is convenient and straightforward with various kits available commercially, such as Exo-Quick (System Bioscience), and Total Exosome Isolation reagents (Thermo Scientific). The essential process requires mixing samples with reagents followed by simple centrifugation steps. The precipitation method is useful for isolating exosomes from low volume samples. Nonetheless the reagents for exosome isolation become expensive if the process is scaled-up for a large sample size (Safdar et al., 2016).
Size exclusion chromatography
Size exclusion chromatography has also been used to isolate exosomes in various biofluids. The large particles, including cellular debris, are removed by centrifugation at low speed, followed by filtration with filters of pore size 0.8 and 0.2 µm. The resultant pellets of EVs are yielded by running through a gel filtration column, followed by ultracentrifugation at around 100,000 ×g for one hour. Despite its prevalent use, the method has several disadvantages. For example, EVs may be damaged during the filtration step. In addition, the method requires the selection of a suitable gel to exclude contaminants during isolation of EVs, and an ultracentrifugation machine is heavy (Böing et al., 2014; de Menezes-Neto et al., 2015; Lozano-Ramos et al., 2015; Szatanek et al., 2015).
Immunocapture based isolation of EVs
Immunocapture based isolation of EVs has also gained large attention mainly because of its ability to maintain the integrity of exosomes, as no repeated centrifugation steps are involved. In brief, immunomagnetic beads are coated with a primary antibody that binds to the specific target proteins on the surface of EVs, followed by incubation with the samples. Subsequently, a magnetic field is applied to separate the captured exosomes on the surface of immunomagnetic beads. In general, exosomal marker proteins such as CD9, CD63, and CD81 are utilized to isolate exosomes. This method consumes relatively less time and enables isolation of a specific population of exosomes based on their surface markers. Nonetheless the major disadvantage is the low elution yield of exosomes captured on immunomagnetic beads, making the downstream analysis a challenge.
Recently, efforts have been made to explore the possibility of combined approaches to isolate EVs to enhance their yield and reduce the time required. Benedikter et al. showed that the combination of ultrafiltration and size exclusion chromatography could efficiently isolate EVs from cell culture conditioned medium (Benedikter et al., 2017).
Several other advanced methods have been developed to isolate exosomes, such as integrated microfluidic device (Liang et al., 2017b), nanoplasmon-enhanced scattering (nPES; Liang et al., 2017a), membrane-mediated exosome isolation (Zhang et al., 2017), and on-chip exosome isolation (Liu et al., 2017a). Liang L-G et al. demonstrated a double-filtration device to isolate EVs from urine samples, detected by a microchip enzyme-linked immunosorbent assay (ELISA) method integrated with a cell phone. This integrated device could distinguish bladder cancer samples from healthy samples by integration with advanced sensing techniques, such as surface plasmon resonance (SPR; Liang et al., 2017b). Another study by Liang K et al. demonstrated a fast, ultrasensitive and low-cost nPES assay for direct quantification of cancer-derived EVs from plasma with a volume as low as around 1 μL. This method captured EVs using specific antibodies in a sensing chip, leading to the generation of a local plasmonic effect, thus increasing the sensitivity and specificity for detection of cancer-derived EVs. This technique was also reported to distinguish pancreatic cancer patients from healthy subjects via detection of an EV marker, ephrin type-A receptor 2 (EphA2), associated with pancreatic cancer for clinical diagnosis and prognosis (Liang et al., 2017a). Liu et al. demonstrated that a size-based EV isolation device, referred to as an exosome total isolation chip (ExoTIC), could isolate EVs with high-yield and high-purity from both cell lines and clinical biofluid samples including plasma, urine, and lavage. Importantly, this method was effective in isolating EVs from samples with small volume, and thus crucial for point-of-care diagnosis of cancer and other diseases (Liu et al., 2017a).
Besides isolation, advanced techniques have been developed to characterize the size and morphology, and identify the cargoes of EVs such as protein, long non-coding RNA (lncRNA), and micro-RNA (miRNA; Xu et al., 2016). The quantification of EVs depends on either the whole protein by using bicinchoninic acid (BCA)-, Bradford-, and detergent compatible protein- assay, or the number of EV particles analyzed by a nano-tracking analyzer (NTA; Koritzinsky et al., 2017). NTA can determine the size range as well as distribution of EVs although other techniques are available such as differential light scattering (DLS), transmission electron microscopy (TEM), flow cytometry, and resistive pulse sensing (RPS; van der Pol et al., 2014). The morphology of EVs can be determined using TEM, cryo-EM, and scanning electron microscopy (SEM; Noble et al., 2020). Other molecular techniques are available to identify constituents of EVs. Immunogold-EM is used for the qualitative detection of specific proteins, whereas Western blot and ELISA are used for quantification of specific proteins. The advantages and disadvantages of various methods used for characterization of EVs are listed in Table 1.
Moreover, some advanced tools, such as SPR and AFM, have been applied to analyze EV-proteins as well as their biophysical properties, such as adhesion force, stiffness, and roughness (Sharma et al., 2014). SPR allows real-time, label-free recognition of proteins (on the surface of EVs) binding to target antibodies that are immobilized on the surface of the sensing chip (Im et al., 2017). AFM has been widely employed to characterize morphology, biomolecular constituents, and biomechanics of EVs, because of its prolific role in the study of biomarkers and therapeutics (Sharma et al., 2018). In addition, Raman spectroscopy has been used to study EVs and is based on measurement of the scattered photons by vibration after exposing samples to a monochromatic laser light. The photons alter their energy by excitation of molecular vibration, resulting in a Raman spectrum that enables determination of the molecular composition of a sample by comparing results with known vibration modes of specific chemical groups. Importantly, this method is label-free (without the need to tag the molecule of interest), does not distort the morphology of EVs, and enables analysis of exosomes in the context of the exosomal membrane lipid/protein content along with other modifications on the surface (Carmicheal et al., 2019).
Artificial intelligence (AI) has recently been applied in EV research (Thakur et al., 2020a, 2020b) for clinical diagnosis and prognosis, as disease specific parent cells can be identified using an AI-based classification approach and datasets of EVs (Thakur et al., 2020a).
MICROFLUIDIC SYSTEMS IN EV RESEARCH
EVs occupy a crucial position amidst the biomarker studies due to their clinical utility for the diagnosis and prognosis of diseases (Lane et al., 2018; Ma et al., 2019). Nonetheless, there is a dearth of robust systems by which to achieve their convenient and efficient isolation, characterization, and examination.
A promising technique to solve this problem is the microfluidic system. A microfluidic system is a device with separation and sensing features. It can perform isolation, detection, and analysis of EVs in one single platform. In particular, the microfluidic technique has a broad range of applications in the development of point-of-care devices for clinical use, and offers a new avenue to use EV-based liquid biopsy for personalized medicine (Contreras-Naranjo et al., 2017). Typically, the microfluidic-based platforms contain various components for isolation of EVs, such as membrane-based filtration, acoustic nano-filtration, immunoaffinity, deterministic lateral displacement (DLD) sorting, and trapping on nanowires (Contreras-Naranjo et al., 2017). In the membrane-based filtration microfluidic system, many techniques have been developed to better facilitate the isolation of EVs, such as pressure-driven filtration, electrophoresis-driven filtration (Davies et al., 2012), electrophoretic isolation on the nano-porous membrane (Cho et al., 2016), and double filtration (Liang et al., 2017b; Woo et al., 2017). Lee, Kyungheon et al. (2015) utilized differential ultrasound forces in an acoustic nano-filter system that was integrated into a microfluidic device to isolate EVs by size in a contact-free manner (Lee et al., 2015). Further, various microfluidic systems have been designed for immunocapture of EVs by targeting their specific surface markers (Kanwar et al., 2014). For example, the antibodies against CD24, CD63 or EpCAM, conjugated on the gold surface with nanohole arrays (reusable nPLEX), can facilitate capture of specific populations of EVs (Im et al., 2014). The capture of anti-CD9, anti-HER2 antibodies on the functionalized gold electrodes could be enhanced by a nano-shearing approach (Vaidyanathan et al., 2014). A study by Mei et al. reported isolation of EVs using antibodies targeting other EV surface proteins, such as insulin like growth factor 1 receptor (IGF-1R), CA125, CD9, CD63, and CD81, using immunomagnetic microbead (He et al., 2014). A microfluidic device was developed to specifically isolate a subpopulation of exosomes from plasma samples and quantitatively detect surface biomarkers with improved sensitivity (He et al., 2014). The application of rapid inertial solution exchange (RInSE) was also employed for the isolation of EVs via EpCAM based affinity-capture microbeads (Dudani et al., 2015). For DLD sorting in the microfluidic system, an array of pillars has been utilized to isolate EVs (Wunsch et al., 2016). For instance, ciliated micropillars were employed in a microfluidic system to trap EVs on nanowires (Wang et al., 2013). Overall, various miniaturization- and functionalization- based EV isolation approaches have augmented yields, consumed less time, and attained better purity.
The integration of the microfluidic technique with other methods has also improved the analysis of EVs. For example, the sequential ExoChip that combines immunoaffinity-based isolation of EVs and fluorescent dye-based staining can isolate and detect EVs in one process (Kanwar et al., 2014). Another study demonstrated the integration of immunoaffinity-based EV isolation and fluorescence immunoassay in sequential nano-interfaced microfluidic exosome (nano-IMEX; Zhang et al., 2016a). Similarly, better analyses of EVs was achieved by a sequential Exodisc that combined double filtration isolation and colorimetric ELISA (Woo et al., 2017). In addition, an integrated microfluidic system for the analysis of miRNA in EVs was developed and combined sequential iMER, immunomagnetic isolation, exosome lysis, RNA capture, and multiplexed RT-qPCR. Advancements in the development of microfluidic systems have solved various issues pertaining to EV-based studies. Nonetheless several challenges remain to be addressed before it can be used in routine clinical practice such as antibody free processing, integration of microfluidic system with downstream analysis, standardization of techniques, and the possibility of commercialization (Contreras-Naranjo et al., 2017).
CONCLUSION AND FUTURE PERSPECTIVES
EVs play important roles in stem cell biology, including intercellular communication in the stem cell-niche and at various stages of embryonic development. In addition, the role of EVs in cancer progression has begun to be elucidated. Studies of the application of EVs as a diagnostic and therapeutic tool have gained attention due to their numerous outstanding advantages. For example, EVs can be isolated from body fluids (such as blood, CSF, and urine) non-invasively, and continuous time-dependent monitoring of a patient’s response is possible with EV-based therapeutics. Because of their small size, EVs can cross physiological barriers, including the BBB, making them useful in drug delivery. The benefits of EVs in therapy of various diseases including cancer have also been studied. Other critical aspects of the application of EVs involve their self-renewal capacity, ability to arbitrate communication between stem cells and niche, and their functions in stem cell differentiation or cell fate determination. The role of EVs in normal physiological processes and disease conditions has also been extensively studied, including malignant progression of cancer. The exploration of various multidimensional roles of EVs have become possible with technological advancements in methods for isolation and characterization of EVs, and the development of advanced microfluid based systems for studies associated with EVs.
The role of EVs has yet to be thoroughly explored. In the study of disease progression via intercellular communication of EVs, the potential of EVs to carry various cargoes to recipient cells warrants attention. In the context of cancer progression, there are several drugs such as paclitaxel, which causes neuropathic pain, or drugs against which drug resistance has developed such as temozolomide for glioma, that could be a cargo for EVs. Furthermore, the exploration of potential changes in the molecular constituents of stem cells, after uptake of EVs released from a different subset of cancer cells, may help future research into the mechanisms that underlie the development of drug resistance.
The use of EVs as a carrier for either genetic materials such as lncRNA, miRNA, or small molecules such as anti-cancer drugs, holds great promise for the development of novel delivery methods. One of the important advantages of EVs as a delivery carrier is their ability to escape the reticuloendothelial system (RES). Nonetheless the synthetic nano-delivery systems are often prone to be captured by RES and therefore very likely to prompt an immune response. In the prevailing scenario, the use of EVs, particularly autologous EVs, has strong potential for targeted delivery after surface modifications with specific ligands against the target receptor, for example, epidermal growth factor receptor (EGFR) in lung cancer and EGFRvIII in glioma.
The application of EVs to track disease progression, including metastatic progression of cancer, is an exciting prospect. The conventional approaches to diagnosis of cancer, such as investigation of molecular changes in tissues via biopsy, imaging techniques like magnetic resonance imaging or computed tomography in glioma, and digital rectal examination in prostate cancer, are limited by their resolution in detecting the development of tumor. In the prevailing scenario, the development of biosensors is required to detect EVs and their constituents with high sensitivity and specificity for tracking disease diagnosis. Additionally, mapping of various proteins on the surface of EVs could be used to potentially demarcate the disease state.
Although there is significant ongoing research associated with EVs, there remains a need to develop a universal protocol for their isolation and characterization, as well as for their clinical application. When available, it could be certified and applicable for good clinical practice (GMP). A specific road map could be developed for the translation of EVs from bench to bedside.
Abbreviations
- EPCs:
-
endothelial progenitor
- ESCs:
-
embryonic stem cells
- EVs:
-
extracellular vesicles
- HSCs:
-
hematopoietic stem cells
- MSCs:
-
mesenchymal stem cells
- MVBs:
-
multivesicular bodies
- NSCs:
-
neural stem cells
- SSCs:
-
somatic stem cells
References
Alberti C, Cochella L (2017) A framework for understanding the roles of miRNAs in animal development. Development 144:2548–2559
Aliotta JM, Pereira M, Johnson KW et al (2010) Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription. Exp Hematol 38:233–245
Anderson RL, Balasas T, Callaghan J et al (2019) A framework for the development of effective anti-metastatic agents. Nat Rev Clin Oncol 16:185–204
André F, Chaput N, Schartz NEC et al (2004) Exosomes as Potent Cell-Free Peptide-Based Vaccine. I. Dendritic Cell-Derived Exosomes Transfer Functional MHC Class I/Peptide Complexes to Dendritic Cells. J Immunol 172:2126–2136
Arslan F, Lai RC, Smeets MB et al (2013) Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res 10:301–312
Aryani A, Denecke B (2016) Exosomes as a Nanodelivery system: a key to the future of neuromedicine? Mol Neurobiol 53:818–834
Asea A, Jean-Pierre C, Kaur P et al (2008) Heat shock protein-containing exosomes in mid-trimester amniotic fluids. J Reprod Immunol 79:12–17
Baquir B, Hancock REW (2017) Exosomes, your body’s answer to immune health. Ann Transl Med 5:81–81
Baskar R, Lee KA, Yeo R, Yeoh K-W (2012) Cancer and radiation therapy: current advances and future directions. Int J Med Sci 9:193–199
Basu B, Ghosh MK (2019) Extracellular vesicles in glioma: from diagnosis to therapy. BioEssays 41:1800245
Battaglia R, Palini S, Vento ME et al (2019) Identification of extracellular vesicles and characterization of miRNA expression profiles in human blastocoel fluid. Sci Rep 9:84
Becker JC (2014) Tumor-educated myeloid cells: impact the micro- and macroenvironment. Exp Dermatol 23:157–158
Beer KB, Wehman AM (2017) Mechanisms and functions of extracellular vesicle release in vivo—what we can learn from flies and worms. Cell Adh Migr 11:135–150
Bellavia D, Raimondi L, Costa V et al (2018) Engineered exosomes: a new promise for the management of musculoskeletal diseases. Biochim Biophys Acta 1862:1893–1901
Benedikter BJ, Bouwman FG, Vajen T et al (2017) Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. Sci Rep 7:15297
Bernard V, Ling J, Maitra A (2018) Heterogeneity of tumor exosomes – role in precision medicine. In: Diagnostic and therapeutic applications of exosomes in cancer. Elsevier, Amsterdam, pp 59–67
Bier E, De Robertis EM (2015) BMP gradients: a paradigm for morphogen-mediated developmental patterning. Science 348:aaa5838–aaa5838
Böing AN, van der Pol E, Grootemaat AE et al (2014) Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles 3:23430
Booth AM, Fang Y, Fallon JK et al (2006) Exosomes and HIV gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol 172:923–935
Boriachek K, Islam MN, Gopalan V et al (2017) Quantum dot-based sensitive detection of disease specific exosome in serum. Analyst 142:2211–2219
Budnik V, Ruiz-Cañada C, Wendler F (2016) Extracellular vesicles round off communication in the nervous system. Nat Rev Neurosci 17:160–172
Bunggulawa EJ, Wang W, Yin T et al (2018) Recent advancements in the use of exosomes as drug delivery systems. J Nanobiotechnol 16:81
Camussi G, Deregibus MC, Bruno S et al (2010) Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int 78:838–848
Cao M, Ning J, Hernandez-Lara CI et al (2015) Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding. Elife. https://doi.org/10.7554/eLife.05242
Cappello V, Marchetti L, Parlanti P et al (2016) Ultrastructural characterization of the lower motor system in a mouse model of Krabbe disease. Sci Rep 6:1
Carmicheal J, Hayashi C, Huang X et al (2019) Label-free characterization of exosome via surface enhanced Raman spectroscopy for the early detection of pancreatic cancer. Nanomed Nanotechnol Biol Med 16:88–96
Chaffer CL, Weinberg RA (2011) A perspective on cancer cell metastasis. Science 331:1559–1564
Chalmin F, Ladoire S, Mignot G et al (2010) Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin Invest. https://doi.org/10.1172/JCI40483
Chang JC (2016) Cancer stem cells. Medicine (baltimore) 95:S20–S25
Chen I-H, Xue L, Hsu C-C et al (2017a) Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. Proc Natl Acad Sci 114:3175–3180
Chen Z, Li Y, Yu H, et al (2017b) Isolation of extracellular vesicles from stem cells. pp 389–394
Chernyshev VS, Rachamadugu R, Tseng YH et al (2015) Size and shape characterization of hydrated and desiccated exosomes. Anal Bioanal Chem 407:3285–3301
Cho S, Jo W, Heo Y et al (2016) Isolation of extracellular vesicle from blood plasma using electrophoretic migration through porous membrane. Sens Actuators B Chem 233:289–297
Choi H, Mun JY (2017) Structural analysis of exosomes using different types of electron microscopy. Appl Microsc 47:171–175
Colombo M, Raposo G, Théry C (2014) Biogenesis, secretion, and iIntercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30:255–289
Conde-Vancells J, Rodriguez-Suarez E, Embade N et al (2008) Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes. J Proteome Res 7:5157–5166
Contreras-Naranjo JC, Wu H-J, Ugaz VM (2017) Microfluidics for exosome isolation and analysis: enabling liquid biopsy for personalized medicine. Lab Chip 17:3558–3577
Corrado C, Raimondo S, Chiesi A et al (2013) Exosomes as intercellular signaling organelles involved in health and disease: basic science and clinical applications. Int J Mol Sci 14:5338–5366
Corrigan L, Redhai S, Leiblich A et al (2014) BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior. J Cell Biol 206:671–688
Costa-Silva B, Aiello NM, Ocean AJ et al (2015) Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol 17:816–826
Cruz L, Arevalo Romero JA, Brandão Prado M et al (2018a) Evidence of extracellular vesicles biogenesis and release in mouse embryonic stem cells. Stem Cell Rev Reports 14:262–276
Cruz L, Romero JAA, Iglesia RP, Lopes MH (2018b) Extracellular vesicles: decoding a new language for cellular communication in early embryonic development. Front Cell Dev Biol. https://doi.org/10.3389/fcell.2018.00094
Danilchik M, Williams M, Brown E (2013) Blastocoel-spanning filopodia in cleavage-stage Xenopus laevis: Potential roles in morphogen distribution and detection. Dev Biol 382:70–81
Davies RT, Kim J, Jang SC et al (2012) Microfluidic filtration system to isolate extracellular vesicles from blood. Lab Chip 12:5202
de la Gomez C T, Goreham RV, Bech Serra JJ et al (2018) “Exosomics”—a review of biophysics, biology and biochemistry of exosomes with a focus on human breast milk. Front Genet. https://doi.org/10.3389/fgene.2018.00092
de Menezes-Neto A, Sáez MJF, Lozano-Ramos I et al (2015) Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals. J Extracell Vesicles 4:27378
De Toro J, Herschlik L, Waldner C, Mongini C (2015) Emerging roles of exosomes in normal and pathological conditions: new insights for diagnosis and therapeutic applications. Front Immunol. https://doi.org/10.3389/fimmu.2015.00203
Deng T, Zhang H, Yang H et al (2020) Exosome miR-155 derived from gastric carcinoma promotes angiogenesis by targeting the c-MYB/VEGF axis of endothelial cells. Mol Ther Nucleic Acids 19:1449–1459
Deregibus MC, Cantaluppi V, Calogero R et al (2007) Endothelial progenitor cell–derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood 110:2440–2448
Desrochers LM, Bordeleau F, Reinhart-King CA et al (2016) Microvesicles provide a mechanism for intercellular communication by embryonic stem cells during embryo implantation. Nat Commun 7:11958
Dignat-George F, Boulanger CM (2011) The many faces of endothelial microparticles. Arterioscler Thromb Vasc Biol 31:27–33
Dimov N, Kastner E, Hussain M et al (2017) Formation and purification of tailored liposomes for drug delivery using a module-based micro continuous-flow system. Sci Rep 7:12045
Draening T, Heigwer J, Juergensen L et al (2018) Extracellular vesicle-delivered bone morphogenetic proteins: a novel paracrine mechanism during embryonic development. BioRxiv. https://doi.org/10.1101/321356
Dudani JS, Gossett DR, Tse HTK et al (2015) Rapid inertial solution exchange for enrichment and flow cytometric detection of microvesicles. Biomicrofluidics 9:014112
Fairchild CL, Barna M (2014) Specialized filopodia: at the ‘tip’ of morphogen transport and vertebrate tissue patterning. Curr Opin Genet Dev 27:67–73
Fang S, Tian H, Li X et al (2017) Clinical application of a microfluidic chip for immunocapture and quantification of circulating exosomes to assist breast cancer diagnosis and molecular classification. PLoS ONE 12:e0175050
Filipe V, Hawe A, Jiskoot W (2010) Critical evaluation of nanoparticle tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res 27:796–810
Fröhlich D, Kuo WP, Frühbeis C et al (2014) Multifaceted effects of oligodendroglial exosomes on neurons: impact on neuronal firing rate, signal transduction and gene regulation. Philos Trans R Soc B Biol Sci 369:20130510
Frühbeis C, Fröhlich D, Kuo WP et al (2013) Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication. PLoS Biol 11:e1001604
Gallagher S, Winston SE, Fuller SA, Hurrell JGR (2008) Immunoblotting and immunodetection. Curr Protoc Mol Biol. https://doi.org/10.1002/0471142727.mb1008s83
Gatti J-L, Métayer S, Belghazi M et al (2005) Identification, proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles1. Biol Reprod 72:1452–1465
Goreham RV, Ayed Z, Amin ZM, Dobhal G (2020) The future of quantum dot fluorescent labelling of extracellular vesicles for biomedical applications. Nano Futur 4:022001
Gradilla A-C, González E, Seijo I et al (2014) Exosomes as Hedgehog carriers in cytoneme-mediated transport and secretion. Nat Commun 5:5649
Greco V, Hannus M, Eaton S (2001) Argosomes: a potential vehicle for the spread of morphogens through epithelia. Cell 106:633–645
Gross JC, Chaudhary V, Bartscherer K, Boutros M (2012) Active Wnt proteins are secreted on exosomes. Nat Cell Biol 14:1036–1045
Gross N, Kropp J, Khatib H (2017) MicroRNA signaling in embryo development. Biology (basel) 6:34
Gualerzi A, Kooijmans SAA, Niada S et al (2019) Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality. J Extracell Vesicles 8:1568780
Gupta S, Knowlton AA (2007) HSP60 trafficking in adult cardiac myocytes: role of the exosomal pathway. Am J Physiol Circ Physiol 292:H3052–H3056
György B, Sage C, Indzhykulian AA et al (2017) Rescue of hearing by gene delivery to inner-ear hair cells using exosome-associated AAV. Mol Ther 25:379–391
Ha D, Yang N, Nadithe V (2016) Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges. Acta Pharm Sin B 6:287–296
Hardij J, Cecchet F, Berquand A et al (2013) Characterisation of tissue factor-bearing extracellular vesicles with AFM: comparison of air-tapping-mode AFM and liquid Peak Force AFM. J Extracell Vesicles 2:21045
Harding C, Heuser J, Stahl P (1984) Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding. Eur J Cell Biol 35:256–263
He L, Zhu W, Chen Q et al (2019) Ovarian cancer cell-secreted exosomal miR-205 promotes metastasis by inducing angiogenesis. Theranostics 9:8206–8220
He M, Crow J, Roth M et al (2014) Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology. Lab Chip 14:3773
Heckman CA, Plummer HK (2013) Filopodia as sensors. Cell Signal 25:2298–2311
Hosseinkhani B, van den Akker N, D’Haen J et al (2017) Direct detection of nano-scale extracellular vesicles derived from inflammation-triggered endothelial cells using surface plasmon resonance. Nanomed Nanotechnol Biol Med 13:1663–1671
Hsu H-J, Bahader M, Lai C-M (2019) Molecular control of the female germline stem cell niche size in Drosophila. Cell Mol Life Sci 76:4309–4317
Huang H, Wang C, Liu F et al (2018) Reciprocal network between cancer stem-like cells and macrophages facilitates the progression and androgen deprivation therapy resistance of prostate cancer. Clin Cancer Res 24:4612–4626
Hur YH, Cerione RA, Antonyak MA (2020) Extracellular vesicles and their roles in stem cell biology. Stem Cells 38:469–476
Ibrahim AG-E, Cheng K, Marbán E (2014) Exosomes as critical agents of cardiac regeneration triggered by cell therapy. Stem Cell Rep 2:606–619
Im H, Shao H, Il PY et al (2014) Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor. Nat Biotechnol 32:490–495
Im H, Yang K, Lee H, Castro CM (2017) Characterization of extracellular vesicles by surface plasmon resonance. pp 133–141
Jeong S, Park J, Pathania D et al (2016) Integrated magneto-electrochemical sensor for exosome analysis. ACS Nano 10:1802–1809
Jiang D, Gao F, Zhang Y et al (2016) Mitochondrial transfer of mesenchymal stem cells effectively protects corneal epithelial cells from mitochondrial damage. Cell Death Dis 7:e2467–e2467
Jiang D, Xiong G, Feng H et al (2019) Donation of mitochondria by iPSC-derived mesenchymal stem cells protects retinal ganglion cells against mitochondrial complex I defect-induced degeneration. Theranostics 9:2395–2410
Jiang X-C, Gao J-Q (2017) Exosomes as novel bio-carriers for gene and drug delivery. Int J Pharm 521:167–175
Johnstone RM, Adam M, Hammond JR et al (1987) Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 262:9412–9420
Jones DL, Wagers AJ (2008) No place like home: anatomy and function of the stem cell niche. Nat Rev Mol Cell Biol 9:11–21
Jung YJ, Kim HK, Cho Y, et al (2020) Cell reprogramming using extracellular vesicles from differentiating stem cells into white/beige adipocytes. Sci Adv 6:eaay6721
Kalluri R, LeBleu VS (2020) The biology, function , and biomedical applications of exosomes. Science 367:eaau6977
Kamerkar S, LeBleu VS, Sugimoto H et al (2017) Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 546:498–503
Kanellopoulou C, Gilpatrick T, Kilaru G et al (2015) Reprogramming of polycomb-mediated gene silencing in embryonic stem cells by the miR-290 family and the methyltransferase Ash1l. Stem Cell Rep 5:971–978
Kanwar SS, Dunlay CJ, Simeone DM, Nagrath S (2014) Microfluidic device (ExoChip) for on-chip isolation, quantification and characterization of circulating exosomes. Lab Chip 14:1891–1900
Kao Y-C, Ho P-C, Tu Y-K et al (2020) Lipids and Alzheimer’s disease. Int J Mol Sci 21:1505
Kato S, Goodman A, Walavalkar V et al (2017) Hyperprogressors after immunotherapy: analysis of genomic alterations associated with accelerated growth rate. Clin Cancer Res 23:4242–4250
Kawakami K, Fujita Y, Matsuda Y et al (2017) Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer. BMC Cancer 17:316
Khan M, Nickoloff E, Abramova T et al (2015) Embryonic stem cell-derived exosomes promote endogenous repair mechanisms and enhance cardiac function following myocardial infarction. Circ Res 117:52–64
Khawar MB, Abbasi MH, Siddique Z et al (2019) An update on novel therapeutic warfronts of extracellular vesicles (EVs) in cancer treatment: where we are standing right now and where to go in the future. Oxid Med Cell Longev 2019:1–21
Kim MS, Haney MJ, Zhao Y et al (2018) Engineering macrophage-derived exosomes for targeted paclitaxel delivery to pulmonary metastases: in vitro and in vivo evaluations. Nanomed Nanotechnol Biol Med 14:195–204
Kirkeby A, Perlmann T, Pereira C-F (2016) The stem cell niche finds its true north. Development 143:2877–2881
Klinov D, Magonov S (2004) True molecular resolution in tapping-mode atomic force microscopy with high-resolution probes. Appl Phys Lett 84:2697–2699
Ko J, Carpenter E, Issadore D (2016) Detection and isolation of circulating exosomes and microvesicles for cancer monitoring and diagnostics using micro-/nano-based devices. Analyst 141:450–460
Ko SY, Lee W, Kenny HA et al (2019) Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake. Commun Biol 2:386
Koles K, Nunnari J, Korkut C et al (2012) Mechanism of evenness interrupted (Evi)-exosome release at synaptic boutons. J Biol Chem 287:16820–16834
Koritzinsky EH, Street JM, Star RA, Yuen PST (2017) Quantification of exosomes. J Cell Physiol 232:1587–1590
Korkut C, Ataman B, Ramachandran P et al (2009) Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless. Cell 139:393–404
Kwizera EA, O’Connor R, Vinduska V et al (2018) Molecular detection and analysis of exosomes using surface-enhanced Raman scattering gold nanorods and a miniaturized device. Theranostics 8:2722–2738
Lane RE, Korbie D, Hill MM, Trau M (2018) Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges. Clin Transl Med 7:14
Le Borgne R, Schweisguth F (2003) Notch signaling: endocytosis makes delta signal better. Curr Biol 13:R273–R275
Lee K, Shao H, Weissleder R, Lee H (2015) Acoustic purification of extracellular microvesicles. ACS Nano 9:2321–2327
Li J, Chen Y, Guo X et al (2017) GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer. J Cell Mol Med 21:838–847
Li J, Sherman-Baust CA, Tsai-Turton M et al (2009) Claudin-containing exosomes in the peripheral circulation of women with ovarian cancer. BMC Cancer 9:244
Li L, Xie T (2005) STEM CELL NICHE: structure and function. Annu Rev Cell Dev Biol 21:605–631
Liang K, Liu F, Fan J et al (2017a) Nanoplasmonic quantification of tumour-derived extracellular vesicles in plasma microsamples for diagnosis and treatment monitoring. Nat Biomed Eng 1:0021
Liang L-G, Kong M-Q, Zhou S et al (2017b) An integrated double-filtration microfluidic device for isolation, enrichment and quantification of urinary extracellular vesicles for detection of bladder cancer. Sci Rep 7:46224
Liégeois S, Benedetto A, Garnier J-M et al (2006) The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans. J Cell Biol 173:949–961
Liu C, Zhao J, Tian F et al (2019) Low-cost thermophoretic profiling of extracellular-vesicle surface proteins for the early detection and classification of cancers. Nat Biomed Eng 3:183–193
Liu F, Vermesh O, Mani V et al (2017a) The exosome total isolation chip. ACS Nano 11:10712–10723
Liu R, Wang X, Curtiss C et al (2018) Monoglyceride lipase gene knockout in mice leads to increased incidence of lung adenocarcinoma. Cell Death Dis 9:36
Liu X, Li Q, Niu X et al (2017b) Exosomes secreted from human-induced pluripotent stem cell-derived mesenchymal stem cells prevent osteonecrosis of the femoral head by promoting angiogenesis. Int J Biol Sci 13:232–244
Logozzi M, Angelini DF, Iessi E et al (2017) Increased PSA expression on prostate cancer exosomes in in vitro condition and in cancer patients. Cancer Lett 403:318–329
Loh KM, van Amerongen R, Nusse R (2016) Generating cellular diversity and spatial form: Wnt signaling and the evolution of multicellular animals. Dev Cell 38:643–655
Lozano-Ramos I, Bancu I, Oliveira-Tercero A et al (2015) Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples. J Extracell Vesicles 4:27369
Luan X, Sansanaphongpricha K, Myers I et al (2017) Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin 38:754–763
Ludwig N, Whiteside TL, Reichert TE (2019) Challenges in exosome isolation and analysis in health and disease. Int J Mol Sci 20:4684
Das S (2014) Extracellular vesicles in heart disease: excitement for the future? Exosomes Microvesicle. https://doi.org/10.5772/58390
Ma C, Jiang F, Ma Y et al (2019) Isolation and detection technologies of extracellular vesicles and application on cancer diagnostic. Dose-Response 17:155932581989100
Ma X, Yu H (2006) Global burden of cancer. Yale J Biol Med 79:85–94
Maas SLN, De Vrij J, Broekman MLD (2014) Quantification and size-profiling of extracellular vesicles using tunable resistive pulse sensing. J vis Exp. https://doi.org/10.3791/51623
Maguire JE, Silva M, Nguyen KCQ et al (2015) Myristoylated CIL-7 regulates ciliary extracellular vesicle biogenesis. Mol Biol Cell 26:2823–2832
Maia J, Caja S, Strano Moraes MC et al (2018) Exosome-based cell-cell communication in the tumor microenvironment. Front Cell Dev Biol. https://doi.org/10.3389/fcell.2018.00018
Mao Y, Wang Y, Dong L et al (2019) Hypoxic exosomes facilitate angiogenesis and metastasis in esophageal squamous cell carcinoma through altering the phenotype and transcriptome of endothelial cells. J Exp Clin Cancer Res 38:389
Martin TA, Ye L, Sanders AJ et al (2013) Cancer invasion and metastasis: molecular and cellular perspective. In: Metastatic cancer: clinical and biological perspectives
Mašek J, Andersson ER (2017) The developmental biology of genetic Notch disorders. Development 144:1743–1763
Masyuk AI, Huang BQ, Ward CJ et al (2010) Biliary exosomes influence cholangiocyte regulatory mechanisms and proliferation through interaction with primary cilia. Am J Physiol Liver Physiol 299:G990–G999
Matusek T, Wendler F, Polès S et al (2014) The ESCRT machinery regulates the secretion and long-range activity of Hedgehog. Nature 516:99–103
McMahon AP, Hasso SM (2013) Filopodia: the cellular quills of Hedgehog signaling? Dev Cell 25:328–330
Melo SA, Luecke LB, Kahlert C et al (2015) Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523:177–182
Menck K, Scharf C, Bleckmann A et al (2015) Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN. J Mol Cell Biol 7:143–153
Merchant ML, Rood IM, Deegens JKJ, Klein JB (2017) Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery. Nat Rev Nephrol 13:731–749
Minciacchi VR, Spinelli C, Reis-Sobreiro M et al (2017) MYC mediates large oncosome-induced fibroblast reprogramming in prostate cancer. Cancer Res 77:2306–2317
Momen-Heravi F (2017) Isolation of extracellular vesicles by ultracentrifugation. In: Methods in Molecular Biology. pp 25–32
Nakase I, Futaki S (2015) Combined treatment with a pH-sensitive fusogenic peptide and cationic lipids achieves enhanced cytosolic delivery of exosomes. Sci Rep 5:10112
Noble JM, Roberts LM, Vidavsky N et al (2020) Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles. J Struct Biol 210:107474
Ohlstein B, Kai T, Decotto E, Spradling A (2004) The stem cell niche: theme and variations. Curr Opin Cell Biol 16:693–699
Osteikoetxea X, Sódar B, Németh A et al (2015) Differential detergent sensitivity of extracellular vesicle subpopulations. Org Biomol Chem 13:9775–9782
Oszvald Á, Szvicsek Z, Sándor GO et al (2019) Extracellular vesicles transmit epithelial growth factor activity in the intestinal stem cell niche. Stem Cells. https://doi.org/10.1002/stem.3113
Othman N, Jamal R, Abu N (2019) Cancer-derived exosomes as effectors of key inflammation-related players. Front Immunol. https://doi.org/10.3389/fimmu.2019.02103
Palmieri V, Lucchetti D, Gatto I et al (2014) Dynamic light scattering for the characterization and counting of extracellular vesicles: a powerful noninvasive tool. J Nanoparticle Res 16:2583
Pan BT, Teng K, Wu C et al (1985) Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes. J Cell Biol 101:942–948
Panáková D, Sprong H, Marois E et al (2005) Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435:58–65
Pang B, Zhu Y, Ni J et al (2020) Extracellular vesicles: the next generation of biomarkers for liquid biopsy-based prostate cancer diagnosis. Theranostics 10:2309–2326
Paolini L, Zendrini A, Radeghieri A (2018) Biophysical properties of extracellular vesicles in diagnostics. Biomark Med 12:383–391
Peinado H, Alečković M, Lavotshkin S et al (2012) Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med 18:883–891
Peinado H, Zhang H, Matei IR et al (2017) Pre-metastatic niches: organ-specific homes for metastases. Nat Rev Cancer 17:302–317
Peng Y, Baulier E, Ke Y et al (2018) Human embryonic stem cells extracellular vesicles and their effects on immortalized human retinal Müller cells. PLoS ONE 13:e0194004
Pennings S, Liu KJ, Qian H (2018) The stem cell niche: interactions between stem cells and their environment. Stem Cells Int 2018:1–3
Plusa B, Hadjantonakis A-K (2014) Embryonic stem cell identity grounded in the embryo. Nat Cell Biol 16:502–504
Quesenberry PJ, Dooner MS, Aliotta JM (2010) Stem cell plasticity revisited: the continuum marrow model and phenotypic changes mediated by microvesicles. Exp Hematol 38:581–592
Raposo G, Nijman HW, Stoorvogel W et al (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183:1161–1172
Ratajczak J, Miekus K, Kucia M et al (2006) Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia 20:847–856
Ringborg U, Bergqvist D, Brorsson B et al (2003) The Swedish Council on Technology Assessment in Health Care (SBU) systematic overview of radiotherapy for cancer including a prospective survey of radiotherapy practice in Sweden 2001–summary and conclusions. Acta Oncol 42:357–365
Ronquist G, Brody I (1985) The prostasome: its secretion and function in man. Biochim Biophys Act 822:203–218
Rosa-Fernandes L, Rocha VB, Carregari VC et al (2017) A perspective on extracellular vesicles proteomics. Front Chem. https://doi.org/10.3389/fchem.2017.00102
Rozenblum P, Radrizzani (2019) Challenges in electrochemical aptasensors and current sensing architectures using flat gold surfaces. Chemosensors 7:57
Russell JC, Postupna N, Golubeva A et al (2020) Purification and analysis of Caenorhabditis elegans extracellular vesicles. J vis Exp. https://doi.org/10.3791/60596
Saenz-Antoñanzas A-I et al (2019) Liquid biopsy in glioblastoma: opportunities, applications and challenges. Cancers (basel) 11:950
Safdar A, Saleem A, Tarnopolsky MA (2016) The potential of endurance exercise-derived exosomes to treat metabolic diseases. Nat Rev Endocrinol 12:504–517
Sagar PF, Wiegreffe C, Scaal M (2015) Communication between distant epithelial cells by filopodia-like protrusions during embryonic development. Development 142:665–671
Salas-Vidal E, Lomeli H (2004) Imaging filopodia dynamics in the mouse blastocyst. Dev Biol 265:75–89
Salomon C, Kobayashi M, Tapia J et al (2015) Exosomes are fingerprints of originating cells: potential biomarkers for ovarian cancer. Res Reports Biochem. https://doi.org/10.2147/RRBC.S58281
Sandfeld-Paulsen B, Aggerholm-Pedersen N, Baek R et al (2016) Exosomal proteins as prognostic biomarkers in non-small cell lung cancer. Mol Oncol 10:1595–1602
Sato YT, Umezaki K, Sawada S et al (2016) Engineering hybrid exosomes by membrane fusion with liposomes. Sci Rep 6:21933
Savina A, Furlán M, Vidal M, Colombo MI (2003) Exosome release is regulated by a calcium-dependent mechanism in K562 cells. J Biol Chem 278:20083–20090
Segura E, Nicco C, Lombard B et al (2005) ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming. Blood 106:216–223
Sharma S, Das K, Woo J, Gimzewski JK (2014) Nanofilaments on glioblastoma exosomes revealed by peak force microscopy. J R Soc Interface 11:20131150
Sharma S, Gillespie BM, Palanisamy V, Gimzewski JK (2011) Quantitative nanostructural and single-molecule force spectroscopy biomolecular analysis of human-saliva-derived exosomes. Langmuir 27:14394–14400
Sharma S, LeClaire M, Gimzewski JK (2018) Ascent of atomic force microscopy as a nanoanalytical tool for exosomes and other extracellular vesicles. Nanotechnology 29:132001
Sharma S, Rasool HI, Palanisamy V et al (2010) Structural-mechanical characterization of nanoparticle exosomes in human saliva, using correlative AFM, FESEM, and force spectroscopy. ACS Nano 4:1921–1926
Sharma S, Zuñiga F, Rice GE et al (2017) Tumor-derived exosomes in ovarian cancer & liquid biopsies for early detection and real-time monitoring of cancer progression. Oncotarget. https://doi.org/10.18632/oncotarget.22191
Sheldon H, Heikamp E, Turley H et al (2010) New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes. Blood 116:2385–2394
Simons M, Raposo G (2009) Exosomes-vesicular carriers for intercellular communication. Curr Opin Cell Biol 21:575–581
Song M, Han L, Chen F et al (2018) Adipocyte-derived exosomes carrying Sonic Hedgehog mediate M1 macrophage polarization-induced insulin resistance via Ptch and PI3K pathways. Cell Physiol Biochem 48:1416–1432
Soo CY, Song Y, Zheng Y et al (2012) Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells. Immunology 136:192–197
Soung Y, Ford S, Zhang V, Chung J (2017) Exosomes in cancer diagnostics. Cancers (basel) 9:8
Stahl PD, Raposo G (2018) Exosomes and extracellular vesicles: the path forward. Essays Biochem 62:119–124
Steeg PS (2016) Targeting metastasis. Nat Rev Cancer 16:201–218
Stronati E, Conti R, Cacci E et al (2019) Extracellular vesicle-induced differentiation of neural stem progenitor cells. Int J Mol Sci 20:3691
Subra C, Grand D, Laulagnier K et al (2010) Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins. J Lipid Res 51:2105–2120
Sun L, Xu R, Sun X et al (2016) Safety evaluation of exosomes derived from human umbilical cord mesenchymal stromal cell. Cytotherapy 18:413–422
Sun Z, Shi K, Yang S et al (2018) Effect of exosomal miRNA on cancer biology and clinical applications. Mol Cancer 17:147
Sverdlov ED (2012) Amedeo Avogadro’s cry: what is 1 µg of exosomes? BioEssays 34:873–875
Szatanek R, Baj-Krzyworzeka M, Zimoch J et al (2017) The methods of choice for extracellular vesicles (EVs) characterization. Int J Mol Sci 18:1153
Szatanek R, Baran J, Siedlar M, Baj-Krzyworzeka M (2015) Isolation of extracellular vesicles: determining the correct approach (Review). Int J Mol Med 36:11–17
Tai Y-L, Chu P-Y, Lee B-H et al (2019) Basics and applications of tumor-derived extracellular vesicles. J Biomed Sci 26:35
Takov K, Yellon DM, Davidson SM (2017) Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes. J Extracell Vesicles 6:1388731
Tanaka Y, Okada Y, Hirokawa N (2005) FGF-induced vesicular release of Sonic hedgehog and retinoic acid in leftward nodal flow is critical for left–right determination. Nature 435:172–177
Teng X, Chen L, Chen W et al (2015) Mesenchymal stem cell-derived exosomes improve the microenvironment of infarcted myocardium contributing to angiogenesis and anti-inflammation. Cell Physiol Biochem 37:2415–2424
Thakur A (2019) The Study of Glioma-derived Exosomes as Biomarkers and Functional Mediators via MCT1, CD147, and CD44. City University of Hong Kong
Thakur A, Mishra AP, Panda B et al (2020a) Detection of disease specific parent cells via distinct population of nano-vesicles by machine learning. Curr Pharm Des. https://doi.org/10.2174/1381612826666200422091753
Thakur A, Mishra AP, Panda B, Majhi B (2020b) Application of artificial intelligence in pharmaceutical and biomedical studies. Curr Pharm Des. https://doi.org/10.2174/1381612826666200515131245
Thakur A, Qiu G et al (2017) Direct detection of two different tumor-derived extracellular vesicles by SAM-AuNIs LSPR biosensor. Biosens Bioelectron 94:400–407
Thakur A, Qiu G, Xu C, et al (2020c) Label-free sensing of exosomal MCT1 and CD147 for tracking metabolic reprogramming and malignant progression in glioma. Sci Adv 6:eaaz6119
Thakur A, Sidu RK, Zou H et al (2020d) Inhibition of glioma cells’ proliferation by doxorubicin-loaded exosomes via microfluidics. Int J Nanomed 15:8331–8343
Thakur A, Zou H, Yang M, Lee Y (2018) Abstract 3720: Augmented loading efficiency of doxorubicin into glioma-derived exosomes by an integrated microfluidic device. In: Cancer Chemistry. American Association for Cancer Research, pp 3720–3720
Théry C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 30:3.22.1–3.22.29
Théry C, Boussac M, Véron P et al (2001) Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 166:7309–7318
Théry C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9:581–593
Théry C, Regnault A, Garin J et al (1999) Molecular characterization of dendritic cell-derived exosomes. J Cell Biol 147:599–610
Tian T, Zhang H-X, He C-P et al (2018) Surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy. Biomaterials 150:137–149
Tian W, Liu S, Li B (2019) Potential role of exosomes in cancer metastasis. Biomed Res Int 2019:1–12
Tonini T, Rossi F, Claudio PP (2003) Molecular basis of angiogenesis and cancer. Oncogene 22:6549–6556
Torregrosa Paredes P, Esser J, Admyre C et al (2012) Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma. Allergy 67:911–919
Tran C, Damaser MS (2015) Stem cells as drug delivery methods: application of stem cell secretome for regeneration. Adv Drug Deliv Rev 82–83:1–11
Vaidyanathan R, Naghibosadat M, Rauf S et al (2014) Detecting exosomes specifically: a multiplexed device based on alternating current electrohydrodynamic induced nanoshearing. Anal Chem 86:11125–11132
Vakhshiteh F, Atyabi F, Ostad SN (2019) Mesenchymal stem cell exosomes: a two-edged sword in cancer therapy. Int J Nanomedicine 14:2847–2859
Val S, Jeong S, Poley M et al (2017) Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology. Pediatr Res 81:911–918
van der Meel R, Fens MHAM, Vader P et al (2014) Extracellular vesicles as drug delivery systems: lessons from the liposome field. J Control Release 195:72–85
van der Pol E, Coumans FAW, Grootemaat AE et al (2014) Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost 12:1182–1192
Vasan N, Baselga J, Hyman DM (2019) A view on drug resistance in cancer. Nature 575:299–309
Viaud S, Terme M, Flament C et al (2009) Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Rα. PLoS ONE 4:e4942
Waldenström A, Gennebäck N, Hellman U, Ronquist G (2012) Cardiomyocyte Microvesicles Contain DNA/RNA and Convey Biological Messages to Target Cells. PLoS ONE 7:e34653
Wang J, Silva M, Haas LA et al (2014a) C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr Biol 24:519–525
Wang L, Hu L, Zhou X et al (2017) Exosomes secreted by human adipose mesenchymal stem cells promote scarless cutaneous repair by regulating extracellular matrix remodelling. Sci Rep 7:13321
Wang RN, Green J, Wang Z et al (2014b) Bone Morphogenetic Protein (BMP) signaling in development and human diseases. Genes Dis 1:87–105
Wang Z, Wu H, Fine D et al (2013) Ciliated micropillars for the microfluidic-based isolation of nanoscale lipid vesicles. Lab Chip 13:2879
Wehman AM, Poggioli C, Schweinsberg P et al (2011) The P4-ATPase TAT-5 inhibits the budding of extracellular vesicles in C. elegans embryos. Curr Biol 21:1951–1959
Whitehead CA, Luwor RB, Morokoff AP et al (2017) Cancer exosomes in cerebrospinal fluid. Transl Cancer Res 6:S1352–S1370
Wiklander OPB, Brennan MÁ, Lötvall J et al (2019) Advances in therapeutic applications of extracellular vesicles. Sci Transl Med 11:eaav8521
Witwer KW, Buzás EI, Bemis LT et al (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2:20360
Woo H-K, Sunkara V, Park J et al (2017) Exodisc for rapid, size-selective, and efficient isolation and analysis of nanoscale extracellular vesicles from biological samples. ACS Nano 11:1360–1370
Wood CR, Rosenbaum JL (2015) Ciliary ectosomes: transmissions from the cell’s antenna. Trends Cell Biol 25:276–285
Wu J, Qu Z, Fei Z-W et al (2017) Role of stem cell-derived exosomes in cancer. Oncol Lett 13:2855–2866
Wu Y, Deng W, Klinke DJ II (2015) Exosomes: improved methods to characterize their morphology, RNA content, and surface protein biomarkers. Analyst 140:6631–6642
Wunsch BH, Smith JT, Gifford SM et al (2016) Nanoscale lateral displacement arrays for the separation of exosomes and colloids down to 20 nm. Nat Nanotechnol 11:936–940
Xia Y, Liu M, Wang L et al (2017) A visible and colorimetric aptasensor based on DNA-capped single-walled carbon nanotubes for detection of exosomes. Biosens Bioelectron 92:8–15
Xu R, Greening DW, Zhu H-J et al (2016) Extracellular vesicle isolation and characterization: toward clinical application. J Clin Invest 126:1152–1162
Yamashita T, Takahashi Y, Takakura Y (2018) Possibility of exosome-based therapeutics and challenges in production of exosomes eligible for therapeutic application. Biol Pharm Bull 41:835–842
Yao X, Wei W, Wang X et al (2019) Stem cell derived exosomes: microRNA therapy for age-related musculoskeletal disorders. Biomaterials 224:119492
Yokoyama S, Takeuchi A, Yamaguchi S et al (2017) Clinical implications of carcinoembryonic antigen distribution in serum exosomal fraction—measurement by ELISA. PLoS ONE 12:e0183337
Yu L-L, Zhu J, Liu J-X et al (2018) A comparison of traditional and novel methods for the separation of exosomes from human samples. Biomed Res Int 2018:1–9
Yu X, Deng L, Wang D et al (2012) Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes: initiated by hypoxia inducible factor 1α, presented by exosomes. J Mol Cell Cardiol 53:848–857
Yuana Y, Oosterkamp TH, Bahatyrova S et al (2010) Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles. J Thromb Haemost 8:315-323
Zhang H, Jiang L-H, Hou J-C et al (2018) Exosome: a novel mediator in drug resistance of cancer cells. Epigenomics 10:1499–1509
Zhang M, Vojtech L, Ye Z et al (2020) Quantum dot labeling and visualization of extracellular vesicles. ACS Appl Nano Mater 3:7211–7222
Zhang P, He M, Zeng Y (2016a) Ultrasensitive microfluidic analysis of circulating exosomes using a nanostructured graphene oxide/polydopamine coating. Lab Chip 16:3033–3042
Zhang W, Yu Z-L, Wu M et al (2017) Magnetic and folate functionalization enables rapid isolation and enhanced tumor-targeting of cell-derived microvesicles. ACS Nano 11:277–290
Zhang Y, Liu Y, Liu H, Tang WH (2019) Exosomes: biogenesis, biologic function and clinical potential. Cell Biosci 9:19
Zhang Y, Yu Z, Jiang D et al (2016b) iPSC-MSCs with high intrinsic MIRO1 and sensitivity to TNF-α yield efficacious mitochondrial transfer to rescue anthracycline-induced cardiomyopathy. Stem Cell Rep 7:749–763
Zhao Z, Yang Y, Zeng Y, He M (2016) A microfluidic ExoSearch chip for multiplexed exosome detection towards blood-based ovarian cancer diagnosis. Lab Chip 16:489–496
Zhou Q, Rahimian A, Son K et al (2016) Development of an aptasensor for electrochemical detection of exosomes. Methods 97:88–93
Zhu Q, Heon M, Zhao Z, He M (2018) Microfluidic engineering of exosomes: editing cellular messages for precision therapeutics. Lab Chip 18:1690–1703
Zhu Q, Ling X, Yang Y et al (2019) Embryonic stem cells-derived exosomes endowed with targeting properties as chemotherapeutics delivery vehicles for glioblastoma therapy. Adv Sci 6:1801899
Zitvogel L, Regnault A, Lozier A et al (1998) Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell derived exosomes. Nat Med 4:594–600
(2018) P30: Uptake of trophoblast extracellular vesicles by neighboring cells. Am J Reprod Immunol 80:71–71
Funding
This study was in part supported by U.S. National Institute of Health (4R00CA226353-02 to H J. Chen), Hong Kong Health and Medical Research Fund (HMRF; No.: 06172956 to Q.L), and Stem Cell and Regenerative Medicine Fund (Guangzhou Women and Children’s Medical Centre, Grant No.: 5001-4001010 to Q.L), and Research Grants Council Theme-based Research Scheme (TRS; No. T12-703-19R to Q.L.).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
Authors declare no conflict of interest/competing interests.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Thakur, A., Ke, X., Chen, YW. et al. The mini player with diverse functions: extracellular vesicles in cell biology, disease, and therapeutics. Protein Cell 13, 631–654 (2022). https://doi.org/10.1007/s13238-021-00863-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13238-021-00863-6