The Role of Exosomes in Cancer Progression

Early detection, characterization and monitoring of cancer are possible by using extracellular vesicles (EVs) isolated from non-invasively obtained liquid biopsy samples. They play a role in intercellular communication contributing to cell growth, differentiation and survival, thereby affecting the formation of tumor microenvironments and causing metastases. EVs were discovered more than seventy years ago. They have been tested recently as tools of drug delivery to treat cancer. Here we give a brief review on extracellular vesicles, exosomes, microvesicles and apoptotic bodies. Exosomes play an important role by carrying extracellular nucleic acids (DNA, RNA) in cell-to-cell communication causing tumor and metastasis development. We discuss the role of extracellular vesicles in the pathogenesis of cancer and their practical application in the early diagnosis, follow up, and next-generation treatment of cancer patients.


Introduction
Extracellular vesicles (EVs) were discovered in 1946 as procoagulant platelet-derived particles in ultracentrifuged pellets from blood plasma [1]; however, the term "extracellular vesicle" was first used later in independent observations [2,3]. We now know that EVs are nano-to micron-sized vesicles covered by phospholipid bilayers. They are classified into three groups: apoptotic bodies (ABs), ectosomes/microparticles (MPs)/microvesicles (MVs), and exosomes (Exs) [4]. Their subtypes include large dense core vesicles, membrane blebs, oncosomes, outer membrane vesicles (OMVs), and prostasomes (exosome-like vesicles) [5][6][7]. According to current nomenclature, MVB-derived EVs are called exosomes, while plasma membrane-derived EVs are microvesicles [8,9]. The contents, membrane composition and size of EVs are highly heterogenous and depend on cellular location and environment. Extracellular vesicles are released by different tissues and cell types. They are found in body fluids, including amniotic fluid, ascites, bile, breast milk, nasal and bronchial lavage fluid, blood plasma, saliva, semen, synovial fluid, and urine, allowing extraction of EVs from various liquid biopsies [6,7]. EVs contain proteins, lipids, DNA, RNA, and microRNA serving as mediators of cell-to-cell communication [10][11][12]. Membranes protect their contents from nuclease and protease degradation and micro-environment changes (e.g., osmolarity and fluctuations in pH) [12]. Apoptotic bodies are released by blebbing of plasma membrane during apoptosis. The second main group of EVs includes vesicles of different size and components. The last main group, exosomes, belongs to the class of intraluminal vesicles (ILVs) contained in multi-vesicular bodies (MVBs) and are released to the extracellular environment by MVBs fusing with the cell membrane [7,13]. from 50-200 nm up to 1 µm in diameter [18,32,33] and contain a portion of the plasma membrane separated by blebbing outward and released into the extracellular space [12]. MVs are found in the blood [34] and in other body fluids, e.g., urine [35], cerebrospinal fluid [36], tears [37], saliva [38] and nasal secretions [39], ascites [40], and semen [41]. They are involved in intercellular communication, signaling pathways and promotion of cell invasion by cell-independent matrix proteolysis. Ectosomes, microparticles, oncosomes, shedding bodies, and shedding vesicles are all referred to as microvesicles [42,43].
Tumor-derived microvesicles (TMVs) and oncosomes originate from cancer cells [44] and an altered release of MVs is associated with cancer progression. Higher numbers of MVs indicate a more severe disease, and a high amount of proteolytic content in MVs correlates with the quick spreading of breast cancer and fibrosarcoma [45]. TMVs transfer bioactive molecules (nucleic acids, lipids, and proteins) to recipient cells causing disease, promoting cancer and providing diagnostic markers. In TMVs, the membrane-type 1 matrix metalloprotease (MT1-MMP) promotes cell invasion of the extracellular matrix (ECM) [46]. Some specific markers might be identified as MV markers, e.g., adenosine diphosphate ribosylation factor 6, CD40 ligand, various integrins and selectins [32,[47][48][49]. ARF6 is involved in tumor formation, growth, and metastasis [47] ( Table 1).
Tumor-derived microvesicles (TMVs) and oncosomes originate from cancer cells [44] and an altered release of MVs is associated with cancer progression. Higher numbers of MVs indicate a more severe disease, and a high amount of proteolytic content in MVs correlates with the quick spreading of breast cancer and fibrosarcoma [45]. TMVs transfer bioactive molecules (nucleic acids, lipids, and proteins) to recipient cells causing disease, promoting cancer and providing diagnostic markers. In TMVs, the membrane-type 1 matrix metalloprotease (MT1-MMP) promotes cell invasion of the extracellular matrix (ECM) [46]. Some specific markers might be identified as MV markers, e.g., adenosine diphosphate ribosylation factor 6, CD40 ligand, various integrins and selectins [32,[47][48][49]. ARF6 is involved in tumor formation, growth, and metastasis [47] ( Table 1).

Exosomes
Exosomes were first reported as a "type of small vesicles" in Pan and Johnstone's sheep reticulocyte-related experiment in 1983 [50], and the name "exosome" was first used (and visually described using transmission microscopy) by Johnstone et al. in 1989 [51]. Among the groups of EVs, exosomes are the smallest (40-100 nm) [52]. They may be verified based on their cup-shaped morphology by negative-staining transmission microscopy and the presence of markers (CD9, CD63, and CD81) [53-55]. Besides tetraspanins, other molecules are also found on the surface of exosomes, e.g., ALIX, TSG101, HSP70, flotillin, LAMP-1, MHC-I, -II [8,12,[55][56][57]. Exosomes originate as intraluminal vesicles by inward budding of endosomal membranes forming multivesicular bodies (MVBs) from internal multivesicular compartments of the endocytic pathway. Following the secretory pathway, exosomes merge with the plasma membrane and are released into the extracellular milieu or fused with lysosomes for degradation [8,9] (Figure 1). Figure 1. Exosome biogenesis. The process of endocytosis results in the formation of an early endosome, followed by a maturation to a late endosome that may bidirectionally exchange vesicles with the Golgi apparatus and the endoplasmic reticulum. Invagination of late endosomal membranes forms the intraluminal vesicles (ILVs) contained in the multivesicular body (MVB). The MVB may fuse with the plasma membrane and release ILVs to the extracellular space as exosomes. On the other hand, the MVB may also be transported to a lysosome for subsequent degradation of its content. Some studies in the 2000s proved that exosomes may deliver RNA such as mRNA and miRNA involved in cell-to-cell communication [11,54]. During their biogenesis, exosomes are packed with various cargo such as nucleic acids (e.g., DNA, mRNA, miRNA, lncRNA, circRNA) [11,58,59], proteins [60,61], lipids [62], metabolites [63,64] (Figure 1). When each component is released, they affect intercellular communication through direct cellto-cell interaction contributing to tumorigenesis [11,[58][59][60][61][62][63] and tumor-derived exosomes filled with RNAs and proteins may transfer oncogenic activity to recipient non-tumor cells [58-60,64] ( Figure 1, Table 1).

Exosomes in Tumors
Some studies described the connection between exosomes and the development and progression of different cancer types. Exosomes may serve as multicomponent biomarkers in tumor diagnostics [71,72].

Exosomes in Tumor Progression
Under physiological conditions, exosomes contribute to normal cellular function, while their altered function may cause cancer. Exosomes induce tumor development by changing the landscape of tumor microenvironments, and immune system activation by changing vascularity and cell polarity. They are also responsible for epithelialmesenchymal transition (EMT) and an interconversion to mesenchymal-epithelial transition (MET) in several human malignancies [73]. Exosomes are involved in promoting tumorigenesis and metastasis and are associated with gaining chemoresistance, but exosomes derived from dendritic cells may be engineered to trigger antitumor immune responses ("dexosomes") [74,75].
Exosomes have different ways to participate in tumor development. They may alter gene expression. Post-translational modifications such as ubiquitination frequently occur on EVs. Some studies showed that a dysregulation of ubiquitination and deubiquitination may lead to various diseases such as cancer, and they are part of the regulation of metabolic reprogramming in cancer cells [76]. Exosomes rich in Wnt5b have been associated with head and neck squamous cell carcinomas, invasive breast cancer, and lung and pancreatic cancer [77].
In addition to their effect on gene expression, they may be involved in posttranslational modifications as explained below. EVs from ovarian carcinoma were found to be enriched in mannose and sialic acid residues [22], i.e., glycosylation is modified.
Phosphorylation is another example: the role of Src-phosphorylation in the angiogenesis of myeloid leukemia is promoted by exosomes. This type of phosphorylation may be therapeutically targeted [78].

Exosomes in Cancer Immunology
Exosomes containing nucleic acids may control the innate and adaptive immune responses [74]. Secreted exosomes may stimulate the anticancer activity of effector CD4+ T cells [4] and promote their proliferation by indirectly activating naïve T cells and B cells by interacting with antigen-presenting cells [79]. Exosomes derived from various cells may release molecules which evoke immune responses in tumor formation. The role of Th17 cells is well-known in ovarian cancer: they may secrete pro-and anticancer factors, and promote angiogenesis [80,81]. Ye et al. (2014) reported that exosomes from nasopharyngeal carcinoma (NPC) cells hindered the proliferation of T cells and the differentiation of Th1 and Th17 cells by reducing the level of interleukin-2 (IL-2), interferon gamma (IFN-γ), and interleukin-17 (IL-17). However, the same exosomes activated Treg cells as a consequence of increasing the levels of interleukin-1b (IL-1b), interleukin-6 (IL-6), and interleukin-10 (IL-10) released from T cells [82]. The Treg/Th17 ratio was elevated in primary and metastatic tumors of patients with epithelial ovarian cancer compared to benign tumors and peritoneum. Inequality of the Treg/Th17 ratio is caused by an exosome-mediated transfer of miR-29a-3p and miR-21-5p from macrophages to T helper (CD4+) cells; the process is suppressed by signal transducer and activator of transcription 3 (STAT3) signaling [83]. Macrophage-derived exosomes of liver cancer cells induced the secretion of IL-6, MCP-1, IL-10, and TNF-α via STAT3 signaling [84]. Transfer of miRNA let-7d via exosomes-as analyzed in TME cells-reduced Th1 proliferation and secretion of IFN-γ [85].

Exosomes in Immunosuppression
Exosomes carry programmed cell death receptor ligand 1 (PD-L1), which may directly prevent the anticancer function of CD8+ T cells in vivo [65,86], so evasion of immune surveillance may be possible by exosomes. Some authors are optimistic about the future of anti-PD-L1 therapy of cancer [87,88]. PD-L1 on the surface of extracellular vesicles is associated with immunosuppression, disease progression of tumor patients and altered response to immunotherapy [65,86].

Exosomes in Angiogenesis and Lymphangiogenesis
Cellular microenvironments have a major impact on cancer progression. Exosomes released by cancer cells precondition tissue environments for local spreading and distant metastasis by delivering inflammatory and other factors [89][90][91]. Exosomes from breast cancer promote adhesion of cells to extracellular matrix proteins. Exosome-induced metastases were reported by several authors. Tumor-derived exosomes may affect factors related to EMT and escaping from immune surveillance (β-catenin, caveolin-1, transforming growth factor beta (TGFβ), etc.) [91].
The growth and spread of tumors are associated with the formation of new blood and lymph vessels. Cells located in a tumor and its microenvironment secrete angiogenic factors leading to tumor angiogenesis and growth factors contributing to lymphangiogene-sis. Tumor cell-derived exosomes loaded with non-coding RNAs are involved in the same processes [92,93]. Several recent studies proved that cancer cell-derived exosomal miRNAs promote angiogenesis and/or lymphangiogenesis using different signaling pathways affecting endothelial cells [94][95][96][97] and non-endothelial cells [98]. Tumor-derived exosomes containing miR-21 or let-7a (under hypoxic stress) increase M2 polarization of macrophages which may stimulate tumor-associated angiogenesis and lymphangiogenesis [99,100]. Exosomal long non-coding RNAs are also reported to be involved in the process [101][102][103]. For example, lncRNA H19 and HOTAIR stimulate angiogenesis by synthesis and secretion of vascular endothelial growth factors [104,105] and other lncRNAs are involved by sponging microRNAs [106,107]. Exosomal circRNA-100338 may regulate angiogenesis to promote metastasis in hepatocellular carcinoma [108].
Other factors delivered by exosomes may also play key roles in promoting angiogenesis. Some studies have shown that exosomes from breast cancer transfer Annexin II (a tumorigenic factor) both in vivo and in vitro [109]. New vessel formation was detected predominantly in hypoxic regions of tumors with a low level of chemoresponsiveness [110].

Exosomes in the Therapy Phase
Radiotherapy promotes the secretion of exosomes, and their content plays a significant role in cancer survival. Irradiation may unexpectedly increase survival of cancer cells by triggering the release of exosomes carrying survivin (an apoptosis inhibitor) [110]. It may also affect the ratio of migratory factors (such as Insulin Like Growth Factor Binding Protein 2 (IGFBP2) and exosome connective tissue growth factor (CTGF) in exosomal cargo [111]. Various forms of stress (e.g., heat and oxidative stress) and some chemotherapeutic drugs (such as proteasome inhibitors like Bortezomib and alkylating agents like melphalan) may induce and then increase exosome release from cancer cells [112][113][114].

Exosomes in Cancer Detection
Exosomes contain nucleic acids, proteins, lipids, and carbohydrates. They are suitable for the detection of different types of cancer non-invasively from liquid biopsies.

DNA Genomic/Nuclear DNA
In cancer patients, more gDNA content in exosomes is derived from cancer cells than from normal cells due to apoptosis or necrosis. Exosomes with nuclear content are secreted by tumor cells in high quantities, allowing for their application as cancer biomarkers. Recent studies revealed the connection between micronuclei and exosomes with nuclear content [129].
DNAs are shuffled into MVBs by tetraspanins and DNA-binding proteins interacting with CD63 [129]. Exosomes are released from MVBs containing molecules derived from them and then release cell-free DNA into plasma [130]. Circulating tumor DNA is partially derived from exosomes of tumor cells, which may serve as biomarkers in cancer diagnosis [131].
Circulating cell-free DNA encapsulated in exosomes in the plasma of gastrointestinal tumor patients may transform normal gastrointestinal cells into tumor cells, in a process known as genometastasis [132][133][134].
Mitochondrial DNA Apart from genomic DNA, mtDNA is also present in exosomes and may show relevant copy number differences between cancer patients and healthy controls, as shown by our group in ovarian carcinoma [117]. Release of mtDNA in exosomes was also characteristic for cancer-associated fibroblasts in a breast cancer model [89].

RNA MicroRNAs
The role of miRNA is highlighted as a marker in diagnosis and monitoring of the progression of many types of cancers [135]. It has been shown that miR-21 is overexpressed in exosomes obtained from patients with esophageal squamous cell carcinoma and glioblastoma [136]. Fabbri and colleagues found that exosomal miR-21 and miR-29a may reduce the overall survival of lung cancer [137]. Anfossi et al. measured the level of miR-21 and found that it is a valuable diagnostic biomarker for breast cancer [138].

Long Non-Coding RNAs
Exosome-derived lncRNAs are emerging as useful cancer biomarkers, and peripheral blood is not the only liquid biopsy from which they are obtained-e.g., urine exosomes carry markers for urothelial bladder cancer, while cervicovaginal lavage yields exosomes containing lncRNA relevant for the diagnosis of cervical cancer [139]. A combination of 2 exosomal mRNAs and an exosomal lncRNA-breast cancer anti-estrogen resistance 4 (BCAR4)-was reported as a robust diagnostic marker set for the screening of colorectal cancer [121]. Plasma lncRNA long intergenic non-protein-coding RNA 152 (LINC00152) is thought to be present exclusively in exosomes. In the diagnosis of gastric cancer, LINC00152 was reported to be more sensitive than established markers [122]. Exosomal Metastasisassociated lung adenocarcinoma transcript 1 (MALAT-1) is overexpressed in non-small cell lung cancer and is thought to increase proliferation and migration capabilities of tumor cells [140].

Circular RNAs
Interestingly, exosomal circRNAs are globally downregulated in most cancer types, but individual tumor biomarkers are usually still present [123]. Their tertiary structure makes them stable and suitable for clinical use. Exosomal circ-0051443 has been reported to be underexpressed in hepatocellular carcinoma [141], while hsa_circ_0065149, circ-KIAA, and hsa_circ_0000419 were downregulated in plasma samples of gastric cancer patients [142][143][144].

Tetraspanins
Tetraspanins such as CD9, CD63, CD81 and CD151 are scaffolding membrane proteins highly enriched in exosomes [145,146]. Logozzi et al. (2009) demonstrated that plasma CD63+ exosomes are significantly increased in melanoma patients [147]. Yoshioka et al. found CD63 to be present in higher levels in exosomes produced by malignant cells, providing evidence that exosomal CD63 could be a protein marker for cancer [20]. CD81 plays a critical role in hepatitis C attachment and cell entry [148].

SNARE Proteins
The main function of SNARE proteins composed of multiple proteins is promoting the fusion of vesicle membranes and plasma membranes [149]. The vesicle associated membrane protein 7 (VAMP7), a member of the SNARE family, is an important component of exosomes involved in their secretion to the extracellular environment [150]. The abnormal lncRNA LINC00511 induces formation of invadopodia by regulating the colocalization of VAMP7 and synaptosome associated protein 23 (SNAP23) and is thus involved in tumor progression as shown in hepatocellular carcinoma (HCC) cells [151].

Rab Proteins
There are more than 60 different Rab proteins in humans. Rab proteins are small GTPase proteins regulating membrane trafficking, intracellular transport, lipid remodeling, fusion, and exosome release [152,153]. RAB11 was the first protein from the RAB family that was shown to be involved in the secretion of exosomes containing TFR and HSC70 from myelogenous leukemia cell lines [154]. Depending on the cell type, Rab5, Rab7, Rab11, Rab27, and Rab35 are involved in vesicle secretion and thus cancer progression. It was observed that ovarian cancer cells significantly increased their exosome release in hypoxia by upregulating Rab27a and downregulating Rab7, LAMP1/2, and NEU-1 [155].

Annexins
Annexins are a group of calcium-and phospholipid-binding proteins highly expressed in exosomes in cancer. Maji et al. demonstrated their participation in breast cancer pathogenesis [109].

Flotillins
Flotillins are membrane-associated proteins involved in scaffolding, signaling, and endocytosis. They are enriched in exosomes and may be used as exosomal biomarkers. Phuyal et al. showed that they affect the composition of exosomes [156].

Proteins Involved in ESCRT Complex
Programmed cell death 6-interacting protein (PDCD6IP) or ALIX is a cytoplasmic protein involved in apoptosis as a binding protein in endosomal sorting complex required for transport (ESCRT) complexes [157]. Specific components are sorted into ILVs by ESCRT complexes (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III, which contain specific proteins, e.g., VPS4, VTA1, and ALIX) involved in the biogenesis of exosomes [158,159]. The hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), which is part of ESCRT-0, also has a significant role in the exosome biogenesis [159]. HRS recognizes ubiquitinated proteins and interacts with STAM (a part of ESCRT-0) [160]. ALIX (ESCRT-III-related protein) may cooperate with syndecan-syntenin and is also involved in exosome formation [161].

Heat Shock Proteins
Exosomes also contain heat shock proteins (HSPs) which are produced under stressful conditions. HSPs were found to play a role in antigen presentation by loading peptides onto the major histocompatibility complex (MHC). Thus, they have the capacity to stimulate antitumor immune responses. Lv et al. found that exosomes from resistant human hepatocellular carcinoma cells may improve tumor immunogenicity by the induction of HSP-specific natural killer (NK) cell responses [162].

Lipids
The lipid composition of exosomes varies and is affected by the cell of origin. Skotland et al. used exosomal lipids as biomarkers and reported that engineering lipid composition of exosomes may yield useful drug delivery vehicles [163].

Glycans
During glycosylation, glycans (a subgroup of saccharides) may be attached to lipids, proteins, and other glycans [164]. They are essential in exosome function [165]. Conserved and unique glycan contents were found in exosomes corresponding to parent cell membranes (e.g., specific sialic acid-containing glycoproteins in exosomes derived from SKOV3 cells) [166,167]. Bisecting GlcNAc-containing N-glycans were found in exosomes derived from SKOV3 and OVM ovarian carcinoma cell lines [167]. These molecules have been associated with a lower occurrence of metastasis in multiple cancer types [168], suggesting that exosomes containing glycans may be used in cancer vaccine development. The glycan properties of cancer cell-derived exosomes make them promising early diagnosis markers such as lectin-conjugated nanoparticles in pancreatic cancer [169].
Nucleic acid, protein, lipid, and carbohydrate contents of exosomes are summarized in Table 2. Table 2. A summary of exosome contents.

Exosomes as Next-Generation Treatment Options
Recently, exosome research has been focused on developing designed drug or nucleic acid delivery to cancerous cells or tissues to improve the effectiveness of cancer therapy   (Table 3). Table 3. Application of exosomes in cancer therapy.
Recent reviews highlighted the possible therapeutic application of exosomes for personalized medicine. Engineered extracellular vesicles loaded with various molecules may find application in EV-based personalized medicine as a new option for tailoring clinical treatment [206][207][208][209].

Conclusions
In this review, we summarized the role of extracellular vesicles in cancer progression focusing on exosomes carrying extracellular nucleic acids (DNA, RNA) from cell to cell, causing tumor and metastasis development. We outlined the roles of liquid-biopsy-derived exosomes in tumor pathology and therapy against cancer. They serve as excellent sources of various markers for early non-invasive detection, classification of cancer and follow up. They are suitable for the targeted delivery of drugs to tumor cells, making them promising next-generation treatment vehicles.