Extracellular vesicles in urologic malignancies—Implementations for future cancer care

Abstract Extracellular vesicles (EVs), a heterogeneous group of vesicles differing in size and shape, cargo content and function, are membrane‐bound and nano‐sized vesicles that could be released by nearly all variations of cells. EVs have gained considerable attention in the past decades for their functions in modulating intercellular signalling and roles as potential pools for the novel diagnostic and prognostic biomarkers, as well as therapeutic targets in several cancers including urological neoplasms. In general, human and animal cells both can release distinct types of EVs, including exosomes, microvesicles, oncosomes and large oncosomes, and apoptotic bodies, while the content of EVs can be divided into proteins, lipids and nucleic acids. However, the lack of standard methods for isolation and detection platforms rein the widespread usage in clinical applications warranted furthermore investigations in the development of reliable, specific and sensitive isolation techniques. Whether and how the EVs work has become pertinent issues. With the aid of high‐throughput proteomics or genomics methods, a fully understanding of contents contained in EVs from urogenital tumours, beyond all doubt, will improve our ability to identify the complex genomic alterations in the process of cancer and, in turn, contribute to detect potential therapeutic target and then provide personalization strategy for patient.

vesicles are released during cell undergoing apoptosis ranged from 50 to 2000 nm, respectively. [7][8][9] Extracellular vesicles serve as an appealing source for the development of biomarkers as their membrane-bound structure to protect against exogenous proteases and RNases. 10,11 The biological function of EVs is performed by cytosolic lipids, proteins, DNA, mRNA, miRNA, lincRNA and other non-coding RNAs, as well as cell membrane. 12 In addition, cancer cell releases more EVs than that normal one does. 13 Herein, we introduce EVs briefly and provide a comprehensive overview of their biophysical properties, roles and applications in the most common urologic neoplasms, including kidney, prostate and bladder, and discuss potential clinical applications in the future.

| E V CL A SS E S , B I OG ENE S IS AND CONTENTS
Our current understanding of EVs indicates that at least four heterogeneous types of EVs have been identified based on their mechanism of formation and distinguished size: microvesicles, exosomes, oncosomes or large oncosomes, and apoptotic bodies (Table 1). In general, the formation of exosomes and microvesicles is two completely different approaches, but they function similarly. Oncosomes or large oncosomes resemble the way of microvesicles via membrane budding. Apoptotic bodies specifically arise resulting in indiscriminate membrane bubbling during apoptosis ( Figure 1).
Exosomes generally form an early endosome by the endocytosis and internalization of cell-surface receptors into membrane-bound vacuoles in the first step, 14 which then matures to generate a late endosome within undergoing several changes, such as the limiting membrane of the late endosome then buds inward and pinches off as the result of the formation of intraluminal vesicles (ILVs), also known as multivesicular bodies, and ILVs traffick to and fuse with the plasma membrane leading in releasing exosomes eventually. 15 ESCRT-0-III plays significant roles in driving exosome formation 16,17 ; in addition, multivesicular bodies could intermediate in the lysosomal degradation pathway. 18 However, the mechanisms related to the fusion of multivesicular bodies with the cellular membrane are uncovered, which may be regulated by several factors including lipid ceramide and Rab GTPase (including Rab5 and Rab7) proteins and ESCRT. [19][20][21] Numerous literatures have indicated that several biomarkers expressed in the exosome differentially compared with another types of EVs, including heat shock proteins (eg HSP60, 70 and 90), tetraspanins (eg CD9, CD63 and CD81), membrane transporters, fusion proteins, ALG-2-interacting protein X (Alix) and tumour susceptibility gene 101 protein (TSG101). 20,22 In contrast, microvesicles with nano-sized with 100-1500 nm are straightforwardly shed from the cellular membrane responding to stimuli or physiological conditions. 23  factor 6 (ARF6) can meditate the freeing of protease-loaded vesicles from the cellular membrane due to the crosstalk with Rho signalling pathways. [24][25][26] Moreover, microvesicles are specifically produced in the cellular membrane regions that are linked to be enriched in cholesterol, ceramide and lipid rafts. 27 TSG101 is also known to interact with accessory proteins Alix and arresting domain-containing protein-1 (ARRDC1) during releasing microvesicles, illustrating that microvesicles sharing some same characteristics with exosomal biogenesis. 28 As described for microvesicles, oncosomes and large oncosomes are generated by plasma membrane budding, with amoeboid-like phe-

| ISOL ATI ON TECHNI QUE S OF E VS
No remarkable consensus is in existence of the best approach for isolation, qualitative and quantitative analysis of EVs. There are listing several methods for the isolation of EVs ( Figure 2) and demonstrate the available disadvantages and advantages as well (Table 2). 31

| CENTRIFUG ATI ON
In recent, differential centrifugation is the most common technique in responding to isolating EVs, and this approach is consisting of three main centrifugation processes: low speed to eliminate a main por-

| IMMUNOAFFINIT Y ISOL ATION ME THODS
Immunoaffinity isolation is another approach to isolate the EVs with increasing purity, owing to selectively exploiting the presence of specific molecules in the small EV surface 37 ; for example, the lipids, proteins and polysaccharides are common substances that exposed on the surface of EVs, as a result, showing potency in being ligands for manifold molecules. Generally, there are five main methods for the isolation of EVs based on immunoaffinity, including antibodies to EV receptors, 38 phosphatidylserine-binding proteins, 39 heparinmodified sorbents 40 and binding of heat shock proteins, 34 as well as lectins. 41 Although along with evident advantages of the EV purified isolation, the expensive costs, and the insufficient efficiency of isolation, and difficulties encountering in the process of isolation the large volumes of EVs, which substantially limits the applicability of immunoaffinity isolation methods.

| MICROFLUIDIC DE VICE S
Microfluidic devices are composed of a network of microchannels with different sizes, which have been implicated for EV isolation from cell culture and various tissue fluids on the basis of the immunoaffinity principle, as well as systems. However, some issues are yet to be removed; for instance, the inputted sample shows great possibility to block channels and the efficacy of isolation of EVs is extremely slow, consequently, decreasing their diagnostic potential, 38 and overcome some of the challenges involving in EVs detection, such as the problem of the small size and lacking in distinct biomarkers, which contributing to get a comprehensive understanding function of their contents (eg protein, RNA and lipid).
Several qualitative and quantitative analysis techniques are currently available (Table 3). For instance, transmission electron microscopy (TEM) could be combined with immunogold staining to represent structural details and delineate the subpopulations of EVs. 42 A study indicates that cryo-electron microscopy might be more suitable for depicting the morphology of EVs as its no fixation or staining. 43 The size, morphology and intactness of EVs also could be determined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). 44 Measuring the size and number distribution of single EVs can be made by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). 45 Both the conventional flow cytometry and novel fluorescence-based flow cytometry could be promising tools to qualitatively and quantitatively analyse the EVs. 46 Western blot, enzyme-linked immunosorbent assay (ELISA) and EVs arrays are used to present purity and enrichment. Micronuclear magnetic resonance [μNMR] system and a photosensitizer-bead detection system (ExoScreen) are other sensitive qualitative and quantitative approaches. [47][48][49] As mentioned above (

| G ENER AL FUN C TI ON S OF E VS IN MALIG NAN CIE S
Bioactive molecules of EVs secreted by both cancer cells and tumour-associated cells provide the essential signals for favouring tumour growth via remodelling the architectures in tumour microenvironments and forming pre-metastatic niches. Different mechanisms of EVs-mediated tumour proliferation and progression will be discussed in the following sections ( Figure 3).

| PROMOTION OF ANG IOG ENE S IS
Tumour progression is a dynamic and multistep process requiring continuous nutrient and oxygen supplied by sufficient blood conducts, while also serving to remove waste materials. The advent of cancer stem cells (CSCs) has provided a novel mechanism for the development and progression of the tumour via differentiating into endothelial cells to contribute to the angiogenesis. 50,51 In addition, a research indicates, for example, that miRNAs, secreted from exosomes, regulate transcription, proliferation, metabolic processing and mRNAs encode vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiopoietin1, Ephrin A3, matrix metalloproteinase-2 (MMP-2), and MMP-9 and growth factors in contrast to CD105-negative CSCs. 19

| EPITHELIAL-TO -ME S EN CHYMAL TR AN S ITI ON
Epithelial-to-mesenchymal transition (EMT) is a developmentally vital reversible process of which fully differentiated cells lose their epithelial features (eg E-cadherin, β-catenin and plakoglobin), acquiring a migratory mesenchymal phenotype (eg N-cadherin and vimentin). EMT also contributes to the metastatic potential of tumours. 52 Exosome mediates that growth in migration and invasion by the way of EMT has been observed in many other studies. [53][54][55] Urothelial cells exhibit the EMT after exposing to tumour-derived exosomes. 56

| FORMATI ON OF PRE-ME TA S TATI C NICHE S
Primary tumours can release some biological factors that migrate to preferred metastatic regions and dynamically remodel these sites before spreading to a distant organ, which means that form predetermined metastatic microenvironments, also referred to as pre-metastatic niches. 57 In general, exosomes display the characteristics of organ tropism, and the process of the construction of pre-metastatic niche involves with initial tumour-derived exosomes releasing into the circulation system and then escaping from the vascular beds to migrate to distant secondary organ. 58,59 During the process, the crucial initial step is how vascular leaking exosomes can target organ tissues; nowadays, it is induced by complicated processes involved combination of stromal cells and released cancer cell-derived exosomes resulting in reprogramming of these cells [60][61][62][63] and activation of several vital signalling pathways, 64,65 which alter the local chemokine repertoire of the tumour microenvironment (TME) and remodel the components of the extracellular matrix (ECM) in turns. 61 Moreover, recent research shows that transferring TGF-β also contributes fibroblasts converse to myofibroblasts, which could secrete insulin-like growth factor 1 (IGF-1), activin A and VEGF to induce tumour progression. 72 The irritation of another signalling pathway by exosomes in the bidirectional crosstalk between cancer cells and normal stromal cells, such as nuclear factor kappa B (NF-κB) and epidermal growth factor receptor (EGFR) signalling, also plays vital roles in the proliferation and migration of tumour. Endothelial cells show enhanced cell motility and tube formation ability after re-educated by tumour-derived exosomes 73 ; moreover, RNA secreted from EVs develops hepatocyte growth factor synthesis through the activation of ERK1/2 and AKT signalling pathways. 74 It is widely believed that tumour-derived EVs impose significant effects in mediating communication between immune and cancer cells of renal cell cancer (RCC), 75 such as immune evasion of tumours. 76,77 MiR-222-3p induces polarization of tumour-associated macrophages by the activation of SOCS3/ STAT3 pathway to facilitate tumourigenesis and cancer progression. 78 Additionally, Rab27a supports exosome could modify the tumour microenvironment via advancing recruitment and differentiation of bone marrow-derived neutrophils to cancer cells. 79 Furthermore, a few studies suggest that tumour-derived EVs facilitate cancer progression by attenuating immune and more specific EVs could diminish the cytotoxicity of natural killer cells and T cell in immunoreaction. [80][81][82] Tumour-derived EVs also could influence the cancer cells themselves via autocrine to irritate the invasion and migration, and reduce adhesion abilities as well via enhancing MMP-9 or chemokine receptor type 4 (CXCR4). 74

| THE B L ADDER C AN CER
Bladder cancer (BCa) is the seventh most commonly diagnosed cancer in the male population worldwide, and the diagnosis for BCa is usually on the basis of cytology, urinalysis and cystoscopy. Cytology is a highly specific test, but low in the sensitivity for the diagnosis of BCa. 106 Cystoscopy is the gold standard to diagnose the BCa, while this method is expensive and invasive, even for flexible cystoscopy, and the risk of developing urinary infections is up to 10% 107 ; non-invasive and reliable biomarkers are therefore required in the future. Given that, urine is an excellently suitable fluid for biomarkers discovery in BCa. The biomarkers (mainly including proteins, mRNA, lncRNA and miRNA) within EVs isolated from BCa were investigated by different research groups, which could be promising molecules to identify the BCa and predict the progression of the BCa ( The author identified seven proteins differentially expressed in the low-risk group (Table 5). 109 Proteome profiling of urinary exosomes indicates H2B1K and alpha 1-antitrypsin as prognostic and diagnostic biomarkers for urothelial bladder cancer, which could be verified in immunohistochemistry (IHC). 110 Additionally, HEXB, S100A4 and SND1 significantly identified in EV derived from the MIBC cell line also are upregulating in urinary EV from MIBC patients when vs to normal groups. 111 There are other proteins could be recognized as potential diagnostic and prognostic markers for BCa. 112

| PROS TATE C AN CER
Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, accounting for 15% of all cancers diagnosed. 128 Although PSA testing contributes to identify and manage PCa in the early phase, it still has some limitations, for example, the specificity of discrimination of benign prostate diseases, such as acute prostatitis and benign hyperplasia. 129 Thus, the more specific and ideal substrate (eg urine, prostatic plasmas and blood samples) for PCa are urgently developed rather than invasive prostate biopsies. 130 Some studies have presented the usefulness of urinary EVs as diagnostic factors ( sequencing reveals the potential values for miRNA served as diagnostic and prognostic biomarkers for PCa within serum or plasma EVs, 41,131,132,[158][159][160][161][162] such as miR-141 and miR-375 in serum, have been correlated with metastatic PCa. 159,163 Another study indicates that exosomal miR-1290 and miR-375 could be as prognostic markers in castration-resistant prostate cancer (CRPC). 164 In recent, several research works demonstrate that the lipids including diacylglycerol and triacylglycerol are differentially enriched in PCa rather than healthy groups. 165,166 Glycomic and metabolomic profiling of urinary EV reveal several small molecule metabolites could be novel biomarkers to predict the development of PCa, for example levels of N-linked glycans, glucuronate, adenosine, d-ribose-5-phosphate and isobutyryl-l-carnitine. 167,168 The intercellular crosstalk through EVs could stimulate tumour progression. Several proteins presenting on and in EVs from PCa cell lines are recognized as significant meditators for the biological communication between cancer cells and tumour microenvironment or surrounding cell, including cytokine CX3CL1, MMPs and transforming growth factor B, play significant roles in the proliferation and differentiation of fibroblasts. 169,170 In addition, integrins ITGA3 and ITGB1 can affect invasion and migration of normal prostate epithelial cells. 138 Several studies suggest that complicate intercellular interactions between cancer cells, osteoclasts and osteoblasts contribute to bone metastasis. 171,172 It is the first protein that has been reported in the EVs orig-  and therapeutics owing to their enormous potencies in several aspects, as described below (Figure 4). 19  of EVs from non-specific sites towards accumulation in desired tissues. Although considerable efforts by engineering EVs to present cell type-specific ligands have been made in guaranteeing rich accumulation in target tissues, one of the major obstacles remains low delivery efficacy. The elucidation to these questions will enhance rationality and reliability to irritate the utility of EV-involving molecular cargoes as cancer diagnostics in the clinical practices. F I G U R E 4 Future implements for EVs in urological cancer. EVs impact the multistep process of cancer; therefore, EVs should be a novel treatment strategy by inhibiting intercellular crosstalk. EVs could serve as promising diagnostic and prognostic biomarkers to dynamically trace the changes in cancer due to their high specificity and sensitivity. In addition, EVs have the potential functions to stably deliver substantial therapeutic cargoes liking miRNAs and siRNAs with stability, few side effects and organ specificity. Furthermore, several studies have reported the potential of EVs derived from dendritic cells used as vaccine vesicles. Copyright 2018, The Jikei University School of Medicine, Fumihiko Urabe 19 are yet unexplored. With the increasing knowledge of their roles and development of the next-generation sequencing, mass spectrometry-based metabolomics and proteomics, we are enthusiastically sure that EVs will contribute to play clinical applications for urological cancer treatment and management in the near future.

ACK N OWLED G EM ENTS
We gratefully acknowledged the help from staffs at the Department of Urology of the Third Affiliated Hospital of Shenzhen University for the data assistance.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
Z.W., S.W. and Z.Z. designed the review and made a retrieval strategy; Z.W. and Y.L. drafted the review text; W.X. and J.C. drafted the tables and figures; and both authors contributed to revision and finalization of the manuscript.

DATA ACCE SS I B I LIT Y
Research data are not shared.

CO N S E NT FO R PU B LI C ATI O N
The patient has given his consent for his case report to be published.