The Exosome Journey: From Biogenesis to Regulation and Function in Cancers

Exosomes are a type of small endosomal-derived vesicles ranging from 30 to 150 nm, which can serve as functional mediators in cell-to-cell communication and various physiological and pathological processes. In recent years, exosomes have emerged as crucial mediators of intracellular communication among tumor cells, immune cells, and stromal cells, which can shuttle bioactive molecules, such as proteins, lipids, RNA, and DNA. Exosomes exhibit the high bioavailability, biological stability, targeting specificity, low toxicity, and immune characteristics, suggesting their potentials in the diagnosis and treatment of cancers. They can be applied as an effective tool in the diagnostics, therapeutics, and drug delivery in cancers. This review summarizes the regulation and functions of exosomes in various cancers to augment our understanding of exosomes, which paves the way for parallel advancements in the therapeutic approach of cancers. In this review, we also discuss the challenges and prospects for clinical application of exosome-based diagnostics and therapeutics for cancers.


Introduction
Extracellular vesicles (EVs) are a collective term containing various types of cell-released membranous structures, including exosomes, microvesicles, microparticles, ectosomes, oncosomes, and apoptotic bodies [1,2]. Among these EVs, exosomes are able to escape from the phagocyte system because of their small size and exert their superiority in intercellular communications. Exosomes are a type of endocytic and heterogeneous membrane-derived vesicles, which could be secreted by multiple cell types to induce or inhibit different cellular and molecular pathways, including immune cells, stem cells, and tumor cells [3,4]. Exosomes used to be regarded as garbage cans for abandoning redundant cell materials. Recently, the roles of exosomes are gradually suggested, including immune regulation, intercellular communications, and biological events.
Cancer is the second leading cause of death all over the world [5]. Continuous improvements in the treatment of cancers, including surgery, chemotherapy, and radiotherapy, have significantly increased the survival, but these strategies cannot effectively control the recurrence and metastasis of cancers [6]. us, novel therapeutic methods are urgently needed. Since exosomes can play key roles in various physiological and pathological processes, it seems likely that they can also affect a variety of pathophysiological processes of cancers, including cancer development, migration, drug resistance, and metastasis. In this review, we comprehensively searched the PubMed database (https://pubmed.ncbi.nlm.nih.gov/) with the combined keywords "exosomes" and "cancers," and focus on the biogenesis, regulation, and function of exosomes in cancers, which are capable of being used as a powerful weapon for the treatment of cancers.

Characteristics and Biogenesis.
In 1981, Trams and his colleagues firstly described exosomes as microvesicles, which contained 5′-nucleotidase activity, released by neoplastic cell lines [7]. However, after that, the concept had been corrected and the endocytic origins of exosomes were proven, which had been regarded as natural nanocarriers. Exosomes are characterized by having a size between 30 and 150 nm, a cupshaped structure, a density of 1.13-1.19 g/mL, and unique double lipid layer [8][9][10]. In addition, exosomes exhibit high biocompatibility and low toxicity. Exosomes are considered as a crucial player in the intercellular communication by transferring cellular contents to recipient cells [11]. ey could transfer the bioactive molecules from cancer cells to cells in tumor microenvironment, thereby facilitating the development and progression of cancers [12]. e bioactive molecules in exosomes are transferred to nearby cells or recipient cells faraway, where they can modulate the signaling pathways involved in the cell proliferation, differentiation, and apoptosis. Exosomes regulate the biological behaviors of cancer cells, tumor microenvironmental cells, and distant recipient cells in this way and participate in the growth, invasion, metastasis, angiogenesis, and drug resistance of cancer cells [13].
Exosomes are commonly obtained from cell culture supernatants and various body fluids, including blood, cerebrospinal fluid, saliva, breast milk, bile, and urine. Exosomes contain a multitude of biomolecules, such as DNA, RNA, mRNA, lipids, metabolites, cytosolic, and proteins, and play essential roles in intercellular communications [14]. ey can be secreted after fusion of multivesicular endosomes with the cell surface or directly sprout from plasma membranes. e biogenesis of exosomes mainly includes four stages, including initiation (membrane formation), endocytosis, formation of multivesicular bodies (MVBs), and sorting (secretion/degradation/recycling) [11]. At first, the cell membrane with ubiquitinated surface receptors is internalized endocytosis of the plasma membrane. en, endosomal sorting complexes, which are necessary for the transport-0 (ESCRT-0), can bind to the ubiquitinated surface receptors via a FYVE domain to be changed into the early endosomes. And MVBs are generated in the form of trans-budding when ESCRT-I and ESCRT-II are assembled into the endosome membranes. At last, ESCRT-III induces the release of exosomes by fusing with the plasma membrane or the degradation by fusing with lysosomes.

Isolation and
Purification. Differential ultracentrifugation method is usually used for the isolation of exosomes. e exosomes were extracted and isolated through multiple centrifugation steps, containing increasing centrifugal strength to sequentially pellet cells, microvesicles, and exosomes [15]. e method is fast and simple, while soluble proteins and nonexosomal particles in exosomes are hardly removed. Based on the centrifugation, ExoQuick precipitation method is also applied to isolate exosomes. After chemically precipitated with ExoPrep solution, the ultracentrifugation is performed. e advantages and disadvantages are similar to the differential ultracentrifugation method. Besides, there are also other techniques, such as ultrafiltration, antibody-coated magnetic beads, commercial precipitation kits, microfiltration techniques, and the stateof-the-art microfluidic technology, used for exosome separation.
Until now, the purest exosomes are isolated through density gradient isolation method by using sucrose or iodixanol. is method can obtain the purest exosomes, but are more time-consuming and more costly. Besides, microfluidic immunoisolation is applied for purification of exosomes by using antibodies against exosomal markers, such as EpCAM and CD63.
is method is centered on exosomes expressing the target surface markers rather than other subpopulations of exosomes (27440105). ere are also other novel detection modalities, such as biosensing and basic proteomics methods. Despite their unique characteristics, there is still a long way in the research of exosome-based assays.

Identification.
Accurately detecting exosomes are important in the biology of vesicles. Currently, optical techniques and nonoptical methods are applied to characterize the vesicles. e optical methods contain nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), fluorescence signals, and flow cytometry. e nonoptical methods include resistive pulse sensing (RPS), micronuclear magnetic resonance (μNMR), nanoholographic imaging, atomic force microscopy (AFM), enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy (TEM), field-effect transistors (FET), lateral flow immunoassay (LFIA), and Raman spectroscopy. e above and emerging exosome-based technologies are helpful and urgently needed for the application of exosomes in the diagnosis and treatment of cancers. With gradually better understanding of exosomes, the corresponding technologies are also improving, which is helpful for developing the exosome-based application.

Exosomal RNAs in Cancers
Exosomes comprise various cellular elements, which are reflected by parental cells and affect the development of cancers. Increasing evidence shows that the functions of exosomes in cancers depend on their cargos and the identification of the components is one of the major challenges in the fields [16]. e uniqueness and enrichment of exosomal nucleic acids (RNAs) have been recently reported to be produced during the process of endosomes endocytose, collecting a large number of noncoding sequences. ere are mainly three types of RNAs, including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), and other noncoding RNAs (ncRNAs) [17,18].

miRNAs.
It is widely accepted that miRNAs are a class of evolutionarily endogenous small noncoding RNAs that are about 20 to 25 nucleotide long sequences at length, which PC miR-125b, miR-130b, and miR-155 Promote neoplastic transformation in adipose derived stem cells [62] miR-196a-5p and miR-501-3p Downregulated in exosomes from PC patients [63] miR-141, miR-375, miR-107 and miR-574-3p Biomarkers for the diagnosis, staging, and prediction of PC [64] miR-1290 and miR-375 Prognostic biomarkers for castration-resistant PC patients [65] can bind to the target mRNAs and negatively regulate the gene expression via post-transcriptional inhibition or target mRNA degradation [19,20]. As reported that, miRNAs are involved in the development and progression of cancers, which have brought increasing attraction [21]. For example, prostate cancer (PCa) cell-derived exosomes promote the angiogenesis, while the exosomes obtained from noncancer individuals are regarded as suppressors in the local environment [22]. Owing to the specific sorting mechanisms, exosomes contain greater concentrations of miRNAs, which are more stable than circulating miRNAs. e stability of miRNAs in exosomes is significantly correlated with the expression levels, which are able to transfer the information to various cellular processes [23]. According to the results of a large cohort including 195 non-small-cell lung cancer (NSCLC) patients and 30 healthy controls, in the research by Dejima et al., the plasma levels of exosomal miR-21 and miR-4257 derived from NSCLC were markedly higher in patients with recurrence than that in patients without recurrence or healthy individuals [24]. Besides, this study also indicated that the higher levels of exosomal miR-21 or miR-4257 are correlated with a worse prognosis with a shorter disease-free survival (DFS) [24]. It has been demonstrated that hepatocellular carcinoma (HCC) cells (Huh7)-derived exosomes restrain the growth and promote the aging of HepG2 cells by transferring miR-122 [25]. Previous studies indicated that breast cancer cell-derived exosomes promoted the growth, metastasis, and autophagy of breast cancer cells by transferring miR-1910-3p [26]. It has been shown that exosomederived miR-200b promotes the proliferation of colorectal cancer, which is transferred to the recipient cells and suppresses the expression of p27 in the target cells [27]. Growing body of evidence indicated that exosomal RNAs play important roles in cancers (Table 1). With the in-depth research on the transportation functions of exosomes, exosomal miRNAs obtain a focus of attention. us, miRNAs play crucial roles in the development and progression of cancers.

lncRNAs.
Other molecules, such as lncRNAs, have also been found in exosomes. LncRNAs contain 200 nucleotide sequences at length, which can regulate the expression of genes or proteins via coordinating epigenetic, transcriptional, and post-transcriptional steps [69]. Numerous studies showed that lncRNAs exert their functions via multiple molecular mechanisms, including binding with DNA to regulate the gene transcription, acting as the competing endogenous RNA (ceRNA) to control the gene expression at post-transcriptional level, combining with proteins, and encoding functional small peptides [70]. Interestingly, lncRNAs could be selectively sorted from exosomes and involved in the intercellular communication in the tumor microenvironment [71].
Exosomal lncRNAs can play important roles in the occurrence and development of cancers, including proliferation, invasion, angiogenesis, and drug resistance, which might act as attractive therapeutic targets and diagnostic biomarkers [72] (Table 2). A recent study showed that in patients with NSCLC, the exosomal lncRNA MALAT-1 was much higher than healthy controls. Besides, the upregulation of exosomal lncRNA MALAT-1 is closely related to an advanced tumor stage and lymphatic metastasis [84]. Although a growing number of evidence reveals that found that lncRNAs could be transferred between various cells, which is related to exosomes, many questions are still doubtful. For example, it is unclear whether nuclear and cytoplasmic lncRNAs exert the similar function that can be loaded into exosomes, and how their sequences, structures, and protein-binding partners affect the sorting. How many copies of lncRNAs are involved in exosomes are needed for phenotypically affecting the recipient cells? ere is a dramatical difference between physiological exosome communication and in vitro treatment with purified vesicle preparations in these aspects, including time and intensity, which require further investigation and careful evaluation. In addition, while lncRNAs are upregulated in specific cancers in comparison with normal tissues and could be detected in exosomes in the circulation of patients with cancers, it is unclear whether the incorporation into exosomes depends on the specific sorting mechanisms in cancer cells [86].

circRNAs.
As a type of exosome-derived noncoding RNAs, exosomal circRNAs have been found to play key roles in the development and progression of cancers and act as novel diagnostic and prognostic biomarkers in cancers. CircRNAs are a class of tissue specific and covalently closed circular noncoding RNA, which is widely present in eukaryote. Exosomal circRNAs are novel frontiers in cancer research, and the important exosomal circRNAs are summarized in Table 3.
ese studies have suggested that exosomal circRNAs might exhibit an important influence on the development and occurrence of cancers, which suggested the potential diagnostic and therapeutic values of exosomal circRNAs. To explore the mysterious connections of exosomes and circRNAs, it might provide a key hint to probe into the biological functions of exosomal circRNAs. In addition, studying the pathogenesis mechanisms of cancers and identifying novel promising diagnostic biomarkers and therapeutic targets are a popular topic in the future.

Exosomal Proteins in Cancers
Exosomes can carry a broad variety of molecules depending on the origins and in vitro culture conditions, such as proteins. It has been shown that exosomes can express a wide range of proteins involved in membrane-related functions, including Rab GTPase, Annexin, cellular adhesion proteins (integrins and tetraspanins), cytoskeletal proteins (actin and myosin), and heat shock proteins (Hsp70) [112]. Recent studies suggest that membrane transport and fusion proteins in exosomes play vital roles in the occurrence and development of cancers, including annexins, RAB5/RAB7, and TSG101. For example, dendritic cells (DCs)-derived exosomes are capable of carrying MHC-I, which can bind to the tumor-derived peptides, and then, the complex activates the immune cells to play crucial antitumor roles [113]. An interesting study has revealed that membrane surface protein TRAIL from exosomes can transfer the apoptosis-related signals to the tumor cells and induce the apoptosis of tumor cells [114]. Moreover, exosomes with SIRPα are capable of binding to CD47 on tumor cells, and promoting the phagocytosis of macrophages and finally inhibiting the cancer progression [115]. In addition to the RNAs and DNA present in exosomes, exosomal proteins could also be used as promising biomarkers (Table 4). It has been reported that exosomal EGFR protein can be regarded as a potential biomarker for the characterization of lung cancer [141,142]. As demonstrated, the diagnostic potentials of 49 exosomal membrane-attached proteins in 336 patients with lung cancer and 127 controls are confirmed. Among these proteins, CD151, CD171, and tetraspanin 8 are reported to be the strongest biomarkers for patients with lung cancer [143].

Diagnosis.
With the aging and growth of the population, the incidence and mortality of cancers are rapidly growing and cancer is one of the most leading causes of death. Cancers seriously endanger human health, and early diagnosis of cancers is required for increasing the survival rates of patients and reducing the mortality. Recently, one of the latest concepts regarding early diagnosis is based on the extracellular vesicles released by cells where the exosomes are at the frontiers. e biomarker potentials of exosomes in liquid biopsies hold huge promise and might revolutionize the diagnostics strategies of cancers. For example, exosomederived miRNAs can act as novel potential biomarkers in the diagnostics and prediction of colorectal cancer [144,145]. It has been found that miR-423-5p is increased in the serum exosomes of patients with gastric cancer (GC), and the levels of miR-423-5p are related to lymph node metastasis and poor outcome [146]. And the levels of miR-126 and let-7a are much higher in exosomes collected from the bronchoalveolar fluid of patients with lung adenocarcinoma than healthy controls [147]. Moreover, patients with pancreatic cancer (PC) exhibit higher level of exosomal miR-191, miR-21, and miR-451a than healthy subjects [148]. Exosomal lncRNA SAP30L-AS1 has been discovered to upregulated in benign prostatic hyperplasia (BPH) and lncRNA SChLAP1 is increased in prostate cancer than in BPH and healthy controls, which is confirmed to possess diagnostic values in distinguishing prostate cancer by the receiver operating characteristic curve [149]. A study containing 246 subjects (126 patients and 120 healthy controls) showed that the expression levels of exosomal lncRNA HOTTIP were much higher in gastric cancer patients than normal controls, which was in close correlation with invasion depth [82].
In addition, the protein levels in exosomes might also act as biomarkers in the diagnosis of cancers. As previously described, the patients with acute myeloid leukemia (AML) exhibit higher levels of plasma exosomes with a specific phenotype containing transforming growth factor beta 1 (TGFβ-1), which has a suppressive effect in the cytotoxic activity of NK cells [150]. According to the results of proteomics analysis by mass spectrometry of urinary exosomes obtained from 16 patients with prostate cancer (PC) and 15 controls, the transmembrane protein 256 is the highest sensitivity of 94% and distinctly enriched with high specificity in patients with PC [151]. As previous detailed, an inhibitor of apoptosis protein (Survivin-2B) has been found to be differentially expressed in exosomes from patients with breast cancer, suggesting its values in the diagnosis and prognosis of breast cancer [152]. e results of gene microarray from blood exosomes of mice with glioblastoma multiforme (GBM) revealed that the levels of DNM3, p65, and CD117 are significantly increased, whereas PTEN and p53 are decreased, which might be served as novel diagnostic markers for GBM [153].
Some studies also focus on the roles of circRNAs in cancer diagnosis. For example, the level of circ-PDE8A has been detected in the plasma exosomes from patients with pancreatic ductal adenocarcinoma (PDAC) and exosomal circ-PDE8A has been found to be related to the progression and prognosis in PDAC patients [109]. Many studies have stressed the roles of exosomes in the diagnosis and prediction of cancers owing to the exosomal molecules, including miRNAs, proteins, and lncRNAs. We believe that indepth understanding of exosomes in the diagnosis of cancers could help to design novel cancer-diagnostic and cancerprognostic tools. Effective diagnostic strategies for cancers based on exosomes are expected in the near future. Despite these advances in exosomes-based diagnostics and prediction of cancers, there are still many challenges needed to be overcome until clinical application. Much research needs to be done about the roles of exosomes in the diagnosis and prediction of cancers in the future.

Treatment.
Regarded as the best-studied natural nanomaterials in the past decades in cancer therapy, exosomes are able to pass through the biological barriers, such Journal of Oncology 5 as the blood-brain barrier (BBB) and blood-tumor barrier, indicating their potentials in the diagnosis and treatment of brain cancers. Mesenchymal stem cells (MSCs)-derived exosomes have been reported to be applied in the treatment of pancreatic cancer (PC) in animal experiments by delivering KRAS, G12D, and siRNAs [154,155]. Besides, exosomes could be applied as novel antitumor vaccines. For example, exosomes obtained from dendritic cells can deliver the melanoma antigen identified by T cells 1 (MART-1) into patients with NSCLC at IIIB/IV stage and then inhibit the   Journal of Oncology progression of NSCLC and improve the survival through activation of immune response, which is revealed by phase II clinical trials [113]. Many targets of anticancer chemical agents are intracellular, which need to cross the cell membranes and then exert anticancer functions. Because of their poor water solubility and easy degradation, the chemical agents are difficult to produce the desired effects or avoid adverse reactions. Exosomes have the advantages of low immune prototype and weak side effects, whose lipid bilayer can play protective roles in the contents in vivo [156]. Besides, exosomes are easy to enter the cells via the interaction between their membrane proteins and target cells.
us, exosomes are regarded as the best natural carriers for chemical agents [115]. Hence, a better understanding of how to engineer and deliver exosomes to specific cells is crucial to improve the cancer therapy potential of exosomes. For example, plant-derived exosomes are easily to be absorbed by the intestine and are used for inhibiting the development of colon cancer by delivering curcumin, confirmed by clinical trials (ClinicalTrials.gov identifier: NCT01294072). It has been confirmed that the treatment with DCs-derived exosomes carrying doxorubicin significantly inhibits the growth of breast cancer cells and exerts no toxicity effects on mouse models [157]. In the study of K. Tang et al., exosomes loaded with cisplatin exert significant anticancer effects on ovarian cancer and cause no side effects on other tissues in mice, including liver and kidney. And this is an advantage in comparison with cisplatin treated alone [158]. Moreover, exosomes from plants can also be used in the adjuvant therapy for cancers. It has been shown that during the treatment of head and neck cancer, the patients suffer from oral mucositis. And grape-derived exosomes are able to ease the oral mucositis and pain, revealed by clinical trials (ClinicalTrials.gov identifier: NCT01668849). Exosomes can extend the circulation half-life and block the drug accumulation in nontarget organs by targeting specific cells.
us, the release of exosomes in biofluids could have major advantages for exploration of the complex mechanisms of tumor progression and treatment response.
To successfully develop novel and efficient therapeutic strategies in the treatment of cancers, exosomes should not be disregarded. And the application of exosomes in clinical treatment of cancers is a challenging but intriguing endeavor that needs further investigation and exploration.

Conclusion and Future Prospective
Increasing evidence concerning exosomes has stressed their important roles in cancer development and clinical application recently. Exosome-derived cargos are involved in the pathophysiological processes of cancers, including cancer development, migration, drug resistance, and metastasis. In this review, we have elaborated upon the characteristics, biogenesis, isolation, purification, and identification of exosomes and summarized the roles of exosomal RNAs (miRNAs, lncRNAs, and circRNAs) and proteins in cancers. Investigation of the molecular mechanisms underlying the biogenesis and biological functions of exosomes will help to design more novel therapeutic methods targeting exosomemediated pathophysiological processes in cancers. It is noteworthy that various exosomal molecules are applied for developing emerging diagnostic strategies and clinical treatments of cancers. Obviously, the specific RNA in exosomes is one of the most robust surrogates of cancers and affects the current states of cancers. us, the corresponding targeted agents for these RNAs and molecules can be designed, which can be further personalized medicine. In addition to the research progress in mechanism, there are also further challenges in clinical application. e current knowledge on the roles of exosomes in cancers is limited; therefore, their potentials in the diagnosis and treatment of cancers require extensively studied.
Although exosome-based cancer treatment exerts great potentials, there are still many problems need to be solved until clinical application. e technologies of production and quality control are flawed. Although some preclinical studies evaluate the roles of exosomes as therapeutic drug delivery carriers in the cancer therapy and suggest their clinical application potentials, few exosomes-based clinical trials have been conducted, which might be the focus in the future research. Besides, several technical limitations, including high quality and bulk exosome preparation based on standard protocol, and exosome target specificity, are the biggest barriers presently impeded exosome-based diagnostic and therapeutic applications. Currently, there is still a long way to overcome the difficulty in the exosome-based therapeutic strategies in the treatment of cancers. Exosomesbased research might need to put more energy into in vivo models and clinical application in the future so that it is helpful to elucidate these questions.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Yuan Li conceived of the review paper with the guidance of Baobing Zhao; Yuan Li wrote the original draft; Yuan Li, Li Meng, Bo Li, Yanxia Li, Tao Shen, and Baobing Zhao reviewed and edited the paper. All authors have read and agreed to the published version of the manuscript.