The multifaceted involvement of exosomes in tumor progression: Induction and inhibition

Abstract As key performers in intercellular communication, exosomes released by tumor cells play an important role in cancer development, including angiogenesis, cancer‐associated fibroblasts activation, epithelial‐mesenchymal transformation (EMT), immune escape, and pre‐metastatic niche formation. Meanwhile, other cells in tumor microenvironment (TME) can secrete exosomes and facilitate tumor progression. Elucidating mechanisms regarding these processes may offer perspectives for exosome‐based antitumor strategies. In this review, we mainly introduce the versatile roles of tumor or stromal cell derived exosomes in cancer development, with a particular focus on the biological capabilities and functionalities of their diverse contents, such as miRNAs, lncRNAs, and circRNAs. The potential clinical application of exosomes as biomarkers in cancer diagnosis and prognosis is also discussed. Finally, the current antitumor strategies based on exosomes in immunotherapy and targeted delivery for chemotherapeutic or biological agents are summarized.


F I G U R E 1
The functions of tumor cell-derived exosomes in cancer development. Tumor cell-derived exosomes carrying biological molecules, such as miRNAs, proteins, and nucleic acids, are involved in various aspects of tumor progression, including angiogenesis, cancer-associated fibroblasts activation, immune escape, epithelial-mesenchymal transformation, pre-metastasis niche formation, and drug resistance through the transportation of cargos within extracellular space. 5 On the other hand, exosomes also play a critical role in the progress of some diseases, especially as the transmitters in tumor microenvironment (TME). 6,7 It has been demonstrated that cancer cells exhibited the significantly increased exosomes secretion compared with normal cells with profound influences on tumor progression and metastasis. 6,7 For example, exosomes derived from tumor cells can alter the properties of normal cells by carrying oncogenic materials and initiate phenotypic changes in TME, thus progressing cancer development. 8,9 Emerging evidences have also proved that exosomes are responsible for the tumor cell expansion, metabolic activity remodeling, tumor angiogenesis, immunosuppressive TME, and acquired drug resistance (Figures 1 and 2). 10 These studies urge the need of corresponding regarding these revealed mechanisms.
Research have shown that exosomes are recognized as important transporters in cancer and exhibit promising prospects for cancer diagnosis (Figure 3). 11 Recently, the applications of exosomes have been focused on biomarkers in diagnosis, underlying functions and mechanisms, and drug delivery systems owing to its unique natural features. 12,13 In this review, we focus on the present studies and the underlying mechanisms of exosomes, especially regarding the involvement in TME. Additionally, we conclude the application exosomes in tumor diagnosis and therapy.

ROLE OF TUMOR CELL-DERIVED EXOSOMES FOR TUMOR PROGRESSION
Exosomes function variably according to the origin of cells. Tumor cells-derived exosomes (TCDEs) contribute to the tumor progression through the induction of tumor cell proliferation, invasion, and migration, and are also proved to be the key attributes of tumorigenicity and TME maintenance. Targeting TME is widely accepted as a promising strategy to effectively suppress tumor progression, and six important aspects of involvement in tumor progression regarding tumor cell-released exosomes have been summarized as follows ( Figure 1 and Table 1).

TCDE and angiogenesis
Angiogenesis is a crucial part which shows a profound influence on promoting tumor progression, consequently leading to poor prognosis. In the preexisting vascular network, new blood vessels are formed by either endothelial cells mediated-sprouting angiogenesis or interstitial tissues-expanded intussusceptive angiogenesis. 14 Besides, the secretion of pro-angiogenic factors by tumor cells triggers the creation of this disorganized, immature, and permeable vascular network, promoting cancer cell invasion and impeding the tumor-killing action of immune cells. 15

F I G U R E 2
The functions of exosomes from other cells in TME in cancer development. Exosomes from other cells in TME, such as mesenchymal stem cells, cancer-associated fibroblasts and macrophages can promote cell proliferation, drug resistance, and tumor metastasis, thus facilitating tumor progression F I G U R E 3 Application of exosomes in cancer diagnosis and therapy. A, Exosomes containing specific RNAs and proteins can be utilized as noninvasive biomarkers for cancer diagnosis and prognosis. B, Exosomes can serve as nanocarriers for drug delivery and can be applied in immunotherapy as cancer therapeutic vaccine Surprisingly, exosomes show great potential in inducing either pro-or anti-angiogenic signaling through the information delivery to endothelial cells. Additionally, exosomes adjust their cargo composition to fine tune the process of blood vessel formation in response to changes in TME. 14 Contents of exosomes, especially miRNAs potentiate this process, and multiple studies have validated the role of exosomal miRNA in the activation of angiogenesis. 16 TCDEs are identified to trigger angiogenesis in papillary thyroid cancer, 17 ovarian cancer, 18 breast cancer, 19 and lung cancer 20 through exosomemediated miRNA signaling. Under hypoxic conditions, glioblastoma cells derived-exosomes carrying miR-182-5p promote tumor angiogenesis and increase vascular permeability, which are supportive for microenvironment. 21 Consistently, glioma stem cells-derived exosomal miR-26a enhances angiogenesis of microvessel endothelial cells in glioma. 22 In cervical squamous cell carcinoma (CSCC), CSCC cell-secreted exosomal miR-221-3p was found to enhance the angiogenesis in the transfer to vessel endothelial cells targeting THBS2. 23 In gastric cancer (GC), TCDEs carrying miR-130a, 16 miR-155, 24 miR-135b, 25 miR-23a, 26 and miR-155 27 were investigated to exert identical effect on promotion of angiogenesis through varying mechanisms. In colorectal cancer (CRC), exosomal miR-183-5p induces angiogenesis by regulation of FOXO1, 28 and miR-1229 promotes angiogenesis by targeting HIPK2. 29 In  83 Symbol "/" represents the unknown cargo. hepatocellular carcinoma (HCC), exosome-mediated secretion of Angiopoietin-2-induced angiogenesis 30 and exosomal lysyl oxidase like 1 (LOXL) promotes cell invasion and metastasis. 31 Most of TCDE-related miRNAs exhibit angiogenic promotion effect, however, some exosomes are the opposite. miR-590-5p acted as an antionco-miR which was found to inhibit angiogenesis and metastasis through the regulation of nuclear factor (NF) 90/VEGFA axis. 32 Several stem cells, including menstrual mesenchymal stem cells (MSCs) and liver stem cells, have been identified as inhibitors of angiogenesis, and secreted exosomes loading miRNA are detected to suppress angiogenesis and tumor growth. 33,34 In breast cancer, researchers discovered that an antiangiogenic agent, docosahexaenoic acid (DHA), triggered the secretion of exosomes and the expression level of miRNAs including miR-23b, miR-27b, and miR-320b, which inhibited tube formation of endothelial cells by decreasing the expression of their respective pro-angiogenic target genes, including PLAU, AMOTL1, NRP1, and ETS2. 35

TCDE and cancer-associated fibroblasts
Cancer-associated fibroblasts (CAFs), a kind of stromal cells, form a part of TME and play an important role in the regulation of the tumor progression. 36 Based on the demonstrated presence of CAFs in malignancy, Paggetti et al unveiled the presence of CAFs in chronic lymphocytic leukemia (CLL) lymph node. What's more, AKT serine/ threonine kinase 1 (AKT) and NF-κB in stromal cell were demonstrated to be essential for induction of the inflammatory phenotype. It is worth noting that, the uptake of cancer exosomes by stromal cells elicits the inflammatory effects which contribute to the CAFs transition. 37 Ringuette et al determined the fibroblast differentiation into CAFs through transforming growth factor beta 1 (TGF-β) via bladder cancer (BC) cells-derived exosomes, suggesting exosomal TGF-β can be considered as a novel molecular mechanism involved in CAF activation in tumor. 38 Previous studies also have shown that exosomes act as a messenger between the communication/crosstalk of tumor cells and CAFs. Therefore, a deep understanding of this interaction may make contribution to the cancer therapy.
Exosome-secreted miRNAs exert the inducing progression effects on tumors. For example, HCC-derived exosomal miRNA-21 induces tumor progression by activation of hepatic stellate cells into CAFs, demonstrating the role of exosome mediator. 39 Another case in melanoma shows that exosomal miR-155-5p induces CAF proangiogenic function via suppressor of cytokine signaling 1 (SOCS1)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway, 40 and exosome-mediated paracrine miR-34a-5p CAF triggers oral cancer cells proliferation and metastasis. 41 Chen et al revealed that exosome-mediated transfer of miR-93-5p from CAFs led to radioresistance in CRC cells through the downregulation of FOXA1 and upregulation of TGF-β3. 42

TCDE and immune escape
Immune system displays a critical role in maintaining tumor development and progression. Exosomes from both non-immune and immune cells participate in immune regulation, such as mediating T cell apoptosis, reducing NKand T cells, and triggering the differentiation of myeloid cells to myeloid derived suppressor cells. 43 To destroy the anti-tumor immunity, tumor-derived-exosomes deliver the suppressive proteins and nucleic acids to immune cells including monocytes, macrophages, dendritic cells (DCs), natural killer cells, T cells, B cells and thus evading immune surveillance. 44,45 programmed death-ligand 1 (PD-L1) is one of these suppressive proteins that exist on the surface of exosomes. 46 Exosomal PD-L1 systemically tends to suppress the anti-tumor immune response, and its genetic blockage promotes T cell activity in the draining lymph node resulting in the induction of systemic anti-tumor immunity and memory. 47 On this basis, investigations revealed that exosomal PD-L1 can be upregulated by interferon-γ, thereby suppressing CD8 + T cells and inducing tumor growth. 48 Besides, it has been revealed that PD-L1-induced immunosuppression is correlated with anti-PD-1 response, implying exosomal PD-L1 can be applied as a biomarker for anti-PD-1 therapy. 48 In addition, compared to soluble PD-L1 which increases in healthy individuals with age, exosomal PD-L1 provides alternative options for cancer diagnosis and prognosis prediction. 49 In parallel, non-small cell lung cancer (NSCLC) cell-derived exosomal PD-L1 fosters tumor growth through immune escape of identical mechanisms. 50 Another study in HCC reported that tumor-derived exosomes could promote the immune-suppressive regulatory B cells (Bregs), a kind of B cells subsets. 51 High mobility group box 1 (HMGB1), a kind of DNAbinding nuclear protein, is expressed on exosome membranes. Interestingly, studies illustrated that Bregs infiltrated tumors, and T-cell immunoglobulin mucin domain-1 (TIM-1) (a marker of Bregs) + Bregs exhibited enhanced suppressive activity against CD8 + T cells. Researchers detected that the expansion of Bregs promoted the advanced disease stage through the exosomal-derived HMGB1 activation. 52 In melanoma, tumor exosomes harboring enriched hsa-miR-3187-3p, hsa-miR-498, hsa-miR-122, hsa-miR149, and hsa-miR-181a/b elicit the immune escape by regulating TCR signaling and TNFα secretion. 53 Furthermore, Haderk et al discovered the protumorigenic skewing mechanisms related to PD-L1 expression in CLL and concluded that CLL-derived exosomes mediated the transfer of non-coding RNAs to monocytes, leading to the immune escape. 54

TCDE and epithelial-mesenchymal transformation
Epithelial-mesenchymal transformation (EMT) is a conserved process characterized as the loss of epithelial features and the possession of mesenchymal phenotype. 8 Prior research generally demonstrates that EMT is a tumor-associated process that may elicit tumor cell invasion, metastasis, and drug resistance. Zhang et al proved that hypoxic bone marrow-derived MSCs (BMSCs)-derived exosomes promoted tumor cell invasion and EMT. 55 Additionally, using miR-193a-3p, miR-210-3p, and miR-5100loading exosomes together exerts better diagnostic accuracy in patients with lung cancer metastasis compared with respective treatment. 55 Exosomes secreted from cancer cells are implicated in inducing EMT in cancer development. Wang et al conducted the study on the correlation between cancer stem cells (CSC) from clear cell renal cell carcinoma and EMT. The outcome revealed that CSC exosomes initiated EMT through the transmission of MIR-19b-3p to CCSCC cells, which was related to the repressed expression of phosphatase and tensin homolog (PTEN). What's more, metastasis exosomes display stronger EMT effect. 56 EMT occurs in melanoma cell-derived exosomes as a consequence of the activation of MAPK pathway, thus leading to metastasis. Decreased let-7, a miRNA modulator of EMT is detected, which may account for this phenomenon. 57 Similar examples are shown in HCCderived exosomes. Highly metastatic HCC-derived exosomes improve migration, chemotaxis, and invasion of low metastatic cells by transferring pro-metastatic molecules. Abrogating the release of these exosomal factors through inhibition of Rab27a, a modulator of exosome secretion, promotes invasion of parental cells. 58 Vps4A, functioning as a tumor suppressor through regulation of exosomal miRNAs in HCC, is demonstrated to mediate the plasma membrane localization and decrease exosomereleased β-catenin, thus inhibiting EMT when it is overexpressed. 59,60 As mentioned above, CAFs act as abettors in tumor progression, including the induction of EMT. 61 Yeon et al observed that MSC-derived exosomes reduced CAFs and suppressed EMT, thereby inducing angiogenesis and maintaining vascular homeostasis, while cancer-derived exosomes that transdifferentiate CAFs promoted EMT. 62 Hu et al examined the elevated level of miR-92a-3p as a consequence of CAFs transferring exosomes to CRC cells. Additionally, increased miR-92a-3p activated Wnt/βcatenin pathway and inhibited mitochondrial apoptosis, leading to EMT and thus promoting metastasis and chemoresistance. 63 Another similar study shows the promotion of EMT in breast cancer caused by CAFs secreted exosomal microRNA-181d-5p and the underlying mechanisms is related to the downregulation of homeobox A5 (HOXA5) and caudal-related homeobox 2 (CDX2). 64 Studies regarding the correlation between CAFs and EMT have been performed, providing understandings on repressing tumor progression.

TCDE and pre-metastatic niche
Pre-metastatic niche in cancers is a prevalent precondition of metastasis. Liu et al elucidated it with six pro-metastasis features including immunosuppression, inflammation, angiogenesis/ vascular permeability, lymphangiogenesis, organotropism, and reprogramming. 65 There is an urgent need to seek novel and effective approaches targeting premetastasis niche to make amends for the delayed detection and prevent further metastasis. The consensus has been that exosomes participate in pre-metastatic niche formation of cancers. 66 Costa-Silva et al found that pancreatic ductal adenocarcinomas (PDAC)-derived exosomes were integrated with Kupffer cells, a kind of macrophage, to stimulate the fibronectin production and induce the formation of pre-metastasis niche. Furthermore, macrophage migration inhibitory factors (MIF), which were released by macrophages in fibrotic microenvironment and enriched in PDACexosomes, could prevent the niche formation when it was blocked. 67 Zeng et al manifested the involvement of CRC-derived exosomal miR-25-3p in promoting premetastatic niche formation through mechanisms related to the angiogenesis and vascular permeability. 68 Toll-like receptor 3 (TLR3) are able to recognize inflammatory signals and promote tumor progression. Liu et al uncovered that deficiency of TLR3 in lung epithelial cells prevented the niche formation, while exosomal RNA reversed this process by activating TLR3, consequently recruiting Neutrophils and favoring the niche formation. 69 Similarly, melanosomes foster tumor niche by reprogramming dermal fibroblast into CAFs through transferring miR-211. 70 Contrary to the aforementioned research, Plebanek et al discovered that non-metastasic exosomes resulted in the inhibition of metastasis. It is identified that premetastatic tumors release exosomes, simultaneously eliciting immune surveillance, resulting in tumor cell clearance at the pre-metastatic niche. 71 It has also been reported that melanoma-derived exosomes facilitate the formation of pre-metastatic niche via induction of vascular leakiness and pro-vasculogenic phenotype of bone marrow progenitors. 72 Another notable investigation based on previous exosome-mediated metastasis theory revealed the crucial role of exosome integrin in promoting metastasis. Hoshino et al provided a rational of exosome integrin predicting organotropism of metastatic cells, which indicated the promising prospect of the application in diagnosis and treatment. 73

TCDE and drug resistance
Acquired drug resistance is a troublesome obstacle that causes limited therapeutic response in cancer patients. 74 Previous studies have documented that exosomes derived from tumor cells or related stromal cells mediate the drug resistance through the exosomal transfer of miRNAs. 75 The miR-21 transferred from macrophages to GC cells was detected to reduce cisplatin chemosensitivity and suppress apoptosis. 76 However, exosome-transmitted miR-128-3p overexpression displays enhanced chemosensitivity in oxaliplatin-resistant advanced CRC through altering target gene expression, while lower expression of miR-128-3p leads to poor drug response. 77 In glioblastoma, exosomal transfer of miR-15a and miR-1238 was substantiated to contribute to chemotherapeuticresistance. 78,79 In multidrug-resistant HCC cells, elevated exosomal miRNA-32-5p and reduced PTEN are associated with poor prognosis. Additionally, miRNA-32-5p targeting PTEN induces multidrug resistance via the PI3K/Akt pathway. 80 EphA2 is overexpressed in tumors and has been recognized as a pivotal mediator of chemoresistance. In pancreatic cancer, exosome-mediated EphA2 transfer is revealed to perform chemoresistance transmission between variable gemcitabine resistance cell lines. 81 Dorayappan et al demonstrated that exosomes were greatly secreted under hypoxic condition, and these hypoxiainduced exosomes increased cisplatin efflux, augmenting metastasis and chemotherapy resistance in ovarian cancer. 82 Another investigation in ovarian cancer has shown that exosomal miR-1246 is potent effector in acquiring chemoresistance via modulating Cav1and p-gp interaction. 83 In addition, the expression of drug targets on exosomes, including PD-L1, CD20, and CTLA-4, may be a novel mechanism for drug resistance. These molecules carried on exosomes can deplete the specific drugs or antibodies to waken the therapeutic effects. 46 Yang et al have proved that the accumulation of exosomal PD-L1 in TME induced therapeutic resistance, and the successful inhibition of these exosomes' secretion could recover the efficacy of anti-PD-1 therapy. 84

Mesenchymal stem cell-derived exosomes
MSCs are considered as crucial regulators of TME and control multiple aspects of cancer progression, including tumorigenesis, angiogenesis, and metastasis. 85 It has been reported that the interactions between MSCs and tumor cells, predominately mediated by exosomes, is the key performer of MSCs functions. On the one hand, exosomes derived from tumor cells transfer biologically active proteins and nuclear acids to MSCs, co-opting MSCs and transforming them into tumor supportive types. On the other hand, MSCs reprogrammed by tumor exosomes become avid producers of their own exosomes, secreting various factors accumulating cancer progression (Table 2). 2, 86 Zhang et al found that exosomes released by bone marrow-derived MSCs (BMSCs) could promote invasion of lung cancer cells by activating STAT3 signaling pathway. The microRNAs, including miR-193a-3p, miR-210-3p, and miR-5100, transferred by exosomes from BMSCs to epithe-lial cancer cells exhibited diagnostic accuracy that discriminate cancer patients from non-cancerous controls. 55 Additionally, MSC-derived exosomes from glioma tissues increase the proliferation and clonogenicity of tumorinitiating stem-like cells, leading to greater tumor burden and decreased survival. 87 Moreover, MSC-derived exosomes are implicated in immune regulation and inhibit the functions of B-lymphocytes by affecting the expression of specific mRNAs. 88 In a recent study, Wang et al demonstrated that exosomes secreted by MSC-differentiated adipocytes promote breast cancer cell growth and protect tumor cells from chemotherapeutic drug-induced apoptosis in vitro. The inhibition of exosomes attenuates the tumor-promoting effects of adipocytes. 89 While MSC-derived exosomes primarily exert pro-tumor effects in cancer development, studies also reported the tumor suppressive activities of exosomes secreted by MSCs. Jiang and colleagues showed that MSC-derived exosomes carrying miR-205 could repress prostate cancer cell proliferation, invasion, and migration, thus impeding cancer progression through inhibiting rhophilin Rho GTPase binding protein 2 (RHPN2). 90 Similarly, MSC-derived exosomal miR-126-3p suppresses the development of pancreatic cancer by targeting ADAM9. 91 In another study, Xu et al found that exosomes containing miR-133b released by MSCs attenuated glioma development via inhibiting the Wnt/β-catenin signaling pathway. 92 Collectively, the diverse cargos transferred by MSC-derived exosomes are the predominant determinant of their effects in tumor progression, and MSCs also varies dependent on local TME. 85

Fibroblast-derived exosomes
Exosomes released by tumor cells promote tumorigenesis through activation of CAFs, which actively remold TME and sustain a cancer permissive state. Conversely, CAFsderived exosomes also significantly affect tumor cells and facilitate tumor progression. 93 A number of studies have demonstrated that CAFs-derived exosomes are involved in tumor cell proliferation, migration, metastasis, and drug resistance. 63,[94][95][96] For example, fibroblast exosomes have been shown to enhance the survival and proliferation of pancreatic cancer cells, while inhibition of exosome release by GW4869 significantly reduces survival of cancer epithelial cells. 94 Hu et al found that exosomes secreted by CAFs are transferred to CRC cells and lead to overexpression of miR-92a-3p, activating Wnt/β-catenin pathway and inhibiting mitochondrial apoptosis, thus promoting metastasis and chemotherapy resistance. 63 In another study, exosomal miR-522 derived from CAFs suppresses ALOX15 expression and lipid-ROS accumulation in cancer cells, decreasing chemo-sensitivity and inhibiting ferroptosis, a novel mode of non-apoptotic cell death. 95 Symbol "/" represents the unknown cargo.

TA B L E 2 Role of exosomes from other cells in TME for tumor progression
CAFs-derived exosomes have also been shown to contain long non-coding RNAs (lncRNAs) that promote the stemness and chemoresistance of CRC. 96

Macrophage-derived exosomes
Macrophages within the TME play a critical role in cancer development, which promote cancer initiation and progression by facilitating cancer cell proliferation, migration, angiogenesis, and immunosuppression. 97 Macrophagederived exosomes orchestrate their communication with cancer cells, significantly affecting the malignant progression.
It has been shown that M2 macrophage-derived exosomes transfer miR-21-5p and miR-155-5p to CRC cells, inducing cancer cell migration and invasion. 98 In addition, exosomes released by macrophages promote progression of PDAC by targeting transforming growth factor beta receptor 3 (TGFBR3), a tumor suppressor gene, and activating TGF-β signaling pathway. 99 It has also been found that M2 macrophage-derived exosomes contribute to cisplatin resistance in GC cells through delivery of miR-21, which activates PI3K/AKT signaling pathway and inhibits cell apoptosis. 76 In another study, the authors demonstrated that macrophage-derived exosomes transfer functional apolipoprotein E to GC cells and promote the migration of cancer cells. 100

APPLICATION OF EXOSOMES IN DIAGNOSIS
In most cases, the poor therapeutic response and prognosis in cancer patients are due to the late detection of diseases, which is largely attributed to the lack of appropriate biomarkers that serve as efficient diagnostic tools. 101 Therefore, considerable attention has been drawn to identifying and validating disease-specific molecules that could be utilized for cancer diagnosis and prediction ( Figure 3). Exosomes containing various proteins, lipids, nucleic acids, and metabolites are considered as the rich source of potential biomarkers with unparalleled advantages. 102 First, almost all cells secrete exosomes, and the multiple cargos carried by exosomes reflect the physiological status of their parental cells. 103 Second, apart from being secreted by different cells in vivo, exosomes also widely exist in various body fluids, such as blood, urine, and saliva. 103 Thus, exosomes are ideal noninvasive biomarkers and easily accessible for clinical detection. Third, due to the unique structure of exosomes, proteins and nucleic acids could be well protected by the lipid bilayer, and important information about disease characteristics is preserved. 101 miRNAs are one of the biological cargos in exosomes that participate in multiple aspects of cancer progression. Emerging evidence suggests that miRNAs are potential biomarkers for cancer diagnosis and prediction due to their stable properties and extreme abundance in exosomes (Table 3). 19 Importantly, with the development of specific and sensitive quantitative techniques, exosomal miRNAs can be detected in small volume samples with high accuracy. 103 Clinical studies have shown that miR-126, a tumor suppressor and metastasis inhibitor, is downregulated in NSCLC patients. Additionally, miR-126 is equally distributed between exosomes and exosome-free serum fractions in healthy people while mainly present in exosomes of NSCLC patients, suggesting the potential of miR-126 as a reliable disease biomarker. 104 Furthermore, Jin et al have identified AC-and SCC-specific miRNAs as diagnostic biomarkers for early stage NSCLC by using next-generation sequencing. The validated miRNAs with diagnostic accuracy such as AC-specific miR-181-5p, miR-30a-3p, miR-30e-3p, and miR-361-5p, and SCC-specific miR-10b-5p, miR-15b-5p, and miR-320b could be promising indicators in the diagnosis of NSCLC. 105 It has also been reported that the expression level of exosomal miR-25-3p is significantly elevated in CRC patients with metastasis than those without metastasis. Mechanism analysis has shown that miR-25-3p regulates the expression of VEGFR2, ZO-1, occludin, and Claudin5 in endothelial cells by targeting KLF2 and KLF4, promoting angiogenesis and pre-metastatic niche formation. 68 Interestingly, Cheng et al have demonstrated that the secretion of colorectal CSC exosomes carrying biomarker miR-146a-5p is dependent on the expression of RAB27B by activation of β-catenin/Tcf-4. 106 In addition, Xue et al identified the differentially expressed exosomal miR-NAs in cholangiocarcinoma and gallbladder carcinoma patients with high-throughput small RNA sequencing. After a larger-scale validation, they found that miR-96-5p, miR-151a-5p, miR-191-5p, and miR-4732-3p were significantly increased in the exosomes of cholangiocarcinoma patients, while miR-151a-5p was increased in the exosomes of gallbladder carcinoma patients. 107 In summary, although a multitude of miRNAs have been identified and validated as potential diagnostic markers for cancer in the basic research studies, much clinical data are also required to confirm their diagnostic effects. 19 In addition to miRNAs, lncRNAs are also attractive candidates in the development of sensitive and specific biomarkers for cancer diagnosis and prediction. These lncRNAs are enriched in exosomes and stable in blood, reflecting the cell status that they origin. Additionally, the different expression level of lncRNAs in tumor tissues and healthy organs suggests their potential clinical application as cancer-specific biomarkers. For instance, the expression level of lncRNA-UCA1 is significantly higher in exosomes of BC patients than in healthy donors and could be used for the clinical detection of BC. 108 While in another study, Zheng et al found that lncRNA PTENP1 was significantly reduced in BC tissues, and normal cell derived-exosomal PTENP1 could be transferred to cancer cells, impeding cancer progression by inhibiting cell migration, invasion, and inducing apoptosis. 109 Moreover, reports have established that a urinary exosome-derived lncRNA panel (MALAT1, PCAT-1, and SPRY4-IT1) could be used as noninvasive diagnostic and prognostic biomarker of BC with considerable clinical value. The upregulation of PCAT-1 and MALAT1 indicates poor recurrence-free survival. 110 Tumor-originated exosomal lncUEGC1 is expressed at a high level in stage I GC patients and GC cells. Notably, it exhibits higher diagnostic accuracy than carcinoembryonic antigen in discriminating early GC patients from healthy individuals and late stage patients. 111 In addition, Zhao el al demonstrated that the expression level of lncRNA HOTTIP is significantly correlated with invasion depth, tumor-node-metastasis (TNM) stage, and overall survival of GC patients, functioning as an independent prognostic factor. 112 Recent studies have reported that exosomal lncSBF2-AS1 confers chemoresistance to glioblastoma cells, and high levels of lncSBF2-AS1 are associated with poor response to temozolomide treatment in glioblastoma patients. 113 In recent years, circular RNAs (circRNAs) are emerged as novel biomarkers for cancer diagnosis due to their universal existence in eukaryotic cells and tissue-and developmental stage-specific expression patterns. 114 Accumulating evidence indicates that circRNAs are regulators of parental gene transcription and exhibit diverse biological functions in the progression of cancer. 115 Exosomal circ-PDE8A derived from plasma of PDAC patients has been reported to promote tumor invasion by upregulating MET (MET proto-oncogene, receptor tyrosine kinase). The expression level of circ-PDE8A is positively corre-lated with lymphatic invasion, TNM stage and negatively correlated with survival rate, acting as a promising indicator for PDAC diagnosis and prognosis. 116 In addition, Li et al found that circ-IARS secreted by PDAC tumor cells could be transferred to human microvascular vein endothelial (HUVECs) cells and facilitate tumor invasion and metastasis. 117 By screening circRNA expression profiles for GC, Tang and colleagues identified a novel circulating biomarker circ-KIAA1244 in the detection of GC. Clinical data analysis demonstrated that low expression level of circ-KIAA1244 indicated lymphatic metastasis and a shorter overall survival time. 118 A recent study revealed that circNRIP1 delivered by exosomes promoted GC progression via the AKT1/mTOR pathway, functioning as a microRNA-149-5p sponge. 119 Finally, proteins within exosomes also possess clinical potential as diagnostic markers. It has been reported that PD-L1 levels in exosomes are correlated with progression of head and neck SCCs (HNSCCs), and exosomal PD-L1 is involved in inducing immune escape that leads to resistance of immunotherapy. 120 Studies have demonstrated that ZIP4, a membrane-located zinc ion transporter, could be carried by exosomes to tumor cells and promotes pancreatic cancer growth. The expression level of ZIP4 varies in different stages of pancreatic cancer, and high ZIP4 expression is correlated with worse survival rates. 121 Although numerous basic studies have shown strong relationship between exosome-associated proteins and cancer progression, the high heterogeneity of exosomeassociated proteins and the limited isolation methods significantly hinder their clinical practice. In addition, further clinical research are also required to verify their application in the diagnosis of cancer or monitoring during treatment. 122 Although exosomes serving as biomarkers hold great promise in cancer diagnosis and prognosis prediction, most of research is currently in the early stage of exploration and lacks of clinical practice. 103 In addition, given the predominance of non-tumor derived exosomes in human body fluids such as platelet-derived exosomes in blood plasma, the accurate separation, identification, and purification of exosomes are still facing considerable challenges. Further research on exosomal proteomics and novel detection methods will undoubtedly accelerate the clinical application of exosomes in cancer diagnosis. 123

Exosome-based immunotherapy
In recent years, immunotherapy is emerged as critically important foci in the field of cancer research, and exosomes have attracted enormous attention in the development of novel immunotherapeutic modalities. 124 Exosomes are proved to be vital mediators of immune regulation in TME. 125 It has been showed that exosomes play significant roles in transfer of antigen and signals to antigen-presenting cells directly or indirectly, thereby promoting the activation of immunity. 43 What's more, the intrinsic immunomodulatory capability of exosomes endows them with high clinical application serving as cancer therapeutic vaccines that are able to induce adaptive and innate immunosurveillance. 124 An impressive study by Lu et al revealed that exosomes derived from αfetoprotein expressing dendritic cells (DEX AFP ) could trigger potent antigen-specific antitumor immune responses and result in significant tumor growth retardation. Moreover, DEX AFP improved the TME and prolonged the survival rates in three different HCC mouse models with antigenic and pathological heterogeneity. 126 Importantly, the loading of exosomes to DCs can augment the vaccine efficacy, which is even superior to lysate-loaded DCs. 127 In addition, the combination of exosome-based therapeutic cancer vaccines with other therapeutic modalities, such as chemotherapy and photothermal therapy, may enhance the efficacy of immunotherapy and exert stronger antitumor effect. Morishita and colleagues designed an exosome-based tumor antigens-adjuvant co-delivery system that exosomes were modified with CpG DNA (CpG-SAV-exo). CpG-SAV-exo induced tumor antigen-specific immune response more effectively than simple coadministration of exosomes and CpG DNA and significantly inhibited tumor growth. 128

Exosome-based drug delivery
With momentous progress in elucidating the biological and functional properties of exosomes, these natural nano-vehicles with good biocompatibility, stability, and safety are recognized as compelling candidate platforms for drug delivery. 125 Meanwhile, exosomes display exquisite target-homing specificity and can be preferentially transferred to tumor cells with reduced side effects. 122 Exosome-based nanoplatforms have been employed to encapsulate and transfer multiple functional molecules in cancer treatment, such as chemotherapeutic drugs, proteins, and nucleic acids, and yielded encouraging preclinical results. 124 For instance, doxorubicin has been successfully encapsulated in exosomes to ameliorate their biodistribution. 129 Exosomes can also be deployed in delivering natural phytochemical compounds that with poor solubility, such as curcumin, to improve their bioavailability and accelerate their clinical translation. 130 Recently, with the rapid development of gene therapy, exosomes hold great promise in the delivery of nucleic acid macromolecules. Kamerkar et al developed an exosomeŋbased nanoplatform carrying short interfering RNA or short hairpin RNA for specific targeting of oncogenic KRAS. The exosomal formulation displayed enhanced circulating stability due to CD47-mediated protection from phagocytosis. 131

CONCLUSION
The past two decades have witnessed the admirable progress of exosomal science and therapeutics. These natural cell-derived vesicles possessing unique structural characteristics are regarded as key mediators in intercellular communications and play an important role in cancer progression. The thorough understanding of these specific mechanisms by which exosome-based communication regulates the malignant behavior of cancer cells and stromal cells in TME can make substantial contributions to the development of next-generation exosomal therapies.
In addition, as one of the most distinguishing hallmarks of exosomes, the variable cargo contents within exosomes carrying abundant, specific, and sensitive biological information are emerged as valid biomarkers in cancer diagnosis and prognosis. The enticing results of exosome basedtherapeutics and diagnosis in experimental settings have granted them a fast-tracking into clinic.
With the rapid development of nanotechnology, exosomes are heralded as attractive candidates in the establishment of novel nanoplatforms for cancer treatment. Compared with conventional synthetic nanocarriers, these exosomal nanoformulations display fascinating advantages in drug delivery and are being rampantly developed. However, some obstacles still lay ahead for the optimal translation of these promising results into clinic, such as the technical challenges of exosome isolation, purification, and identification; and tremendous efforts are required to overcome the existing limitations to achieve clinical benefit.

C O N F L I C T O F I N T E R E S T
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.