Extracellular Vesicles in Cancer Immune Microenvironment and Cancer Immunotherapy

Abstract Extracellular vesicles (EVs) are secreted by almost all cells. They contain proteins, lipids, and nucleic acids which are delivered from the parent cells to the recipient cells. Thereby, they function as mediators of intercellular communication and molecular transfer. Recent evidences suggest that exosomes, a small subset of EVs, are involved in numerous physiological and pathological processes and play essential roles in remodeling the tumor immune microenvironment even before the occurrence and metastasis of cancer. Exosomes derived from tumor cells and host cells mediate their mutual regulation locally or remotely, thereby determining the responsiveness of cancer therapies. As such, tumor‐derived circulating exosomes are considered as noninvasive biomarkers for early detection and diagnosis of tumor. Exosome‐based therapies are also emerging as cutting‐edge and promising strategies that could be applied to suppress tumor progression or enhance anti‐tumor immunity. Herein, the current understanding of exosomes and their key roles in modulating immune responses, as well as their potential therapeutic applications are outlined. The limitations of current studies are also presented and directions for future research are described.

exosomes, are frequently used as markers to identify exosomes. Tetraspanins, composed of four transmembrane domains, were firstly identified in B cellderived extracellular vesicles. [38] Lipid rafts, such as glycosylphos phatidylinositol anchored pro teins (LBPA) and flotillin, are highly enriched in exosomes. In addition, metabolic enzymes, such as GAPDH, enolase 1, PKM2, and PGK1, and molecules involved in signal trans duction, such as protein kinases, 1433, and G proteins, have been detected (Table 1; most of the listed proteins are also pre sent in MVs). [6,39] Apart from proteins, exosomes also contain RNA, including mRNAs, microRNAs (miRNAs), noncoding RNAs, and mitochondrial DNA [40,41] (Figure 1a).Actually, the mRNA and miRNA are the first classes of nucleic acids iden tified in exosomes. [40,[42][43][44] Interestingly, small fragments of singlestranded DNA and large fragments of genomic, double stranded DNA encompassing all chromosomes were also reported in exosomes. [45,46] However, it is worth noting that a recent study overturned the conclusion that small extracellular vesicles contain DNA in which the active secretion of cyto solic DNA has been shown to occur through an amphisome dependent but exosomeindependent mechanism. [6] This research indicated that dsDNA in the extracellular environment is not associated with exosomes or any other type of small EVs, Fangfang Zhou is a professor at the Institutes of Biology and Medical Science, Soochow University. Prior to joining Soochow University, she was a research assistant professor at Leiden University Medical Center. Her research interests are cellular signal transduction, including the molecular mechanisms of TGF-β, Wnt, NF-κB, and other signal pathways, as well as the effects of disorder in cell signaling in human diseases, especially in the development and progression of tumors. At present, she, together with her team, is devoted to the study of the interaction and molecular mechanisms of cancer and immunity.
but is present intracellularly in CD63positive compartments of a size consistent with MVEs. In this way, it proposes a model of autophagy and MVEdependent, but exosomeindependent, active secretion of dsDNA. The debate on this issue is likely due to the fact that early studies often did not discriminate between MVs and exosomes. So there is much confusion on the presence of DNA in EVs and it will be of utmost impor tance to correctly identify the compartment and secretion mechanisms of EVs. Unexpectedly, exosomes could also con tain intact metabolites, including amino acids, lipids, and TCAcycle intermediates. [47] After exosomes bind to target cells via ligands or adhesion molecules, a proportion remains Adv. Sci. 2019, 6,1901779 Figure 1. Molecular composition, biogenesis, secretion, and uptake of the exosomes. a) Exosomes contain complex contents including proteins, mRNA, miRNA, ncRNA, and DNA. TSG101 and Alix are involved in the formation of internal vesicles of MVBs. The tetraspanins such as CD9, CD63, and CD81, are the markers currently used to characterize exosomes. b) Exosomes originate from ILVs in MVBs. Firstly, proteins are transported from the Golgi or internalized from the cell surface, and nucleic acids should be endocytosed and transferred into the early endosomes. Then early endosomes maturate into late endosomes/MVB, which follow either the secretory or the degradative pathway. Microvesicles are released after formation by budding from the cytomembrane. Once released, exosomes can interact with recipient cells by direct signaling through ligand/receptor molecules on their respective surfaces. Exosomes can also be taken up by recipient cells via different manners such as direct fusion of their membrane, endocytosis, macropinocytosis, and even phagocytosis (right). Thus, exosomes function as a mode of intercellular communication and molecular transfer.

Biogenesis, Secretion, and the Uptake of Exosomes
Exosomes are generated by inward budding of the endosomal membrane, resulting in the accumulation of intraluminal vesi cles (ILVs) within large multivesicular bodies (MVBs). [20,39,55,56] In contrast, microvesicles are directly generated through the out ward shedding or budding vesicles of the plasma membrane. [57] Therefore, exosomes are basically derived from the endocytic pathway of donor cells: the transmembrane proteins such as internalized receptors or proteins that are transported from the Golgi, such as MHC cl ass II molecules, should be endocytosed first then transferred into the early endosomes, which maturate and differentiate into late endosomes/MVBs. Once the MVBs fuse with the plasma membrane, exosome release into the extracellular environment by exocytosis [20,[58][59][60][61] (Figure 1b). The endosomal sorting complex required for transport (ESCRT) is the most wellestablished driver of early endosomes maturation and MVB formation. [62][63][64][65] The ESCRT machinery consists of the ESRT0, I, II and III complexes and sorts ubiquitinated intracellular cargos, which are destined for lysosomal degrada tion, into MVBs. [66][67][68][69] It has been demonstrated that sorting cargo into multivesicular endosomes (MVEs) did not depend on the function of the ESCRT machinery, but required the sphingolipid ceramide. As a result, the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. [70] In addition to this, the mechanisms of exosome secretion have been extensively studied and the Rasrelated proteins in brain (Rab) family, including Rab11, Rab27A, and Rab27B, are well accepted as key regulators in exosome secretory pathway. [71][72][73][74] Rab27A has been shown to be involved in the fusion of the MVB to the plasma membrane and the size of MVEs was strongly increased upon Rab27a depletion, [75] whereas MVEs were redistributed towards the perinuclear region by knocking down of Rab27b. [75] It was recently discovered that deletion of Rab27A led to loss of exosomal PDL1 thus blocked tumor growth through stimulating antitumor immunity. [76] Moreover, it has been suggested that lysosomal function can regulate exosome biogenesis by altering the fate of MVBs. [77] The reduction of NAD + dependent deacetylase Sirtuin 1 (SIRT1) expression was shown to decrease the protein level of the lys osomal vacuolartype H+ ATPase proton pump (ATP6V1A), resulting in a reduction of MVBs targeted for lysosomal deg radation and the enlargement of MVBs fused with the plasma membrane to release exosomes. [78] And the pseudokinase mixed lineage kinase domainlike (MLKL), which triggers necroptosis upon its phosphorylation by the protein kinase RIPK3, has been show to contributes to endosomal trafficking and generation of EVs. [79] This study also shows that the release of EVs con taining RIPK3phosporylated MLKL antagonizes its necroptotic function, serving as a mechanism of selfrestraint. [79] During exosome secretion, pyruvate kinase type M2 (PKM2) has been reported to promote exosome release from tumor cells by phos phorylating synaptosomeassociated protein 23 (SNAP23), which enables the formation of the soluble Nethylmaleimide sensitive fusion factor attachment protein receptor (SNARE) complex to allow exosome release. [80][81][82] Exosome secretion can also be modulated by cell interactions. In the process of rapid and efficient antigen presentation and immune activa tion, peptideMHC class II complexes (pMHCII) on the B cells associates with T cell antigen receptor (TCR) on antigenspecific T cells, which in turn allow the CD4 T cells to recognize and activate B cells, meanwhile stimulating pMHCII to escape intra cellular degradation and increase the secretion of pMHCII into B cell exosomes, leading to constant stimulation of T cells. [83,84] Exosomes that released into TME and body fluids could be taken up by recipient cells. Therefore, various biomolecules derived from exosomes can functionally regulate multiple cel lular processes in their target cells. [40] Exosomes can interact with their recipient cells by direct signaling via the interac tion of ligand and receptor molecules on their respective sur faces. They can also be taken up by recipient cells through direct fusion of their membrane in different manners such as lipid raft, calveolae, and clathrindependent endocytosis, macropinocytosis and phagocytosis. [52][53][54][85][86][87] In many cases, exosomes are fused with membrane and internalized together with phagocytic tracers. Dynamin2 (Dyn2), a key regulator of phagocytosis, is essential for exosome uptake. [54] However, other reports suggest that exosomes are mainly internalized through nonclathrin dependent, lipid raftmediated endocy tosis rather than membrane fusion. [86] The uptake of exosomes is negatively regulated by the lipid raftassociated protein cave olin1 (CAV1) that depends on the ERK1/2HSP27 signaling. [86] The transmission of exosomes in the body is known to have tissue and organspecificity. Different integrins that expressed on TEXs is proved to dictate exosome adhesion to specific cell types and extracellular matrix molecules in particular organs. [88] However, it is still under intense investigation and remains largely unclear what are the components in the exosomes that determine their organspecific location or cell type specificity. Likewise, much less is known about the difference between exosomes and MVs uptake. Collectively, the knowledge of vesicles biogenesis, secretion and uptake is not complete and deserve further exploration.

Exosome Purification and Characterization
In order to provide insights into the physiological function of exosomes, the purification and quantification of exosomes is critical to meet the demands of basic research and clinical applications. There are mainly five groups of exosome isolation techniques: ultracentrifugation (UC), sizedependent methods such as ultrafiltration and size exclusion chromatography (SEC), densitybased separation, immuneaffinity capture methods and precipitation ( Table 2). [89][90][91][92] Each technique bases on one particular trait of exosomes, such as their morphology, density, size, or surface proteins. A list of advantages and dis advantages of each exosome isolation technique is summa rized in Table 2. Among them, UC is the most traditional and widely accepted technology. In the UC process, a lowspeed centrifugation step (500 × g for 10 min) is firstly used to dis card floating cells, and a subsequent higherspeed centrifuga tion step (2000 × g for 20 min) is applied to remove the dead cells. Then, a higherspeed centrifugation step (10 000 × g for 30 min) is needed to eliminate larger microvesicles and debris. A final ultracentrifugation (120 000 × g for 70 min, twice) allows collection of the precipitated exosomes. [91,93] For more details, we refer the reader to our recent review (https://doi.org/10.1002/smtd.201800021). Exosomes are commonly purified from cell culture super natants or blood plasma and identified by physical and mor phological characteristics. [8,94,95] Typically, western blot, flow cytometry (FACS), and mass spectra analysis identify complex proteins in exosomes from different sources. [96] Moreover, exosomes can be characterized by NTA, [97] resistive pulse sensing (RPS), FACS, and EM. Comparison of these characteri zation technologies, along with their advantages and disadvan tages, are shown ( Table 3).

Functions of TEXs in Immune Environment
In the TME, immune cells including T cells, B cells, macro phages and dendritic cells frequently infiltrate the tumor tissue and interact with tumor and stroma cells. Via secreting TEXs, tumor cells could deliver immunestimulatory or immune suppressive signaling molecules therefore regulate the develop ment, maturation, and antitumor capacity of targeted immune cells [3,26,98,99] (Figure 2).
TEX can carry multiple tumor antigens, which are efficiently taken up and crosspresented by MHCI molecules on dendritic cells in a human in vitro model system. [100] It is likely that TEXs may contain specific receptors or ligands for efficient uptake by antigen presenting cells (APCs). However, the in vivo relevance of TEXs needs to be validated. These tumor antigenloaded DCs can increase the tumor antigenspecific CD8 + cytotoxic Tlym phocytes (CTLs), thus enhancing immune responses. [100,101] Of notable interest, the direct activation of T cells by cancer exosomes has not been reported, CD8 + cytotoxic Tcell stimu latory function of cancer exosomes requires uptake and pro cessing tumor antigens by DCs. [100,102,103] In addition, TEXs that also bear HSP70, as well as other specific tumor antigens, promote the migratory and cyto lytic activity of NK cells and TNFα production by macro phages [104,105] (Figure 2a). Bcl2associated athanogene 4 (Bag4), as an antiapoptotic protein, was found to interact with HSP70 not only in the cytosol but also on the plasma mem brane. [106] HSP70/Bag4positive exosomes from pancreas and colon carcinoma stimulate migration and cytolytic activity in NK cells which could be completely abrogated by HSP70spe cific antibody. [104] Subsequent research indicated that HSP70 was released into the extracellular environment in a mem brane associated form which more effectively induce TNFα Adv. Sci. 2019, 6,1901779   production as an indicator of macrophage activation, as com pared with free recombinant protein. [105] However, for the most part, TEXs have been shown to pro mote immunosuppressive and protumorigenic effects [107][108][109][110][111] (Figure 2b). In fact, in the TME, TEXs carrying native tumor antigens may not "efficiently" transfer these antigens to DCs for processing and crosspresentation. More recent evidences support the conclusion that TEXs could assist cancer cells and reflect the aims and functions of the parent cancer cell: that is, to survive, grow and metastasize. For example, TEXs of melanomas were demonstrated to reprogram bone marrow progenitor cells toward a provasculogenic phenotype in the premetastatic niche. [99] TEXs derived from prostate cancer cells express fas ligand (Fasl also known as CD95L), a T cell killing  molecule that induces the apoptosis of T cells, thus act as sys temic antigen presenting death signals of CD8 + T cells. [112,113] Similarly, TEXs bearing TRAIL also induce apoptosis of acti vated antitumor T cells. [114] As a critical cytokine that mediates suppression of CD8 + T cells and the proliferation of Foxp3 + reg ulatory T cells, [115] Transforming growth factorβ (TGFβ) was shown to be transmitted via breast cancer exosomes. [116] Fur thermore, TEXs expressing ligands for NKG2D could reduce the cytotoxicity of natural killer (NK) cell and CD8 T cells by down regulating their surface NKG2D expression. [117,118] In addition, TEXs can also inhibit dendritic cell maturation by repressing the differentiation of myeloid precursors into DCs and the gen eration of myeloidderived suppressor cells (MDSCs). [110,111] A similar study also showed that HSP72 bearing TEXs activate STAT3 in MDSCs in a TLR2/MyD88dependent manner and thus mediate T cell-dependent immunosuppressive functions of MDSCs. [119] Likewise, exosomes derived from hypoxic epi thelial ovarian cancer cells that enriched with miRNAs, such as miR213p, miR125b5p, and miR181d5p, potently induce the polarization of M2 macrophages with a protumor phenotype. [120] And the exosomes derived from colon cancer cell was shown to be enriched with miR1246 that can reprogram neighboring macrophages into a tumor supportive and antiinflammatory state. [121] As discussed above, TEXs can cause immune suppres sion by promoting the differentiation of inhibitory immune cells, including Treg, MDSCs, and M2like TAMs.

Mechanisms of TEXs in Modulating Innate and Adaptive Immunity
Functions of exosomes are determined by their specific con tent, in other words, depending on the cargos that are specifi cally delivered. It is possible that different TEX subtypes which containing specific context under certain physiological condi tions mediate immunostimulatory or immuninhibitory activity.
Although the specific mechanisms by which tumor exosomes regulate host immunity are complicated and largely unknown, we summarized and discussed several recent important research findings to explain the enormous hetero geneity of immunomodulatory mechanisms in this section (Figure 3). Programmed deathligand 1 (PDL1), a membrane bound ligand on many cancer cells, can bind programmed death1 (PD1) receptor on T cells to suppresses antigen derived activation of T cells and elicit the immune checkpoint response. [122,123] It has been found that PDL1 is also located on the surface of TEXs from plasma samples of patients with a variety of cancers. [124] Two recent studies found exosomal PDL1 could play critical immunosuppressive roles in mela noma and prostate cancer [76,125] (Figure 3a). The circulating exosomal PDL1 suppresses the function of CD8 + T cells thus facilitate tumor growth. Remarkably, in patients with metastatic melanoma during antiPD1 therapy, the responders dis played a larger increase of exosomal PDL1 in comparison to the Adv. Sci. 2019, 6,1901779 Figure 3. Mechanisms of TEXs in modulating innate and adaptive immunity. a) Tumor cell-derived exosomal PD-L1 can be transferred to CD8 + T cells, leading to the immunosuppression and immune escape in melanoma and prostate cancer. b) LATS1/2 deficient tumor cells secrete nucleic-acid-rich extracellular vesicles, which induces anti-tumor immune responses via type I interferon. c) Tumor cell-derived exosomal EGFR can be transferred into host macrophages to reduce their production of type I interferon and inhibit antiviral immunity. d) Primary tumor-derived exosomal small nuclear RNAs can be transferred to the lung epithelial cells, leading to the activation of TLR3, production of chemokine, and recruitment of neutrophils. Thus, tumor-derived exosomal small nuclear RNAs can elicit a pro-metastatic inflammatory microenvironment by suppressing innate and adaptive anti-tumor immunity.
nonresponders. Therefore, the circulating exosomal PDL1 could be considered as a biomarker for the clinical outcomes of antiPD1 therapy. [125] This was confirmed by another inde pendent study which showed that exosomal PDL1 can sup presses T cell function and promotes tumor progression by inducing a systemic immunosuppression. Suppression of Exosomal PDL1 by depletion of Rab27A and nSMase2, two important exosomal biogenesis genes, induces systemic anti tumor immunity and memory. [76] It is worth noting that in addition to PDL1, tumor exosomes should contain other pro teins or RNAs that also exert immunosuppressive functions, awaiting further investigations. Moreover, by delivering dif ferent signals, TEXs can broadly affect the proliferation, apop tosis, cytokine production, and reprogramming of T cells. [126] TEXs are able to induce apoptosis of activated CD8 + effecter T cells by activating the Fas/Fas ligand pathway and promote the expansion of Treg cells, thus contributing to immune sup pression and tumor escape. [127] Both the large tumor suppressor 1 (LATS1) and LATS2 are key kinases in Hippo pathway that controls organ develop ment and mainly play tumorsuppressive roles by targeting YAP/TAZ for proteasomal degradation. [128][129][130] Unexpect edly, loss of LATS1/2 inhibits tumor growth and metastasis by enhancing immunogenicity of tumor cells and antitumor immune responses. The nucleicacidrich extracellular vesi cles (50-200 nm in diameter) secreted from LATS1/2 deficient tumor cells stimulate the host TLRMYD88/TRIF nucleicacid sensing pathways and promote the production of type I inter feron, thus inducing adaptive immune responses. [131] Further investigations are essential to explore the signaling mecha nisms involved in unidentified proteins or nucleic acids in extracellular vesicles (Figure 3b). Several proteomic studies also identified Hippo pathway to be associated with endocytosis and vesicle trafficking, [132][133][134] implying that Hippo pathway may regulate extracellular vesicles biogenesis.
Exosomes from normal human urinary are enriched for innate immune proteins thus function as immune effectors that contribute to host defense within the urinary tract. [135] However, based on our research, exosomes derived from lung cancer cells mainly antagonize the host innate immunity com pared with exosomes derived from normal lung fibroblasts. [136] Our results shown that tumor exosome from lung cancer cells were able to transfer activated epidermal growth factor receptor (EGFR) to the host macrophages, in which the exosomal EGFR engaged with the macrophageintrinsic signaling pathway that reduced their production of type I interferons (IFNs) and anti viral immunity. In macrophages, the Serine/Threonine kinase MEKK2 serves as effecter that could be activated by TEXdeliv ered EGFR. The stimulated MEKK2 directly phosphorylated Ser173 on IRF3, a transcription factor crucial for IFNβ induc tion. This triggered K33linked polyubiquitination of IRF3 on its nuclearlocalization sequence (NLS) thus blocked its dimeri zation, nuclear translocation, and transcriptional activity. [136] This study explained the reason why tumors can interfere with the innate antiviral system via exosomes and identify a mecha nism by which cancer cells can dampen host innate immunity (Figure 3c). In addition to this, TEXs contribute to metastasis through educating the premetastatic niche. The small nuclear RNAs enriched in the TEXs, via activating Tolllike receptor 3 (TLR3) in lung epithelial cells, could induce production of chemokines such as CXCL1, CXCL2, CXCL5, and CXCL12, therefore promote recruitment of neutrophil. [137] Once recruited in the niche, neutrophils can switch to have tumor promoting roles thus drive metastasis [138,139] (Figure 3d). In line with this study, neutrophils were shown to elicit a prometastatic inflam matory microenvironment by suppressing both innate and adaptive antitumor immunity. [139][140][141] We speculate that the TEXs derived from different cancer cells or the same cancer type but under different pathological conditions might be selective for recipient cells and function in specific molecular pathways. However, who or what determines which immune cell could be targeted by TEXs is an unresolved issue. Future studies are needed to elucidate the dual role of TEXs in cancerimmune progression. Based on our prelimi nary observations, the components in TEXs are dynamically altered and closely related to the degree of malignancy of donor tumor cells. Thus it is likely that in the early stage of tumor, TEXs barely contain immunesuppressive molecules but carry relevant tumor antigens to initiate immune responses via DCs. While in the late stage of tumor progression, malignant tumor cells could suppress the host's innate and adaptive immunity by releasing exosomes carrying abundant immunosuppressive factors such as PDL1 and EGFR.

Stroma Cells in the TME Support Tumor Progression via Secreting Exosomes
The development of cancer metastases at distant organs requires disseminated tumor cells' adaptation to, and coevo lution with the different microenvironments of the metastatic sites. Stromal cells in the tumor microenvironment regulate cancer progression, therapy resistance, inflammatory responses via interaction with cancer cells. [142] Exosomes derived from stromal fibroblasts contains unshielded RN7SL1 RNA, which is 5′triphosphorylated. Upon transfer to breast cancer cells, unshielded RN7SL1 RNA activates the viral RNA pattern rec ognition receptor (PRR) RIGI, resulting in STAT1 activation and ISG induction. Then STAT1 amplifies the NOTCH3 tran scriptional response, leading to tumor growth, metastasis, and therapy resistance. Upon transfer to immune cells, unshielded RN7SL1 drives an inflammatory response by increasing the percentage of myeloid DC populations which express matura tion and activation markers [143] (Figure 4a). Advanced ovarian cancer frequently spreads to the visceral adipose tissue of the omentum. Exosomes derived from cancerassociated adi pocytes (CAAs) and fibroblasts (CAFs) contain high level of microRNA21 (miR21). By regulating apoptosis proteaseacti vating factor1 (APAF1) and MMP1 expression in the target ovarian cancer cells, exosomal miR21 derived from neigh boring stromal cells can confer chemoresistance and an aggres sive phenotype (Figure 4b). Except for nucleic acid, exosomes from CAFs also contain intact metabolites, including amino acids, lipids, and TCAcycle intermediates, which could be uti lized by cancer cells for central carbon metabolism and tumor growth under nutrient deprivation or nutrient stressed condi tions. [47] Regardless of the types of tumor, brain metastasis has a particularly poor prognosis with high morbidity and mortality.
Patients with brain tumor barely manage for survive more than a year and few effective treatment are currently available. Of note, tumor cells disseminate to the brain often show loss of PTEN, but not to other organs. Exploring results revealed that brain astrocytederived exosomes mediate an intercel lular transfer of PTENtargeting microRNAs to the metastatic tumor cells. Furthermore, such adaptive PTEN loss in brain metastatic tumor cells induces an increased secretion of the chemokine CCL2, facilitating the recruitment of IBA1 + /CCR2 + myeloid cells at the micrometastasis site and enhancing the outgrowth of brain metastatic tumor cells [29] (Figure 4c). It is well recognized that patients who develop resistance to drug have limited therapeutic options in the clinic. At present, suni tinib resistance appears to be the major challenge for advanced renal cell carcinoma (RCC). Recent studies have described the role of exosomes in the dissemination of drug resistance. Drug resistance is a major challenge for advanced RCC. It has been reported that highly abundant lncARSR (lncRNA activated in RCC with sunitinib resistance) is present in sunitinibresistant RCC cellderived exosomes. The exosomal lncARSR can be transferred to sensitive cells and facilitate AXL and cMET expression in RCC cells by competitively binding miR34/miR 449, thereby disseminating sunitinib resistance [144] (Figure 4d).
These results show that stromal or cancerassociated normal cells reprogrammed in the TME utilize exosomal miRNAs or lncRNAs to induce resistance of tumor cells to drugs or chemotherapy. Based on this research and the fact that tumor exosomes are rich in RNA, we can be sure that there must be similar tumor resistance mechanisms transmitted by exosomal RNA, which needs to be resolved in the future. These identi fied exosomal RNAs are also therapeutic targets to overcome drug or chemotherapy resistance, enhancing the clinical ben efits in patients. Thus, inhibition of specific RNA to block com pensatory signaling pathways could resensitize resistant cancer cells to drug or chemotherapy and it is necessary to identify novel targets for resistance prevention and therapy. In addi tion, preventing the exosomal transfer of miRNA is another new strategy for conferring EVinduced resistance to drugs or therapies. In summary, elucidating the molecular mechanism of EVinduced resistance could contribute to the development of rationally designed combination cancer therapies.

Exosomes from Distinct Immune Cells Play Divergent Roles in Cancer Immunity
Exosomes derived from multiple types of immune cells broadly modulate antigen presentation and T cells function by playing immunestimulatory or immunesuppressive roles, leading to highly efficient antitumor immunity or tumor immune tol erance [30,[145][146][147] (Figure 5). It was first reported in 1996 that EpsteinBarr virustransformed B cells secreted vesicles car rying MHC class II that could be presented to antigenspe cific T cells, inducing antigenspecific MHC IIrestricted CD4 T cell responses [12] (Figure 5a). In line with this, dendritic cell derived exosomes also contain MHC I/II and other tumor anti gens that stimulate antitumor immune response. [148] Besides, exosomes from IL10treated DCs suppress inflammatory and Adv. Sci. 2019, 6,1901779  autoimmune responses. [149] Exosomes derived from TGFβ1 genemodified bone marrow DCs have immunosuppressive function in inflammatory bowel disease by inducing regula tory T cells and decreasing the proportion of Th17 in lympho cytes. [150] As outlined above, exosomes from APCs, such as B cells and DCs, contain specific peptides and antigens involved in activating antigenspecific T cells. [151,152] However, free exosomes in vitro are not able to stimulate naive T cells, sug gesting this process requires TCR crosslinking and T cell costimulation. [153,154] Indeed, it increases the T cell stimula tory capacity after the interaction of exosomes with dendritic cells. [152,153] Moreover, DCderived exosomes also accumu late proteins such as CD80, CD86, and intercellular adhesion molecule 1 (ICAM1, also called CD45) which are involved in T cell costimulation [48,[155][156][157] (Figure 5a). Except for APCs, macrophagesecreted exosomes could transfer their surface antigens to DCs in a ceramidedependent manner thereby promoting the activation of CD4 + T cells [31] (Figure 5a). These findings revealed that exosomes function to mediate collabora tion between macrophages and DCs for antigen presentation. In contrast, regulatory T (Treg) cells, a subset of CD4 + T cells, can suppress other T cells through cellcontact dependent manner (cytolysis and inhibitory receptor engagement) or cellcontact independent manner (IL2 consumption and sup pressive cytokine secretions, such as TGFβ and IL10). [158] It is conceivable that exosomes derived from Treg cells also play immunosuppressive roles, similar with their donor cells. [159] Exosomes from Treg cells contain CD25, CTLA4, and CD73.
Adv. Sci. 2019, 6, 1901779 Figure 5. Exosomes from distinct immune cells play divergent roles in regulating cancer immunity. a) B cell-derived exosomes bearing MHC II activate CD4 + T cells. DC-derived exosomes containing tumor-derived antigens, costimulatory molecules, and proteins, can promote the activation of CD4 + T cells and CD8 + T cells. Macrophage-derived exosomes bearing MHC I can be transferred to DCs, thereby enabling them to activate antigen-specific CD4 + T cells. b) Treg-derived exosomes containing CD73 can inhibit T cell activation. CD8 + T cell-derived exosomes carrying MHC I also mediate immune suppression by inhibiting the antigen presentation of DCs.
CD73positive Treg exosomes could convert extracellular deno sine5monophosphate to adenosine. Once adenosine binds to its receptors on activated effector T cells, it leads to suppres sion of cytokine production and Tcell responses. [159] In addi tion to proteins, specific miRNA also contributes to the Treg exosome mediated suppression. For example, microRNA Let7d could be preferentially packaged into Treg exosomes and trans ferred to T helper 1 (Th1) cells, resulting in suppressed Th1 cell proliferation and IFNγ secretion. [160] Th1 cells, a subtype of Naïve CD4 T cells, are capable of producing IFNγ which plays a central role in antitumor immunity. [161] Furthermore, whether Treg cells package different RNA species or proteins in exosomes and deliver them to different Th cells such as T helper 2 (Th2) is yet to be clarified (Figure 5b). During cog nate T cellDC interactions, several proteins, including MHC and costimulatory molecules, are transferred from DCs to CD4 T cells, downregulating immune responses. One study has showed that exosomes derived from CD8 + T cells can be endocytosed by APCs through MHCI/TCR interactions and this inhibit DCs mediated antigenspecific CD8 + CTL responses (Figure 5b). [162] Overall, the current research on immune cell exosomes lags far behind the direction of tumor cell exosomes. To explore the composition, characteristics and functional pro teins of different immune cell exosomes is of great significance for understanding the functions of immune cells, particularly may explain how immune cells can "communicate" over long distances.

Exosomes in Cancer Immunotherapy
Compared with other nanocarriers, exosomes have high sta bility in circulation and intrinsic ability of horizontal cargo transfer and are less toxic and immunogenic. Due to the pres ence of CD47, a widely expressed integrinassociated trans membrane protein on exosomes, they can effectively avoid phagocytosis by the circulating monocytes, thus promoting the delivery of their cargos. CD47 interacts with its ligand signal regulatory protein α (SIRPα, also known as CD172a) on macrophages, which induces a "don't eat me" signal that inhibits phagocytosis. [163] On the other hand, unlike liposomes, exosomes contain the plasma membranelike phospholipids and membraneanchored proteins, which could contribute to their diminished clearance from the circulation. Due to their capacity to cross the bloodbrain barrier, exosomes are also employed to be a novel strategy in the treatment of brain tumor. [164,165] Therefore, the idea of using exosomes as a vehicle in clinical practice is promising and inspiring. [166,167] The modi fied exosomes are designed for clinical applications through artificially optimizing the integration of specific loadings such as tumor drugs and tumor targeting RNAi. [168,169] Paclitaxel is extensively applied as antitumor drugs for various tumor types including breast cancer. [170] Limited by the poor aqueous solubility of paclitaxel, it is an urgent need to develop new methods for increasing solubility and improving therapeutic efficacy. Solution was made by using exosomes as drug delivery carriers, in which paclitaxel packaging exosomes were observed to efficiently kill tumor cells. [171] Similarly, exosomes loaded with chemotherapeutic drugs such as curcumin, methotrexate and cisplatin have promising antitumor effects in treatment of a variety of can cers (Figure 6a). [172][173][174] In addition to delivering drugs, using exosomes as a siRNA delivery vehicle to silence oncogenes in tumor cells has been explored recently. [168] The oncogenic acti vation of GTPase KRAS are commonly occurred in pancreatic cancer, [175,176] but the nucleic acids targeting KRAS have low stability and uncontrollable release in the blood circulation thus it remains a challenge to generate an effective therapy by targeting of KRAS. A reported, exosomes derived from normal human foreskin fibroblast can function as efficient carriers of KRAS siRNA, which significantly suppresses pancreatic tumors progression and enhances overall survival in mouse models [177] (Figure 6a).
Owing to the lipid bilayer membrane, engineered exosomes can express transmembrane proteins on their surface and thus actively participate in tumor immunotherapy. By engaging SIRPα, CD47 limits the ability of macrophages to engulf tumor cells, which acts as a major phagocytic barrier. [163] Based on this finding, exosomes designed to harbor SIRPα variants could function as immune checkpoint blockade that antagonizes the interaction between CD47 and SIRPα thus induce augmented tumor phagocytosis, leading to prime effective antitumor T cell response [163] (Figure 6a). In recent years, checkpoint blockade antibodies against PD1 or PDL1 have shown unprecedented clinical responses. [178] Similar to the PD1/PDL1 blocking antibodies, cell membrane derived nanovesicles, as a bioen gineering strategy, presenting PD1 receptors on their mem branes could enhance antitumor responses through disrupting the PD1/PDL1 immune inhibitory axis [179] (Figure 6a). More over, exosomebased tumor antigenadjuvant codelivery system for cancer immunotherapy is another engineering strategy to improve immunogenicity. For instance, engineered melanoma tumor cellderived exosomes containing endogenous tumor antigens (gp100 and TRP2) and immunostimulatory CpG DNA could induce potent antitumor effects. [180] Similarly, engineered myeloma cellderived exosomes expressing endogenous P1A tumor antigen and HSP70 are capable of stimulating dendritic cell maturation and Tcell immune responses [181] (Figure 6a).
Additionally, using immune cellderived exosomes to enhance antitumor immunity is another research hotspot. Multiple studies showed that exosomes from dendritic cells loaded with tumor antigen are able to activate the cytotox icity of tumor antigenspecific CD8 + T cells and enhance antitumor responses in animal models and human clinical trials. [101,148,182,183] Exosome from M1, but not M2 macrophages, enhances activity of lipid calcium phosphate (LCP) nanopar ticleencapsulated Trp2 vaccine thus causes a stronger antigen specific cytotoxic T cell response, suggesting they could be used as a vaccine adjuvant. [184] Exosomes from NK cells exert cyto toxic activity against different human tumor cells. [146] The extra cellular vesicles from activated CD8 + cells also prevent tumor progression by depleting the mesenchymal tumor stromal cells [185] (Figure 6a). Not surprisingly, exosomes from the above mentioned immune cells should be employed in cancer immu notherapy in the near future.
Currently, liquid biopsy has emerged as a noninvasive and convenient approach for cancer diagnosis and prognostic monitoring. As mentioned above, due to the high stability and sufficient concentration in the circulation, exosomes have advantage in liquid biopsy compared with other sources such as circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). [186] In addition, exosomes contain a variety of con tents, such as protein and miRNAs, which can be implicated as biomarkers for diseases. [187,188] For example, GPC1 + circu lating exosomes may serve as a potential noninvasive diag nostic biomarker to detect early stages of pancreas cancer. [189] Overwhelming studies have pointed out that exosomes contain high levels of miRNAs that contribute to immunoregulation, chemoresistance, and cancer metastasis in multiple tumor types. [99,190,191] Numerous studies show that the exosomal RNAs can be used as diagnostic biomarkers as well. The miRNA sig natures of TEXs show considerable promise as potential circu lating diagnostic biomarkers in many types of cancer such as glioblastoma ovarian cancer and prostate cancer, [188,192,193] as well as inflammatory liver diseases. [194,195] In addition to miRNAs, circular RNAs (circRNAs) were more abundant in exosomes derived from cancer cells and patients serum, which may serve as a new class of exosomebased cancer biomarkers. [196,197] Interestingly, we noticed that exosomes isolated from preg nant women plasma and serum contain both maternal and Adv. Sci. 2019, 6, 1901779 Figure 6. Exosomes in cancer immunotherapy. a) As exosomes have high stability in circulation and good capacity to transfer horizontal cargo, they have been explored as delivery carriers loaded with drugs or tumor targeted RNAi in different diseases. In addition, exosomes can be employed as immune modulators by expressing proteins such as SIRPα, PD1, or tumor antigen peptides. The exosomes derived from immune cells including DCs, macrophages and CD8 + T cells are demonstrated to stimulate anti-tumor immune responses. Importantly, large-scale generation of good manufacturing practice-grade (GMP-grade) and clinical-grade exosomes are generated for clinical applications. b) Exosomes bearing GPC1, PD-L1, or certain miRNA could be valuable as cancer biomarkers. c) Inhibition of exosomes biogenesis, release, and uptake is another strategy of cancer immunotherapy.
fetal origin DNA, [198] which proposes a possible application of exosomes for clinical utility in prenatal screening and diagnosis (Figure 6b).
TEXs prefer to promote tumor progression, thus blocking the biogenesis and release of TEXs seems to be a potential anti tumor strategy. GW4869, an inhibitor of nSMase2, has been discovered to block the ceramide synthesis. [70] Treating tumor bearing mice with GW4869 reduces the lung metastasis and its combination with cisplatin and gefitinib increased the anti tumor effects. [199] As the most frequently used genetic targets to downregulate exosomes production, Rab27a knockdown could inhibit exosomes secretion thus lead to a reduction of tumor growth. [200,201] TSG101, a protein involved on endosomes traf ficking, was also thought to be a potential therapeutic target to interfere with exosomemediated communication in cancer. [202] Furthermore, Cytochalasin D, an inhibitor of different endo cytosis, can inhibit exosomes uptake, which also downregu late the exosomes biogenesis. [203] In summary, antagonizing the synthesis, release and uptake of tumor exosomes benefits cancer therapy (Figure 6c).

The Mechanisms Underlying the MVBs' Sorting Remains Mysterious
There is no doubt that functions of exosomes are determined by their specific content, in other words, depending on the cargos that are specifically delivered. Although many proteins have been identified in exosomes, little is known about how they were chosen and sorted into the exosomes, what particular post translational modification (PTM) is required for or contributes to exosomal accumulation of proteins. Practically, some but not all of the internalized membrane proteins are frequently found to be phagocytosed into endosome and finally secreted into exosomes, which is likely to be the result of a multistep protein trafficking/sorting that continuously occurred during transport of intracellular vesicles. The MVB sorting process plays a crit ical role in facilitating the degradation of membrane proteins within the hydrolytic lumen of the lysosome/vacuole. Although in the past 10 years, the basic framework by which MVB sorting to lysosome has been elucidated, [204] but how MVB sorting could be switched for producing exosomes remain mysterious. The mechanisms that sort MVB to the plasma membrane and the lysosome are largely unclear, but the existence of a decision point between those two fates suggests that inhibition of one pathway will increase the other. [205,206] Cells might compensate for lysosome malfunction by disposal of potentially toxic cargos into exosomes, thus future studies of the molecular mecha nisms underlying the MVBs' trafficking may advance current understanding on how pathogenic proteins, lipids or infectious agents accumulate outside of cells. [206]

Exploration of DNA in EVs
Exosomes contain a small amount of DNA, including single stranded DNA, doublestranded DNA, genomic DNA, mitochondrial DNA, and even reversetranscribed complemen tary DNAs. [45,46,207] Whether the source of DNA in exosome is nucleus, mitochondria, or cytoplasm is still unknown. Unlike other exosomal cargoes, it is not clear whether selective pack aging of specific DNA into exosomes exists. What is the func tion of exosomal DNAs also needs further explanation. For example, TEXs produced by irradiated mouse breast cancer cells (RTTEX) transfer dsDNA to DCs and stimulate STING dependent activation of type I IFN (IFNI), resulting in eliciting tumorspecific CD8 Tcell responses. [208] Similarly, recent study has shown that T cellderived exosomes contain genomic and mitochondrial DNA (mtDNA) which is transmitted from the T cell to the DC to induce antiviral responses. [209] Transfer of exo somal DNA activates the cGAS/STING cytosolic DNAsensing pathway and increases the expression of IRF3dependent inter feron regulated genes in DCs, [209] and transfer of mtDNA acts as an oncogenic signal promoting an exit from dormancy of therapyinduced cancer stemlike cells. [210] Moreover, exosome derived from senescent cells contain chromosomal DNA frag ments discarded as cellular garbage bags from cells. [211] These results indicated that exosome secretion might play crucial roles in maintaining cellular homeostasis by removing harmful cyto plasmic DNA from cells, which prevents ATM/ATRdependent DNA damage response and aberrant innate immune responses.
Although there are many studies on exosomal DNA, the heterogeneity of extracellular vesicles and nanoparticles, as well as differences in purification strategies make the analysis of exosomes confusing. As mentioned above, a recent study breaks the general view that exosomes are the carriers of extra cellular DNA secretion. [6] This work suggests that doublestranded DNA (dsDNA) and DNAbound histones do not exist in exosomes or any other type of sEV. In view of the increasing interest in extracellular DNA as a marker of disease in liquid biopsy, it is necessary to reas sess the actual measurement results. However, compared with traditional exosome isolation methods, the revised exosome iso lation with greater precision used in this study is more costly and less efficient. Therefore, there is a great need for more standardized isolation and purification techniques of exosomes, or even a revision of the current classification and nomencla ture. [212] In summary, the heterogeneity of EVs and the pres ence of nonvesicular extracellular nanoparticles pose major obstacles to our understanding of the composition and func tional properties of different secretory components. A more accurate understanding of the correct extracellular components of RNA, DNA, and proteins and their secretion mechanisms are essential for identifying biomarkers and designing future drug interventions.

The Challenges in Exosome-Based Therapy
Although exosomes have made great achievements in applica tions, challenges still remain. Since exosomes can be utilized as clinical biomarkers, vaccines, or drug delivery devices, more accurate and standardized purification method is urgently needed. Besides, for an achievement of better immunotherapy or vaccination based on exosomes, the antigenloading effi ciency of exosomes must be improved. Another challenge is to generate largescale production of exosomes for clinical applica tion. Although a process for production of good manufacturing practice (GMP)grade exosomes derived from mesenchymal stem/stromal cells has been reported, this technique still requires expansion to other different cell types. [213] Moreover, what are the most suitable cells for producing clinicalgrade exosomes remains to be further investigated. In addition, superlative exosomebased therapy could combine with other antitumor treatments, which will be broad potential. Through the study of exosomes, more widespread therapeutic applications can be proposed.