Exosomes: Diagnostic Biomarkers and ... - ACS Publications

Review Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Exosomes: Diagnostic Biomarkers and Therapeutic Delivery Vehicles for Cancer Liangdi Jiang,†,‡,§ Yongwei Gu,†,§ Yue Du,†,‡ and Jiyong Liu*,† †

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Department of Pharmacy, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China ‡ College of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, Shandong 250355, China ABSTRACT: Exosomes are described as nanoscale extracellular vesicles (EVs) secreted by multiple cell types and extensively distributed in various biological fluids. They contain multifarious bioactive molecules and transfer them to adjoining or distal cells through systemic circulation, participating in intracellular and intercellular communication, and modulating host−tumor cell interactions. Recent research has indicated that exosomes obtained from different biological fluids and their contents (proteins, nucleic acids, glycoconjugates, and lipids) can serve as biomarkers for cancer diagnosis, prognosis, and therapeutic response. Furthermore, the discovery of exosomes as therapeutic delivery vehicles has drawn much attention in antineoplastic drug delivery. They can be utilized for therapeutic delivery of proteins, genetic drugs, and chemotherapeutic drugs. Herein, this review summarizes the biogenesis, structure, and components of exosomes, focusing primarily on their two possible applications as diagnostic biomarkers and therapeutic delivery vehicles for cancers. KEYWORDS: exosomes, cancer, biomarkers, diagnosis, delivery vehicles, therapy

1. INTRODUCTION Exosomes, the smallest extracellular vesicles (EVs) with a diameter of 30−150 nm and an endosome origin, can be actively secreted by extensive cell types such as erythrocyte, dendritic cells (DC), epithelial and endothelial cells, oligodendroglial cells, neural cells, mesenchymal stem cells (MSCs), and tumor cells.1,2 Exosomes are distributed in most physiological fluids, including blood, synovial fluid, urine, pleural fluid, saliva, in-blood serum, bronchoalveolar lavage fluid, amniotic fluid, and breast milk.3,4 Enriched with biologically active substances such as cytoplasmic proteins, RNA, lipids, and cellular metabolites, exosomes can act as “cellular postmen” of biological information for intra- and intercellular communication.5 Exosomes can also transfer these biomolecules from parental cells to recipient cells, involved in pathophysiological processes and playing a fundamental role in tumor biology. Recently, there is a great interest in comprehensive functions of exosomes as circulating biomarkers and novel delivery vehicles in life-threatening diseases, especially in cancers. Accumulating evidence has manifested that exosomes enjoy potential value as circulating biomarkers for various type of tumors because of their ubiquitous presence in physiological fluids, extraordinary stability in the circulation, and similarity to original cells contents. It has been reported that tumorderived exosomes (TEXs) play a key role in the oncogenesis and progression of tumor malignancy by establishing or © XXXX American Chemical Society

altering tumor microenvironment (TME), promoting tumor angiogenesis and invasion, inhibiting the host antitumor responses, and mediating immune escape and multidrug resistance.6,7 TEXs carry various endogenous cargos that partially mimic the contents and reflect the pathophysiological status or signaling alterations of the parental cells, which makes them potential biomarkers for cancer early diagnosis. In recent years, several nanoscale drug delivery systems are under intense investigation to improve drug efficacy, reduce toxicity, enhance specificity for given targets, and extend the circulation time of drugs. In particular, natural and synthetic polymers or liposomes have gained much attention.8 Although these polymers and liposomes have good quality as delivery vehicles, they still have some inherent disadvantages, including poor biocompatibility, lower cycle stability, rapid phagocytosis, and increased toxicity.3 Exosomes can be loaded with exogenous payloads in vivo or in vitro, preserve payloads during circulation, be internalized into recipient cells, and release the drugs, making them potential therapeutic drug delivery vehicles.9 As natural delivery vehicles, exosomes provide more attractive features than polymers or liposomes for therapeutic delivery, including but not limited to good Received: Revised: Accepted: Published: A

April 16, 2019 June 24, 2019 June 26, 2019 June 26, 2019 DOI: 10.1021/acs.molpharmaceut.9b00409 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Review

Molecular Pharmaceutics

Figure 1. Biogenesis of exosomes. Early endosomes are formed by the inward invagination of the cell membrane. Then with the participation of the Golgi, they mature into late endosomes and MVBs. This process is dependent on either the ESCRT or ESCRT-independent machinery. Inward invagination of MVBs membrane forms ILVs that have incorporated nucleic acids, cytoplasmic proteins, lipids, and surface ligands from the plasma membrane. Eventually, ILVs contained in MVBs are secreted as exosomes into the extracellular environment under the promotion of transport and docking of RAB27 protein or fusing with lysosomes for degradation.

promoting enzymatic deubiquitination of the cargo proteins.2 ESCRT-III drives scission and separation of vesicles and gets disassembled by VPS4 adenosine triphosphatase (AAA+ATPase).2,3 Several studies have shown that exosomes can still form despite ESCRT inhibitors, suggesting the presence of an ESCRT-independent pathway. Lipids, such as ceramides, exert an initiating function on the process of exosomes inward budding into MVBs to form ILVs.17,18 Both the ESCRTdependent and independent pathway are implicated in controlling the formation and cargos sorting of exosomes. And the secretory quantity of exosomes differs due to the selective secretion of original cells. However, these selective secretion mechanisms and why certain MVBs subsequently fuse directly with lysosomes for degradation of vesicular materials versus fusing with the plasma membrane for release still remain under in depth investigation.19,20 2.2. Exosomes Structure and Components. As shown in Figure 2, the structure of exosomes consists of an aqueous core and lipophilic shell. A cup-shaped morphology caused by collapse during drying is often mistakenly deemed as the typical characteristic of exosomes. However, quickly frozen and vitrified exosomes observed by cryo-electron microscopy actually exhibited an almost perfect rounded shape.5 Exosomes contain multifarious physiologically active substances such as proteins, nucleic acids, glycoconjugates, and lipids. The compositions of exosomes differ depending on the parental cells, pathophysiological conditions, and environmental stimulations. Of the targeting and fusion proteins normally present on the surface of exosomes, the most abundant are integrins and tetraspanins. Exosome-specific tetraspanins, such as CD9, CD63, and CD81, regulate

biocompatibility, relatively high stability, minimal or no inherent toxicity, and an intrinsic ability to target cells.3,10 Herein, we summarized recent advances in the TEXs as new biomarkers for cancer diagnosis and exosomes as delivery vehicles for neoplasms therapy.

2. EXOSOMES: BIOGENESIS, STRUCTURE, COMPONENTS 2.1. Exosomes Biogenesis. As shown in Figure 1, exosomes biogenesis starts with the inward invagination of the endosomal system. The process of exosomes production starts from early endosomes that then mature to late endosomes and multivesicular bodies (MVBs). The membrane of MVBs germinates into internal lumen to produce many intraluminal vesicles (ILVs) during the above process.11 Eventually, the MVBs fuse with the lysosome for degradation, or fuse with the cell membrane and secret the ILVs (exosomes) into the extracellular space in an exocytosis manner.12 Rab GTPases, such as RAB14, RAB22, RAB27, and RAB37, are the coordinators of vesicle traffic and are responsible for regulating the formation of ILVs, the fusion of MVBs with cell membrane, and transportation of vesicles.13 Endosomal sorting complex required for transport (ESCRT) primarily regulates the formation and cargo sorting of exosomes.14 The ESCRT system comprises ESCRT-0, -I, -II, and -III which can recognize ubiquitinated cargos, vacuolar protein sortingassociated protein 4 (VPS4), ALG 2 interacting protein X (ALIX), and tumor susceptibility gene 101 (TSG101). ESCRT-0 participates in cargo clustering and recruitment of ubiquitinated proteins for internalization.15,16 ESCRT-I and -II take charge of initiating the inward budding process and B

DOI: 10.1021/acs.molpharmaceut.9b00409 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Review


Figure 2. Structure and composition of exosomes. The structure of exosomes consists of an aqueous core and a phospholipid bilayer. Exosomes contain a range of molecular cargos. General: Tetraspanins (CD9, CD63, CD81); Integrins (α6β4, αvβ5); ICAM-1 (intercellular adhesion molecule 1); MHC I, II (major histocompatibility complexes I and II); Membrane transport/fusion proteins (Rabs, Flotillins); Nucleic acids (miRNAs, lncRNAs, circRNAs, mRNAs, DNA); Lamp 1/2 (lysosomal-associated membrane glycoprotein 1/2); TSG101; ALIX; Cytoplasmic proteins (Syntenin, enzyme); HSP (heat shock protein); HSC (heat shock cognate protein); Surface receptor; Cholesterol; Ceramide; Sphingomyelin; Phosphatidylserine.

3. POTENTIAL OF EXOSOMES IN TUMOR DIAGNOSIS Early diagnosis and effective therapy are essential for prolonging the survival of cancer patients. However, a notable problem in cancer diagnosis is the lack of biomarkers that are specific, highly stable, and noninvasive or minimally invasive. The carried proteins, nucleic acids, glycoconjugates, and lipids of TEXs in body fluids could reflect the pathophysiological states of their parental cells. Furthermore, these exosomal contents can also serve as promising biomarkers for cancer early diagnosis by general or proprietary detection techniques (Figure 3). 3.1. Exosomal Proteins as Biomarkers. Since cancerrelated proteins are abundant in exosomes and the membranous structure of exosomes protects luminal proteins from degradation by extracellular proteases,34,35 proteins from TEXs are becoming diagnostic and prognostic biomarkers for cancers. Exosomal proteins for cancer diagnosis are summarized in Table 1a. Pancreatic cancer with high invasiveness and metastasis is considered to be the most deadly cancer in the world.36 Surgical resection is still the most effective treatment method. However, most patients had locally advanced or varying degrees of metastasis at the time of diagnosis, leading to unresectable tumors.37 Glypican-1 (GPC-1), an overexpressed and membrane-anchored proteoglycan, was abundantly present on pancreatic cancer cell secretory exosomes. GPC1+ exosomes were specifically and sensitively detected in

intercellular signal transduction. In addition, tetraspanins also modulate various biological functions of adhesion molecules, such as mediating interactions between exosomes and recipient cells.21 Integrins have been shown to be involved in exosomemediated metastasis processes. For example, α6β4 and α6β1 can be transferred horizontally to the lung; αvβ5 can be transferred horizontally to the liver, promoting cell migration and metastasis.22 It is widely shared that the lipids of the exosomal membrane originate from the parental cell plasma membrane. However, exosomes also contain lipids from the Golgi,23,24 which are different from the lipid components of the parental cell membrane. Cholesterol, sphingomyelins (SM), ceramide, lysobis phosphatidic acid, lysophosphatidylcholines, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines (PS), phosphatidylinositols, sphosphatidic acid, and phosphatidylglycerol have been found in the exosomes membrane.24 Exosomes also contain functional nucleic acids, including mRNAs,25 circular RNAs (circRNAs),26 microRNAs (miRNAs),27 Piwi-interacting RNAs (piRNAs),28 tRNAs,28 tRNAderived small RNAs (tsRNAs),29 small noncoding RNAs (sncRNAs),30 rRNAs,30 long noncoding RNAs (lncRNAs),31 mitochondrial DNA (mtDNA), 32 and double-stranded (dsDNA).33 C


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Figure 3. Tumor-derived exosomes as diagnostic biomarkers for cancers. TEXs contain abundant physiologically active molecules such as proteins, nucleic acids, glycoconjugates, and lipids, representing an ideal origin of biomarkers. Furthermore, TEXs cargos show dynamic changes or abnormal expressions during the treatment of cancer patients, which can be applied to real-time monitoring and response prediction. (A) Proteins analysis is performed through MS or Western Blot for qualitative and quantitative profiling of oncoproteins. (B) DNA diagnosis is primarily processed by next-generation sequencing (NGS), making individualized treatment rooted in mutational profiles possible. RNA diagnosis (miRNAs, lncRNAs, circRNAs, and mRNA) in TEXs can be processed via Real-time Polymerase Chain Reaction (RT-PCR). (C) Glycoproteins can be qualitatively and quantitatively analyzed by PolyGPA while exosomal lipid components are typically analyzed by MS.

spectrometry (MS).43 Further study suggested that the expression level of certain miRNAs may be negatively correlated with the expression level of TRIM in GC; that is, the up-regulation of miRNAs corresponds to the downregulation of TRIM. Protein phosphorylation is the most fundamental and universal mechanism that controls a variety of cell physiological functions and is indispensable in cellular signal transduction.44 However, until now, few phosphoproteins have been identified as potential biomarkers to distinguish disease from health, and the specific role of phosphorylated proteins in biological fluids for disease diagnosis remains indefinable. Fortunately, a recent study demonstrated that phosphoproteins in plasma exosomes are very feasible as disease biomarkers and may become one of the alternatives for cancer screening and monitoring technology.45 Furthermore, critical lung cancer (LC) phosphoproteins, extracellular regulated protein kinases (ERK) and epidermal growth factor receptor (EGFR), are expressed in LC-derived exosomes and maintain their initial phosphorylation status.46 After treatment of cancer cells with Tyrosine Kinase Inhibitors (TKIs), alterations in protein phosphorylation could be observed in their secretory exosomes. Therefore, changes in protein phosphorylation of exosomes could monitor the therapeutic response of TKIs to LC.46 Exosome proteomics research has indicated that TEXs play a paramount role in tumorigenesis, maintenance of tumor survival, proliferation, and metastasis.47 However, there are still difficulties in the study of exosomes proteomics, especially the quality requirements of exosomes. As an important

pancreatic cancer patients’ serum. Furthermore, it was observed that the levels of GPC-1+ exosomes were relevant to tumor load and the preoperative and postoperative survival of patients. Melo et al. provided an important basis and exemplified the strength of utilizing exosomal proteins for pancreatic cancer diagnosis. 38 In addition, the serum cytoskeleton-associated protein 4 (CKAP 4) levels were higher in pancreatic ductal adenocarcinoma (PDAC) patients than healthy individuals by ELISA using newly generated antiCKAP 4 monoclonal antibodies (mAbs). CKAP 4-containing exosomes in serum may also represent an alternative biomarker for PDAC.39 Nonsmall cell lung cancer (NSCLC) is the most common type of all lung cancers that are considered to be the most malignant tumors with the highest morbidity and mortality worldwide.40 A study found that exosomal expression levels of alpha-2-HS-glycoprotein (AHSG) and extracellular matrix protein 1 (ECM1) significantly elevated in the serum of NSCLC patients. It was further found that the combination of carcinoembryonic antigen (CEA) with AHSG or ECM1 can achieve more accurate diagnostic value than CEA alone. The trinity of AHSG, ECM1, and CEA had a higher diagnostic capacity than either of them alone and improved the diagnostic potential of NSCLC.41 In many groups, including Hispanic and Asian Americans, gastric cancer (GC) with high incidence and mortality rates is one of the main causes of tumor death and a global public health concern.42 Recently, Fu et al. found that the tripartite motif-containing 3 (TRIM3) protein levels were decreased in exosomes from GC patients’ serum and GC tissues by mass D


E

Serum

Diffuse Large B Cell Lymphoma (DLBCL) CRC PCa

PDAC GC HCC

circ-PDE8A circ-KIAA1244 circ-deubiquitination

(circ-DB) mRNAs

PDAC

GBM Breast Cancer BC Early Gastric Cancer (EGC)

Plasma

NSCLC

Cancer tissues and plasma Plasma Plasma Adipocytes and plasma

Serum Serum Urine Plasma

Serum Serum

Plasma Plasma

Serum

HCC CRPC PDAC

Cell culture supernatants

Plasma

Serum Serum LC cell lines Serum Plasma

Serum Serum

Source of Exosomes

Melanoma

NSCLC GC LC Pancreatic Cancer Colorectal Cancer (CRC) Epithelial Ovarian Carcinoma

Pancreatic Cancer PDAC

Cancer type

circRNAs circ-IARS

miR-6803-5p miR-1246 lncRNAs HOTAIR HOTAIR MALAT1, PCAT-1 and SPRY4-IT1 lncUEGC1

miR-1290 and miR-375 miR-10b, miR-21, miR-30c, miR-181a, miR122 and let7a Adenocarcinoma (AC)- and squamous cell carcinoma (SCC)-specific exosomal miRNAs miR-99a-5p and miR-125b-5p

(b) Nucleic Acids miRNAs let-7a, miR-138, miR-125b, miR-130a, miR449a, miR-196b, miR-199a-3p, miR-1 and miR-205 miR-18a, miR-221, miR-222 and miR-224; miR-101, miR-106b, miR-122 and miR-195

AHSG and ECM 1 TRIM3 Phosphoproteins Zinc transporter 4 (ZIP4) Copine III (CPNE3) FGA, GSN, FGG, and LBP

(a) Proteins GPC-1 CKAP4

Cargos

Table 1. Exosomal Cargos as Biomarkers for Cancers

The level of circ-PDE8A was high in patients with PDAC. The expression of circKIAA1244 in GC plasma exosomes was lower compared to the normal controls. The level of exosomal circ-DB was increased in HCC patients suffering higher body fat ratios.

Plasma exosomal circ-IARS levels were significantly increased in patients with metastatic PDAC.

HOTAIR levels were higher in serum of GBM patients. HOTAIR expression was upregulated in breast cancer patients. The expression of PCAT-1 and MALAT1 was upregulated in NMIBC patients. The expression of lncUEGC1 was significantly up-regulated in EGC patients and GC cells-derived exosomes.

miR-6803-5p level was dramatically elevated in CRC patients. miR-1246 was downregulated in PCa clinical tissues but highly expressed in PCa cells-derived exosomes.

The miR-99a-5p and miR-125b-5p expressions were remarkably upregulated in DLBCL patients.

The miR-18a, miR-221, miR-222, and miR-224 levels were remarkably higher in HCC patients than chronic hepatitis B (CHB) or liver cirrhosis patients; the miR-101, miR-106b, miR-122, and miR-195 levels were lower in HCC patients than CHB patients. Elevated miR-375 and miR-1290 were stringently relevant to shorter OS in CRPC patients. The levels of miR-10b, -21, -30c, and -181a were high in PDAC and were low in CP and normal samples; let-7a and miR-122 were low in PDAC. There were 11 high-level expressed and 13 low-level expressed miRNAs in ACC patients; 6 high-level expressed and 8 lowlevel expressed miRNAs in SCC patients.

The expressions of exosomal miR-1 and miR-205 of A375 melanoma cells were lower while the expressions of other microRNAs were higher.

The FGA and GSN expressions were greatly upregulated; the FGG and LBP expressions were downregulated in patients with cancer.

The abundance of GPC-1 was considerably increased in patients with pancreatic cancer. CKAP4 was highly detected in serum from mice bearing CKAP4-expressing PDAC cells and preoperative patients with CKAP4-positive PDAC. The levels of AHSG and ECM1 remarkably elevated in NSCLC patients. Serum exosomal TRIM3 was significantly decreased in GC patients and its expression was down-regulated in cancer tissues. Aberrant EGFR and ERK protein phosphorylation from LC cell-derived exosomes was detected. ZIP4 was dramatically upregulated in malignant pancreatic cancer cells. The level of plasma exosomal CPNE3 was notably elevated in CRC tissues.

Observation

140 141 142

72

60 61 63 139

52 138

51

50

55 56

54

53

137

41 43 46 135 136

38 39

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Review 86 Urine PCa

The contents of cholesterol and phosphatidylcholine were different between urine- and PCa cell-exosomes. PS and LacCer showed the highest significance.

81 144 82 Plasma Plasma PC-3 cell lines Breast Cancer NSCLC PCa

1,453 unique glycopeptides, including 447 from exosomes, were identified, representing 164 glycoproteins in exosomes. Plasma exosomal MUC1 level of NSCLC patients was 1.5-fold higher than that of healthy individuals. Glycosphingolipids, such as HexCer, LacCer, and SM, were highly enriched in exosomes from PCa cells.

78 79 Plasma Serum Pancreatic Cancer PCC and PGL

The elevated level of exoDNA was stringently associated with progression of pancreatic cancer. PCC and PGL exosomes contained genomic dsDNA that reflects the mutation status of susceptible genes.

76 143 Serum Serum HCC Pancreatic Cancer

The levels of hnRNPH1 were remarkably higher in HCC patients. The serum levels of WASF2 mRNA were highly correlated with pancreatic cancer risk.

research method in the postgene era, proteomics should be combined with genomics and transcriptomics to comprehensively conduct research on exosomes for their applications. 3.2. Exosomal Nucleic Acids as Biomarkers. Exosomes not only contain a variety of functional nucleic acids, but some of them (such as miRNAs, lncRNAs, circRNAs, mRNAs, and DNA) can also serve as promising biomarkers for neoplasms diagnosis (Table 1b). Moreover, the double-layer lipid membrane of exosomes can prevent the degradation of internal nucleic acids. Therefore, exosomal nucleic acids have a good clinical application prospect in cancer diagnosis. 3.2.1. miRNAs. Recently, miRNAs have emerged as a crucial regulatory factor in tumor growth and transformation.48 There are extensive data suggesting that exosomal miRNAs play an essential part in cancer biology and could be potential diagnostic and prognostic biomarkers for cancers.49−53 A study on exosomal miRNAs indicated that they have significant functions in hepatocellular carcinoma (HCC) and might be HCC novel serological biomarkers that are more sensitive than simple miRNAs.54 Furthermore, the elevated levels of exosomal miR-1290 and miR-375 in plasma of castration resistant prostate cancer (CRPC) patients were related to their poor overall survival (OS).55 Exosomal miRNAs are also more sensitive diagnostic biomarkers than carbohydrate antigen (CA). As an example, exosomal miRNAs signatures are more advantageous than CA 19-9 or GPC-1 alone in diagnosticating pancreatic cancer and distinguishing between PDAC and chronic pancreatitis (CP).56 Notwithstanding the shortcomings and limitations of exosomal miRNAs in tumor diagnosis, such as the strong variation in exosomal miRNAs levels,57 new ideas and methods for cancer diagnosis are provided. In the future, more types and different development stages of tumors ought to be embraced to increase the robustness and trustworthiness of exosomal miRNAs assays. 3.2.2. lncRNAs. Recently, it was reported that exosomal lncRNAs could be a potential biomarker for diagnosing and prognosticating cancers. For instance, the level of serum exosomal lncRNA-UCA1 in bladder cancer (BC) patients was significantly elevated compared with healthy individuals. In addition, hypoxic BC cells reconstituted TME by secreting exosomes containing carcinogenic lncRNA-UCA1 and by epithelial-mesenchymal transition (EMT) to drive tumor progression.58 Another study has manifested that exosomal lncRNAs-ZFAS1 was highly up-regulated in GC patients and was stringently associated with lymphatic metastasis and TNM staging.59 It was first reported that changes in serum exosomal HOX transcript antisense intergenic RNA (HOTAIR) level could be detected in glioblastoma multiforme (GBM) patients to monitor therapeutic response and recurrence and as effective biomarkers for the responsiveness of GBM to bromodomain and extraterminal inhibitor and possibly targeted therapy against tumors.60 Likewise, the expression level of exosomal HOTAIR may indicate the survival of breast cancer patients and may be a diagnostic indicator.61 Nonmuscle invasive bladder cancer (NMIBC) with higher recurrence and mortality accounts for 75%−85% of all BC in the world.62 Considering the complexity of the pathophysiological processes of BC, no single lncRNAs but a panel consisting of several lncRNAs can stand as combinatorial biomarkers. Zhan et al.63 established a three-lncRNAs panel to diagnosticate BC by analyzing urinary exosomal lncRNAs. It was found that the up-regulated expressions of PCAT-1 and

hnRNPH1 WASF2 DNA exoDNA dsDNA (c) Glycoconjugates Glycoproteins Mucin 1 (MUC1) HexCer, LacCer, and SM (d) Lipids Cholesterol, Phosphatidylcholin, PS, and LacCer

Cargos

Table 1. continued

Cancer type

Source of Exosomes

Observation

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F


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3.2.5. DNA. Exosomal DNA (exo-DNA) has gradually become a rising star in cancer diagnosis and treatment. Using exo-DNA as a workable way of liquid biopsies can achieve longitudinal monitoring of cancers to obtain predictive and prognostic evaluation related to therapeutic response and stratification.78 Williams et al.33 definitively confirmed that dsDNA was present in tumor-associated exosomes. When comparing serum exo-DNA and tumor tissue DNA in patients with pheochromocytoma (PCC) and paraganglioma (PGL), Wang et al.79 found that dsDNA fragments were present in patients’ exosomes and the exosomal dsDNA in PCC and PGL was in high consistence with the paired oncogenome. The finding definitively demonstrated that exosomal dsDNA could act as a noninvasive genetic marker for diagnosis and preoperative evaluation of PCC and PGL. Exo-DNA has a shorter half-life, which can accurately reflect the tumor state in real time. Besides, exo-DNA detection has potential clinical application value in minimal residual lesions and early warning of recurrence, which contributes to manage cancer better and benefit more patients. 3.3. Glycoconjugates. 3.3.1. Glycoproteins. In current FDA-approved protein tumor markers, glycoproteins account for more than half of the total markers. However, lacking oligosaccharide-specific antibodies and good analytical tools hindered the further development of newly discovered glycoproteins as disease biomarkers. Analysis of the glycoproteome in exosomes can largely eliminate the interference of high abundance plasma or serum components, which allows us to detect glycoproteins in a wide dynamic range and discover low-level glycoproteins at high sensitivity.80 As a novel high throughput method introduced, polymer-based reverse phase glycoprotein array (PolyGPA) can be used to quantify glycoproteins in array format simply and sensitively and then can be applied to identify superior exosomal glycoproteins biomarkers for breast cancer.81 The identification and research of glycoproteins as potential markers are not only helpful to elucidate the mechanism of the occurrence and development of malignant tumors and their biological characteristics, but they are also of important theoretical and practical significance for malignant tumors diagnosis. 3.3.2. Glycolipids. Glycoconjugates also contain glycolipids. Glycolipids from exosomes have also been considered to be potential cancer biomarkers elsewhere. For example, glycosphingolipids such as hexosylceramides (HexCer) and lactosylceramides (LacCer) were abundantly enriched in PCa cells-derived exosomes.82 Glycoconjugates in exosomes constitute a promising strategy to identify novel cancer markers and increase the specificity and sensitivity of existing clinical biomarkers (Table 1c). They may be useful tools for significant advances in the field of personalized medicine. 3.4. Lipids. Lipids play crucial and diverse roles in normal cells and cancer cells. Certain classes of lipids also reflect cellular physiology of cancer cells. Abnormal lipid metabolism is closely related to cancer progression and metastasis.83 Lacking optimal tools for PCa clinical diagnosis, many patients face the problems of under- or overtreatment.84 According to a previous report, 10 urinary phospholipids have been identified as possible diagnostic biomarkers for PCa.85 More importantly, another study has revealed the exosomal lipid composition in PCa patients and healthy individuals by a MS quantitative lipidomic analysis. The research showed that there were several

MALAT1 were related to poor recurrence-free survival (RFS) of NMIBC. Moreover, the overexpressed PCAT-1 can be used to independently prognose the RFS of NMIBC. This research shed light on the considerable practical application value of exosomal lncRNAs in BC diagnosis and prognosis. Emerging evidence also implied that lncRNAs play a paramount role in regulating TME, tumorigenesis, and tumor development.64 However, the pathophysiological role of exosomes and lncRNAs in the TME still requires further investigation. 3.2.3. circRNAs. Recently, circRNAs, well-expressed and stable RNA molecules with important regulatory functions and tissue/developmental-stage-specific expression,65 have attracted great interest in tumor biology.66 Emerging studies have shown that circRNAs could be ideal biomarkers for cancers.67−70 Lately, exosomal circRNAs may also be available indicators for cancer diagnosis. Li et al. originally confirmed that more than 1,000 intact and stable circRNAs are present and widely expressed in human serum exosomes.71 According to a report, circ-IRAS accesses human umbilical vein endothelial cells (HUVECs) through pancreatic cancer cells secretory exosomes. Additionally, when studying the effects of circ-IARS on tumors, Li et al. observed that overexpression of circ-IARS triggered a series of tumor-promoting behaviors, including a significant up-regulation of oncogene such as Ras homologue gene family member A (RhoA), a down-regulation of fibronectin such as Zonula occludens-1 (ZO-1), elevated expression of F-actin and macula adherens, and increased permeability of endothelial monolayer.72 Exosomal circRNAs are easy to be detected due to stable structure, extensive expression, conserved sequence, and abundant content, thus providing additional evidence for conventional diagnostic methods and holding great promise as diagnostic markers for cancer. The inconsistent abundance of circRNAs in cells and their exosomes indicates that the molecular transfer mechanism between them is complicated. However, the regulatory mechanisms of circRNAs production and the biological mechanism of molecular selection remain largely unknown.26 Further studies are needed on the gene regulatory mechanisms and expression patterns of circRNAs other than sponges that adsorb miRNAs. 3.2.4. mRNAs. Although the current research on mRNAs for tumor diagnosis is limited, it is also a direction worth studying. hnRNPH1 is a mRNAs-binding protein and splicing factor that controls splicing events.73 Several research results showed that the level of hnRNPH1 was high in esophageal squamous cell carcinoma (ESCC) and prostate cancer (PCa).74,75 Some findings also indicated that exosomal hnRNPH1 may represent a well-accepted “liquid biopsy” indicator. For instance, Xu et al. observed that the hnRNPH1 levels in HCC patients’ serum were greatly elevated compared with other groups and were correlated with the Child-Pugh classification, lymphatic metastasis, TNM staging, and OS.76 According to the above research, exosomal long RNAs (exoLRs) are stable, trackable, and suitable for routine tumor detection. The combinatorial exoLRs (lncRNAs, circRNAs, and mRNAs) profile could effectively differentiate patients with cancer from healthy individuals and may be considered as molecular markers with high diagnostic efficiency for HCC.77 Therefore, the identification of cancer-specific exoLRs profiles is promising in molecular diagnosis and subtyping for malignancy. G


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Figure 4. Exosomes as drug delivery vehicles for cancers. Exosomes with nanoscale molecular structure, good biocompatibility, and transportation characteristics for drugs can deliver proteins drugs, genetic drugs, and chemical drugs in cancer therapy.

important drugs for treating diseases because of their special pharmacological activities. Protein drugs were shown to be effective in the treatment of malignant tumors. For example, caspase-1 (ICE) can be loaded into AAV serotype 1 vectors to inhibit schwannoma growth.87 Likewise, exosomes can also act as delivery carriers for protein drugs, stabilizing activities and achieving targeted administration to tumor tissues. Survivin is an inhibitor of apoptosis protein that maintains apoptosis resistance. As its dominant-negative mutant, Survivin-T34A was proven to block Survivin, activate caspase, and induce apoptosis. According to a related report, exosomes obtained from the transfected melanoma cells without tetracycline contain Survivin-T34A. When exosome-delivered Survivin-T34A was used alone or in combination with Gemcitabine, apoptosis of multiple pancreatic cancer cell lines was significantly increased compared to Gemcitabine alone.88 An analogous study reported an enzymatic exosome system that harbors a native glycosylphosphatidylinositol (GPI)-anchored PH20 hyaluronidase (membrane protein therapeutics), which can penetrate deeply into cancer focus by degrading hyaluronan in the tumor extracellular matrix rapidly and permanently, increasing the gap between tumor vascular endothelial cells, thereby enhancing the macromolecular permeability of tumor blood vessels and inhibiting tumor growth.89 Exosomes, a novel delivery shuttle for membrane protein therapeutics, were highly advantageous over other nanoparticles, particularly protein nanocage. It was found that the therapeutic effect of engineered exosomes

content differences in cholesterol and phosphatidylcholine between urinary exosome and PCa cell-derived exosomes.86 This pioneering study showed initially that urinary exosomal lipids are hopeful noninvasive biomarkers for PCa (Table 1d). Proteins, nucleic acids, glycoconjugates, and lipids in exosomes can act as diagnostic and prognostic biomarkers for cancer to some extent. However, the specificity and accuracy of these bioactive substances for diagnosis need to be enhanced. Furthermore, the sensitivity and reliability of detection techniques may become disadvantages for their clinical translation. To overcome these potential obstacles, it is necessary to understand more about the mechanism of active substances sorting into exosomes and optimize extraction and detection techniques, which may contribute to the development of biomarkers for auxiliary diagnosis and prognostic and therapeutic evaluation for cancer.

4. POTENTIAL OF EXOSOMES IN THERAPEUTIC DELIVERY Recently, due to the superior characteristics over natural or synthetic polymers and liposomes, exosomes have attracted considerable attention in the scientific community as putative drug delivery vehicles for proteins and genetic and chemotherapeutic drugs (Figure 4, Table 2). 4.1. Delivery of Protein Drugs. Protein drugs mainly include enzymes, peptides, cytokines, cytoskeletal proteins, and transmembrane proteins. They have become a class of H


VEGF-siRNA BCR-ABL-siRNA

Breast Cancer Chronic Myeloid Leukemia (CML) Liver Cancer

I

Breast Cancer

Brain Cancer

Lewis Lung Carcinoma NSCLC Pancreatic Adenocarcinoma LC GBM

GBM Glioma Breast Cancer Ovarian Cancer Melanoma Breast Cancer Breast Cancer and Ovarian Cancer CRC

HCC Breast Cancer Breast Cancer

Doxorubicin or Paclitaxel Curcumin

Bovine milk Embryonic stem cells

Paclitaxel Paclitaxel

A33-positive LIM1215 cells and xenograft tumor model 3LL-M27 cell lines 3LL-M27 cells CFPAC-1 cell lines

GSC cell lines 9L cells and rats bearing 9L gliosarcoma MCF-7 cells SKOV3 cells DC2.4 cells and B16BL6 tumor-bearing mice MDA-MB-231 and MCF-7 cell lines Same cell lines

MHCC97H cells HCC70 cell lines 4T1 and TUBO cell lines

HepG 2 cells

MDA-MB-231 cells LAMA84 and K562 cells

HepG2 and PLC cells

U-87 MG cells SKBR3 and MDA-MB231 cells A549 cells

FaDu cells Reproductive cancer cells

PC3 cell line and PC3 tumor-bearing mice HT29 cells HeLa cells SGC-7901 cell lines PANC-1 and BxPC-3 cells

MIA PaCa-2 cell lines

Target

A549 xenografts U87 and U251 cell lines, subcutaneous and orthotopic GBM model U-87 MG and bEND.3 cells Transgenic zebrafish embryos and a xenotransplanted zebrafish cancer model B16, TS/A, and 4T.1 cells NK cells

RAW 264.7 cells RAW 264.7 cells SR4987 MSCs lines

MSCs Marrow stromal cell MDA-MB-231 cells HEK293 and SKOV3 cells B16BL6 cells imDC STOSE and MDA-MB 231 cell lines LIM1215 cells

LX2 cells HEK293 cells MSCs

Bone marrow mesenchymal stem cells Primary dendritic cells HEK293T and IL3LHEK293T cells HEK293T cells

bEND.3 cells HEK 293T cells Raw Milk

Paclitaxel Paclitaxel Paclitaxel

Doxorubicin

miR-335-5p let-7a miR-142-3p inhibitor miR-124a miR-146b miR-9 and miR-155 CRISPR/Cas9 CpG-DNA Doxorubicin Doxorubicin

miR-26a

GRP78-siRNA

HCC

Brain Cancer Breast Cancer LC

HEK 293 cells HeLa and HT1080 cells

HEK 293T cells HEK293T cells CD63-GFP-HeLa cells HEK293T cell lines BJ fibroblasts

PH20 hyaluronidase SIRPα Saporin HGF-siRNA KrasG12D-siRNA

TRPP2-siRNA RAD51- and RAD52-siRNA VEGF-siRNA TPD52-siRNA KRASG12S-siRNA

Melanoma cell lines

Donor

Survivin-T34A

Exosomal cargo

Head and Neck Cancer Fibrosarcoma

Advanced Pancreatic Cancer PCa Colon Adenocarcinoma Cervical Cancer GC Pancreatic Cancer

Cancer type

Table 2. Exosomes as Therapeutic Delivery Vehicles for Cancers

Significantly decreasing growth of xenotransplanted cancer cells and zebrafish cancer model. Restoring the strongest effect to the cytotoxic function of NK cells.

Significantly inhibiting tumor xenografts growth in nude mice. Significantly improving the curative effects of PTX in GBM.

Exhibiting an excellent tumor targeting ability and suppressing tumor growth. Significantly inhibiting pulmonary metastasis growth. Increasing survival of NSCLC patients. Exhibiting a significant antiproliferative activity to cancer cells.

Significantly reducing viability and clonogenicity of GSC cells. Significantly reducing growth of glioma xenograft models. Remarkably downregulating PTEN and DUSP14 in tumor cells. Inhibiting cancer cell proliferation and reducing cancer cell viability. Exhibiting strong antitumor effects in B16BL6 tumor-bearing mice. Inhibiting tumor growth without overt toxicity. Inhibiting tumor proliferation.

Upregulating miR-26a expression in HepG2 cells and inhibiting migration and proliferation of cancer cells. Inhibiting HCC cell proliferation and inducing tumor shrinkage. Significantly inhibiting tumor growth. Efficiently delivering anti-miR-142-3p and restraining cancer proliferation.

Selectively targeting tumor tissues and inhibiting tumor growth. Targeting CML cells and suppressing cancer growth.

Efficiently inhibiting tumor growth. Significantly inducing tumor growth inhibition. Significantly improving the bioactivity of exosome- encapsulated saporin. Suppressing proliferation and invasion of cancer cells. Significantly inhibiting growth of pancreatic cancer mouse models and increasing their OS. Markedly suppressing TRPP2 expression and EMT in FaDu cells. Inducing gene knockdown and reducing the viability and proliferation of cancer cells. Suppressing the expression of VEGF in U-87 MG cells. Binding to cancer cells and delivering siRNA successfully. Significantly inhibiting proliferative activity of A549 cell and tumor xenografts in animals. Targeting GRP78 in HCC cells and inhibiting tumor progression.

Significantly increasing apoptosis in pancreatic cancer cells.

Outcome

124

154

152 153

120 121 123

151

108 149 150 110 111 115 116

104 105 107

103

147 148

146

101 145 10

98 99

89 90 129 96 97

88

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130 131 148 Withaferin A (WFA) Celastrol Imatinib LC and Breast Cancer NSCLC CML

Bovine milk Bovine milk HEK293T and IL3 LHEK293T cells

β-Elemene Breast Cancer

MCF-7/Docetaxel (Doc) and MCF-7/Adriamycin (Adr) cells A549, H1299, MDA-MB-231, and T47D cell lines A549 and H1299 cells LAMA84, and Imatinib-resistant K562 cells MCF-7 cells

SPION and Cur

U251 cell lines

containing signal regulatory protein α (SIRPα) was better than that of SIRPα-conjugated ferritin nanocage formulations.90 Passive targeting is currently the most studied targeting method of exosomes as proteins delivery system for neurological diseases and cancer therapy.91−93 However, the targeting efficiency of passive targeting is significantly lower than that of active targeting. What is more clinically needed is the active targeting that can achieve precise targeting drug delivery and reduce nontarget tissue and organ damage. Therefore, active targeting drug delivery with exogenous proteins of exosomes will be the main direction of future research. 4.2. Delivery of Genetic Drugs. 4.2.1. siRNA. Exosomemediated interfering RNAs delivery is an effective method for tumor RNA interference (RNAi) therapy. A relevant study indicated that exosomes can deliver exogenous siRNA as a therapeutic agent across different biological barriers in vivo.94 In 2011, Alvarez-Erviti et al.95 first developed an efficient, tissue-specific, and nonimmunogenic siRNA exosomal delivery platform. siRNA against BACE1 was successfully encapsulated into exosomes from autologous dendritic cells that were engineered to express lysosomal-associated membrane glycoprotein 2b (Lamp2b) bound specifically to rabies virus glycoprotein (RVG) peptide by electroporation. Systemic administration of RVG peptide-targeted exosomes can specifically deliver GAPDH-siRNA into neuron, microglia, and oligodendrocytes, thus leading to a specific gene knockdown. Exosome-packaged hepatocyte growth factor (HGF) siRNA can also be transmitted into GC cells and significantly downregulated HGF expression.96 The results showed that exosomes encapsulated with HGF siRNA possess excellent antineoplastic ability in vivo and in vitro. Another investigation reported that fibroblast-like mesenchymal cells-derived exosomes were engineered to transport siRNA specific for oncogenic KrasG12D. Subsequently, the engineered exosomes (iExosomes) showed enhanced targeting to carcinogenic Kras compared to liposomes.97 Treatment with iExosomes inhibited tumor growth in multiple pancreatic cancer mouse models.97 Analogously, exosomes can effectively deliver transient receptor potential polycystic 2 (TRPP2) siRNA into FaDu cells. After treatment of FaDu cells with exosome/TRPP2 siRNA conjugates, Wang et al. observed a series of tumorsuppression effects, such as marked inhibition of TRPP2 protein expression and EMT.98 RAD51- and RAD52-siRNA can be loaded into exosomes for delivery into fibrosarcoma cells, inducing gene knockdown and reducing the viability and proliferation of cancer cells.99 In another study, iRGD peptide expressed on exosomes membrane promoted fusion of exosomes with BC cells and delivery of siRNA against Survivin into tumor cells.100 A study also demonstrated that vascular endothelial growth factor (VEGF)-siRNA could be delivered into the zebrafish natural brain endothelia cell secretory exosomes for glioblastoma and astrocytoma therapy.101 siRNA is currently known to be mainly involved in RNAi phenomenon and regulates gene expression in a specific way, which is one of the effective methods of gene therapy. However, there are still certain obstacles and challenges in utilizing exosomes for siRNA delivery in vivo, including nonspecific gene silencing caused by siRNA, limited siRNA silencing effects, evaluation of siRNA encapsulation efficiency, purification of exosomal siRNA formulation, potential genotoxicity and pharmacokinetic and pharmacodynamic

More effective in treating LC and breast cancer models. Inhibiting proliferation and metastasis of NSCLC. Inhibiting growth and migration of cancer cells.

127

126

Significantly improving therapeutic effect of glioma and reducing side effects. Significantly reversing the chemoresistance of breast cancer.

125 Inducing apoptosis in pancreatic cancer cells.

PANC-1 and MIA PaCa-2 cells Raw 264.7 cells Curcumin

Pancreatic Adenocarcinoma Glioma

Cancer type

Table 2. continued

Exosomal cargo

Donor

Same cells

Target

Outcome

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Molecular Pharmaceutics studies in vivo. The solutions to these problems need to be further explored. 4.2.2. miRNA. Recent attention in oncologic therapeutic areas has pointed to the fact that exosomes could also act as natural biological shuttles for miRNA molecules, specifically transferring them into different cancer cells, suppressing the expression of target gene and halting tumor progression.102 Engineered or natural exosomes could serve as natural nanocarriers for targeted delivery of miRNA for oncotherapy. For example, Apo-A1 modified exosomes loaded with miR-26a can be targeted for delivery to liver cancer cells expressing scavenger receptor class B type 1. The expression of miR-26a in HepG2 cells was observed to be upregulated, and miR-26aloaded exosomes showed antimigration and antiproliferation effects on cancer cells.103 Exosomes derived from stellate cells can provide miR-335-5p for HCC cells, arrest HCC cells migration, and shrink tumors.104 This study informed a novel therapeutic strategy for stellate cell-derived exosomes can be encapsulated with therapeutic miRNA and delivered in vivo. Ohno et al.105 utilized let-7a-carrying exosomes decorated with GE11 peptide for targeted delivery to breast cancer cells expressing EGFR. The targeted peptide-decorated exosomes bound to tumor cells with higher efficiency and showed superior antitumor effects compared to blank exosomes. Furthermore, MSCs-derived exosomes could be perceived as a reliable and scalable platform to deliver specific therapeutic miRNA, representing a new therapeutic strategy for cancers.106−108 The above-mentioned studies provide an illustration that we can design targeting ligands that can bind to specific receptors found on cancer cell membranes on the surface of the exosomes encapsulated miRNA, exerting the function of gene silencing in their recipient cells or suppression of target genes expression and initiating programmed death pathways within cancer cells to achieve synergistic effects simultaneously. 4.2.3. DNA. To date, siRNA, and miRNA have been successfully loaded into exosomes. Besides, the potentiality of exosomes for therapeutic delivery of DNA or plasmids has also been developed. Lamichhane et al.109 have reported that exogenous linear DNA can be loaded into exosomes by electroporation and transferred to recipient cells. Alternatively, TEXs function as a novel shuttle that can transfer CRISPR/ Cas9 plasmids into SKOV3 cells efficiently, inducing apoptosis in ovarian cancer cells.110 Recently proposed genetically engineered TEXs contain immunostimulatory CpG DNA and endogenous tumor antigens, representing a promising exosome-based tumor antigens−adjuvant codelivery system that may be suitable for cancer immunotherapy.111 Exosomes are particularly capable of delivering these genetic drugs specifically and safely after systemic administration, representing promising vectors for gene therapy targeting tumors. However, it remains challenging to target specific tissues or cells while avoiding nonspecific delivery, especially in the liver and spleen, and to ensure the stability and reproducibility of exogenous agents carrying genetic drugs. This is the primary improvement strategy for current gene delivery therapy. 4.3. Delivery of Chemical Drugs. Most studies concentrated on the physiological and functional effects of exosomal delivery (i.e., the delivery of proteins and small RNA molecules) on cancer cells and normal cells.112 More importantly, the amphiphilic properties of exosomes allow them to compartmentalize and dissolve both native and

incorporated hydrophilic and hydrophobic chemotherapeutic agents.113,114 4.3.1. Doxorubicin (DOX). As a broad-spectrum antitumor drug, DOX has a strong cytotoxic effect. Regrettably, its cardiotoxicity has a certain impact on clinical application. Therefore, a vector that specifically targets tumors and is not taken up by cardiomyocytes is necessary to be constructed for decreasing the cardiotoxicity of DOX. Exosome can be used as a suitable vehicle to deliver DOX to achieve the decreasing toxicity and increasing efficacy.115,116 For instance, exosomes were produced by immature dendritic cells (imDC) engineered to express Lamp2b protein fused to αv integrinspecific iRGD peptide. Then iRGD exosomes (iRGD-Exos) were loaded with DOX via electroporation, with an acceptable entrapment efficiency of 20%. Intravenously injected iRGDExos delivered DOX specifically to breast tumor focus, inhibiting tumor proliferation and reducing cardiotoxicity when compared to free drug.115 This study indicated that exosomes decorated with corresponding targeting ligands can achieve the goal of therapeutic and targeted delivery of DOX to tumor sites, possessing broad application prospects in future clinical therapeutics. 4.3.2. Paclitaxel (PTX). Paclitaxel (PTX) is a commonly used chemotherapeutic agent with superior specificity. It is also an ideal natural antitumor drug with the best antineoplastic effect worldwide. Despite providing potent antitumor activity, PTX has finite clinical applications because of its low water solubility, myelosuppression, neurotoxicity, and hypersensitivity. Paclitaxel liposome117,118 and paclitaxel albumin119 have been developed to encapsulate paclitaxel, which can reduce the therapeutic dose and toxicity of the drug and improve patient tolerance. Exosome-paclitaxel (exoPTX) has been studied to overcome or reverse multidrug resistance (MDR) in cancer cells. For example, compared with PTX or Taxol, exoPTX can significantly increase the drug cytotoxicity to sensitive and resistant cancer cells, especially to effectively increase MDR of the resistant MDCKMDR1 cancer cells. It is noteworthy that exoPTX can bypass the P-glycoprotein (P-gp) efflux protein either by endocytosis-mediated transport or by fusion with plasma membrane and accumulate in target cells, inhibiting transport function and efflux activity of P-gp and reversing MDR in cancer cells.120 Later, the exoPTX formulation was optimized with incorporated aminoethylanisamide-polyethylene glycol (AA-PEG) vectorized ligand to target sigma receptor overexpressed on LC cells.121 The results demonstrated that AA-PEG-exoPTX possessed a relatively large drug loading capacity, robust accumulation in target cells, and superior therapeutic efficacy compared to other control groups.121 MSCs have been envisioned as an available delivery platform for antineoplastic agents.122 MSCs-derived exosomes are also capable of packaging and delivering anticancer active drugs (i.e., PTX) to inhibit tumor proliferation.123 Compared with MSCs, the secreted exosomes are smaller in size, less complex, and easier to be produced and stored, which can overcome many difficulties in stem cell therapy and may be a new strategy to replace stem cell therapy. Even if the anticancer effects of the exosome-loaded chemotherapeutic agents are improved and the side effects are reduced, the full efficacy of the drug and the complete cure of the cancer cannot be achieved. Different properties of exosomes from different sources, differences in drug-loading techniques, alterations of therapeutic agents on the fluidity and K


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or eliminate any potential systemic and immunogenic toxicity from regular use of milk-derived exosomes. Besides using milk to address the problem of insufficient exosome production, exosome mimics are also an ideal alternative. The representative exosome-mimetic nanovesicles that resemble exosomes but with 100-fold higher yield could traffic DOX to malignant tumor and reduce tumor growth, representing a great research direction.132 More attractively, exosome-mimetic nanovesicles not only can serve as an effective platform with high application prospects for chemical drugs but also as therapeutic nanocarriers for RNAi delivery, inducing functional knockdown or knockout responses in recipient cells.133 Exosomes are considered as a natural and unique nanodelivery system that is very effective in delivering chemotherapeutic drugs to tumors, representing a promising therapeutic approach for future oncotherapy. However, applying exosomes as drug delivery vehicles may present a downside. Given that exosomes can be actively incorporated by target cells and contain many endogenous cargos such as protein and miRNAs, these critical components may induce the transformation of normal stromal cells into cancerassociated fibroblasts, thereby playing a role in tumor promotion.134 Therefore, sequential studies are supposed to focus on revealing the sorting and packaging mechanisms of exosomes, discovering new types of payloads delivered by exosomes, and determining the appropriate drug-loading technology and condition for each type of exosomes to avoid any physiological toxicities and side effects caused by endogenous cargos and by exogenous payloads.

function of exosomal membrane basic skeleton, low encapsulation efficiency of drugs, and limited accumulation in tumor cells after systemic administration all influence the efficacy of exosomal formulations. As far as the drugs are concerned, the aqueous solubility, stability, intracellular release, and bioavailability should be further improved. With the development of different exosomal dosage forms of DOX and PTX, these potent antineoplastic agents will move toward high activity, low toxicity, and more refined targeting. 4.3.3. Other Chemical Drugs. Natural compounds with many active functions, such as dietary polyphenols, can regulate the biological function of exosomes, which have become a research hotspot in recent years.1 In 2007, when investigating the pharmacological actions of six polyphenols (curcumin, genistein, quercetin, calycosin, biochanin A and baicalein) on the cytotoxicity recovery of natural killer (NK) cells restrained by breast cancer-derived exosomes, Zhang et al. observed that curcumin restored the cytotoxic effect of NK cell function most strongly.124 Likewise, pancreatic cancer-derived exosomes carrying curcumin were proven to induce apoptosis in recipient cells, whereas exosomes without curcumin promoted the survival of tumor cells.125 Glioma-targeting exosomes with therapeutic effects were obtained through conjugation of neuropilin-1-targeted peptide with the exosome membrane by click chemistry.126 Then, superparamagnetic iron oxide nanoparticles (SPION) and curcumin (Cur) were synchronously loaded into exosomes by electroporation. These engineered exosomal carriers can smoothly cross the blood−brain barrier (BBB) when applied to glioma cells and orthotopic xenograft models. Additionally, SPION-mediated magnetic flow hyperthermia and Curmediated treatment also exhibited a synergistic antineoplastic activity.126 By coculturing drug-resistant breast cancer cell lines with exosomes loaded with β-Elemene (β-ELE, 50 μmol/L), exosomes-delivered β-ELE upregulated miR-34a expression and its target gene PTEN in drug-resistant cells and downregulated the expression of drug-resistant gene miR-452 and P-gp, significantly reversing the drug resistance of breast cancer.127 Generally speaking, exosomes are uptaken by recipient cells through endocytosis; but the cytoplasmic release of intracellular payloads is inefficient.128 This issue could be addressed by combining cationic lipids and a pH-sensitive fusogenic GALA peptide to boost the cellular uptake of exosomes and stimulate the cytosolic release and transport of payloads.129 Some studies concerning exosomes as drug delivery vehicles have utilized milk and bioinspired exosome mimetic nanovesicles to produce large-scale exosomes with no significant side effects and have made some progress. In a recent study, different natural drugs such as withaferin A (WFA), anthocyanidin, and docetaxel were encapsulated in bovine milk-derived exosomes.130 The results demonstrated that drugs loaded in milk exosomes all showed enhanced efficacies compared to unencapsulated drugs. Moreover, exosomesdelivered WFA also demonstrated potent antitumor effects against LC and breast cancer. Analogously, celastrol was loaded in milk-derived exosomes to effectively hinder the pervasion and invasion of NSCLC in a time- and concentration-dependent manner. Meanwhile, experiments in vivo showed that oral administration of exosomes containing celastrol has no significant effect on liver and kidney functions.131 However, further research is needed to reduce

5. CONCLUSIONS AND FUTURE PERSPECTIVES Since the biological effects of exosomes have been discovered, the functional roles of these secretory vesicles in human health and cancer have been increasingly explored. Exosomes, small secretory vesicles with abundant contents and biological functions, have become the topic of tumor research lately. Microexosomes with macro-roles as multifaceted regulators in various physiological and pathophysiological processes provide new strategies for diagnosis and therapy of neoplastic diseases. In diagnosis, exosomes act as potential biomarkers that could reflect cancer staging to a certain extent. Therefore, they can be used for auxiliary diagnosis, efficacy testing, and prognosis prediction of cancers. The stable, trackable, active, and realtime biological properties are the key factors to make exosomes the next generation of “STAR” in cancer diagnosis. In therapy, exosomes not only contain abundant biologically active molecules but also serve as therapeutic vehicles for exogenous drugs and maintain the stability of the drug in vivo. Exosomes can utilize the congenital precise communication system to transfer the carried active constituent to target sites for therapeutic effects and can break the traditional drug delivery modes, therefore becoming important tools to promote the development of personalized medicine. The stable, target, active and real-time pharmaceutical advantages are critical factors to make exosomes the next generation of “STAR” in translational medicine. However, most of the exosome therapies for cancer are still in the primary stage of research. There is still a long way to go before the exosomes can be used as therapeutic vehicles in clinical treatment. The translation of exosome surface structure and physiological knowledge into the generation of synthetic exosome mimetics that can carry various payloads and is L


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(9) Lai, R. C.; Yeo, R. W. Y.; Tan, K. H.; Lim, S. K. Exosomes for drug delivery - a novel application for the mesenchymal stem cell. Biotechnol. Adv. 2013, 31 (5), 543−551. (10) Aqil, F.; Munagala, R.; Jeyabalan, J.; Agrawal, A. K.; Kyakulaga, A. H.; Wilcher, S. A.; Gupta, R. C. Milk exosomes - Natural nanoparticles for siRNA delivery. Cancer Lett. 2019, 449, 186−195. (11) Hessvik, N. P.; Llorente, A. Current knowledge on exosome biogenesis and release. Cell. Mol. Life Sci. 2018, 75 (2), 193−208. (12) Keller, S.; Sanderson, M. P.; Stoeck, A.; Altevogt, P. Exosomes: From biogenesis and secretion to biological function. Immunol. Lett. 2006, 107 (2), 102−108. (13) Stenmark, H. Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol. 2009, 10 (8), 513−525. (14) Hurley, J. H. The ESCRT complexes. Crit. Rev. Biochem. Mol. Biol. 2010, 45 (6), 463−487. (15) Barile, L.; Vassalli, G. Exosomes: Therapy delivery tools and biomarkers of diseases. Pharmacol. Ther. 2017, 174, 63−78. (16) Katzmann, D. J.; Stefan, C. J.; Babst, M.; Emr, S. D. Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J. Cell Biol. 2003, 162 (3), 413−423. (17) Trajkovic, K.; Hsu, C.; Chiantia, S.; Rajendran, L.; Wenzel, D.; Wieland, F.; Schwille, P.; Brugger, B.; Simons, M. Ceramide triggers budding of exosome vesicles into multivesicular Endosomes. Science 2008, 319 (5867), 1244−1247. (18) Buschow, S. I.; Nolte-’t Hoen, E. N. M.; van Niel, G.; Pols, M. S.; Ten Broeke, T.; Lauwen, M.; Ossendorp, F.; Melief, C. J. M.; Raposo, G.; Wubbolts, R.; Wauben, M. H. M.; Stoorvogel, W. MHC II in Dendritic Cells is Targeted to Lysosomes or T Cell-Induced Exosomes Via Distinct Multivesicular Body Pathways. Traffic 2009, 10 (10), 1528−1542. (19) Futter, C. E.; Pearse, A.; Hewlett, L. J.; Hopkins, C. R. Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J. Cell Biol. 1996, 132 (6), 1011−1023. (20) Pant, S.; Hilton, H.; Burczynski, M. E. The multifaceted exosome: Biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities. Biochem. Pharmacol. 2012, 83 (11), 1484−1494. (21) Batrakova, E. V.; Kim, M. S. Using exosomes, naturallyequipped nanocarriers, for drug delivery. J. Controlled Release 2015, 219, 396−405. (22) Hoshino, A.; Costa-Silva, B.; Shen, T. L.; Rodrigues, G.; Hashimoto, A.; Mark, M. T.; Molina, H.; Kohsaka, S.; Di Giannatale, A.; Ceder, S.; Singh, S.; Williams, C.; Soplop, N.; Uryu, K.; Pharmer, L.; King, T.; Bojmar, L.; Davies, A. E.; Ararso, Y.; Zhang, T.; Zhang, H.; Hernandez, J.; Weiss, J. M.; Dumont-Cole, V. D.; Kramer, K.; Wexler, L. H.; Narendran, A.; Schwartz, G. K.; Healey, J. H.; Sandstrom, P.; Labori, K. J.; Kure, E. H.; Grandgenett, P. M.; Hollingsworth, M. A.; de Sousa, M.; Kaur, S.; Jain, M.; Mallya, K.; Batra, S. K.; Jarnagin, W. R.; Brady, M. S.; Fodstad, O.; Muller, V.; Pantel, K.; Minn, A. J.; Bissell, M. J.; Garcia, B. A.; Kang, Y.; Rajasekhar, V. K.; Ghajar, C. M.; Matei, I.; Peinado, H.; Bromberg, J.; Lyden, D. Tumour exosome integrins determine organotropic metastasis. Nature 2015, 527 (7578), 329−335. (23) Laulagnier, K.; Vincent-Schneider, H.; Hamdi, S.; Subra, C.; Lankar, D.; Record, M. Characterization of exosome subpopulations from RBL-2H3 cells using fluorescent lipids. Blood Cells, Mol., Dis. 2005, 35 (2), 116−121. (24) Subra, C.; Laulagnier, K.; Perret, B.; Record, M. Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies. Biochimie 2007, 89 (2), 205−212. (25) Valadi, H.; Ekstrom, K.; Bossios, A.; Sjostrand, M.; Lee, J. J.; Lotvall, J. O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 2007, 9 (6), 654−U72. (26) Dou, Y. C.; Cha, D. J.; Franklin, J. L.; Higginbotham, J. N.; Jeppesen, D. K.; Weaver, A. M.; Prasad, N.; Levy, S.; Coffey, R. J.; Patton, J. G.; Zhang, B. Circular RNAs are down-regulated in KRAS

suitable for all types of tumors may be an interesting concept to produce large quantities of nanocarriers. The ideal exosome biomimetics not only have the characteristics of exosomes themselves but have the advantages of a high loading capacity, universal applicability, nontoxicity, and ease of modification and administration. Furthermore, there are a series of problems that scientist must solve: the relationships between exosomes and tumorigenesis and progression, the improvement of exosome drug loading methods, and targeted modification technology. It is believed that with the tremendous support of proteomics, genomics, nanotechnologies, high-throughput sequencing technology, and bioinformatics data analysis technologies, exosomes will promise an unparalleled prospect in the diagnosis and therapy of cancers in the coming years.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +86-21-64175590. ORCID

Jiyong Liu: 0000-0003-0444-957X Author Contributions

§ L.J. and Y.G. contributed equally to this work and should be considered as co-first authors.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the projects of National Natural Science Foundation of China (No. 81573613, 81873011), the Science and Technology Commission of Shanghai Municipality (No. 16401901900, 18401931500), the Development Fund for Shanghai Talents (No. 201658), the Outstanding Talents Program of Shanghai Municipal Health Commission (No. 2018BR27) and the Open Project Program of State Key Laboratory of Natural Medicines (No. SKLNMKF201809).

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REFERENCES

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