Exosomal delivery of TRAIL and miR‑335 for the treatment of hepatocellular carcinoma (Review).

1. Introduction

Liver cancer is the sixth most prevalent form of cancer worldwide and accounts for the third most frequent cause of cancer-associated mortality ( 1 , 2 ) ( Fig. 1 ). Hepatocellular carcinoma (HCC) is the most predominant form of malignant liver cancer, which is responsible for ~90% of all non-metastatic liver cancers and has been reported to be associated with cirrhosis and hepatitis B or C virus (HBV/HCV) infection ( 3 , 4 ). Other risk factors include obesity, iron overload, alcohol consumption, diabetes, fatty liver disease and smoking ( 5 ).

However, according to statistics, only 25% of patients with HCC are diagnosed in the initial stages at the onset of the disease ( 6 ). A probable reason for this may be the absence of initial symptoms and frequent overlap with other diseases, thus making it difficult to distinguish HCC from other clinical conditions. The survival rates of cancer patients may be markedly enhanced by timely and precise diagnosis at the initial stages ( 7 ). As with the late detection of advanced-stage HCC, the diagnosis of cancer at a late stage suggests that the patient has reached a stage that is non-curative. Henceforth, the chances of survival are greatly minimized, and such patients are placed under palliative care due to the high rate of metastasis and relapse, as occurs in patients with late-stage HCC ( 8 , 9 ).

At present, the most common strategies for the treatment of HCC include ablation, surgical resection, liver transplantation, chemotherapy, transarterial chemoembolization (TACE), radiotherapy and combination therapy depending on the disease staging and patient's profile ( Fig. 1 ) ( 10 ). However, these conventional therapies have multiple limitations that compromise the quality of life of patients receiving these therapies. For example, undesired effects of radiotherapy and the development of resistance to chemotherapy due to long-term treatment and transplantation lead to long-term immunosuppressive therapy ( 11 ). Although substantial advancements have been made over the past decade in the management and treatment of HCC, including liver resection or transplantation and ablation, only ~15% of patients with early-stage HCC without cirrhosis are eligible for surgical removal. TACE is another available treatment option for patients with intermediate-stage HCC that results in a 23% increase in the 2-year survival rate when compared to traditional treatment therapies ( 12 ).

Most HCC cases are diagnosed predominantly in the late stages of the disease, which renders both surgical (resection and transplantation) and locoregional treatment (chemoembolization) inadequate for the overall survival of patients. As a result, there is an urgent need for the development of an effective therapy for patients with advanced-stage HCC.

In the SHARP randomized controlled trial, sorafenib as a monotherapy was shown to be efficacious for advanced HCC. With a high safety profile, the sorafenib-treated group exhibited an overall survival rate of 10.7 months compared to 7.9 months for the placebo group ( 13 ) ( Fig. 2 ). Sorafenib is currently the only approved prescribed option for the treatment of patients with advanced-stage HCC. Patients with HCC who have not responded to earlier treatments are recommended to use sorafenib, which received authorization by the US Food and Drug Administration (FDA) in 2007 ( 14 ). The anticancer effect of sorafenib is based on its ability to obstruct cell proliferation and angiogenesis, which inhibits tumor growth ( 15 ). However, sorafenib treatment is beneficial to only a limited number of patients and is often accompanied by drug resistance within 6 months of commencing the treatment. Moreover, the use of sorafenib is also associated with side-effects, such as nausea, alopecia and hypertension ( 14 ).

It took >10 years following the approval of sorafenib before a second first-line targeted drug for HCC was developed. As per the outcome from REFLECT trial ( 16 ), a randomized phase III non-inferiority trial reported by Kudo et al ( 16 ), led to the approval of lenvatinib to be used as the first-line treatment for advanced HCC.

In terms of overall survival, this randomized phase III trial in 2018 demonstrated that lenvatinib was not inferior to sorafenib showing overall survival of 13.6 months compared to 12.6 months for the sorafenib-treated group ( 16 ). A recent milestone in the development of HCC first-line drug development was achieved in 2020 when the FDA approved bevacizumab plus atezolizumab, an antibody combination strategy, as a first-line treatment for patients with unresectable HCC on the basis of safety and efficacy determined in the IMbrave150 trial ( 17 ). In this phase III study, 501 patients with HCC who had not previously received systemic treatment were compared to the effectiveness of bevacizumab coupled with atezolizumab against sorafenib. By significantly improving the overall survival by 12.6% at 12 months, the combination treatment significantly outperformed sorafenib monotherapy ( 17 ). Although all three drugs approved by the FDA for first-line therapy had led to an improved response and survival rate, they are all associated with multiple adverse effects ( Fig. 2 ).

Despite advancements being made in several first- and second-line drugs for the treatment of HCC, the use of these drugs still presents the issue of a compromised lifestyle with the provision of less benefit overall. The quality of life of patients receiving therapy with these drugs does not appear to be improving, and there is also the issue of the high costs of these drugs. Moreover, these targeted therapies are associated with the drawback of an inadequate objective response rate (ORR) and adaptive/acquired resistance ( 18 ). Furthermore, the long-term usage of such chemotherapeutic drugs may pose the issue of toxicity, as well as drug inefficacy. Therefore, it is imperative that a novel treatment strategy be developed with the aim of developing targeted therapy that can be applied to patients with HCC at any stage of treatment without posing any side-effects with longer durability.

The present review article illustrates the innovative exosome-based therapy as a delivery agent of two potential anticancer candidates, i.e., tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and microRNA (miRNA/miR)-335. The summary and discussion of scientific investigations highlights the immense potential of harnessing the ability of exosomes for developing an effective anticancer drug therapy for patients with liver cancer and their role in addressing the issue of drug resistance development conferred by existing chemotherapeutics.

2. MicroRNA-335: A novel candidate for anticancer treatment

miRNAs are non-coding RNAs whose function is to perform post-transcriptional gene regulation. They play a key role in tumor development by modulating the expression of various oncogenes and tumor suppressor genes ( 19 ). miRNAs are poorly regulated in multiple types of cancers and, as per their function, they function either as oncogenes or tumor suppressors. Oncomirs, such as miR-21 stimulate tumor growth by impeding tumor suppressor genes ( 20 ). On the contrary, tumor suppressor miRNAs, such as miR-145 restrict the progression of tumors by blocking oncogenes ( 21 ). Furthermore, miRNAs are involved in several biological processes, including cell proliferation, apoptosis, metastasis and angiogenesis ( 22 , 23 ), which are the key features in cancer progression.

miRNAs as a novel class of regulatory factors are crucial to the biological and pathological processes in several cases of human solid tumors ( 24 ). According to numerous recent studies, as a tumor suppressor gene, miR-335-5p has been linked to the emergence and growth of several tumors, including colorectal cancer ( 25 ), non-small cell lung cancer ( 26 ) and HCC ( 27 ). Furthermore, hepatic stellate cell-derived miR-335-5p exosomes may be used as prospective miRNA biomarkers in HBV-related HCC ( 28 ) and thus have potential therapeutic significance in HCC ( 29 ).

Previous research has demonstrated that miR-335 is a suppressor of tumor formation, invasion and metastasis, and is responsible for the regulation of apoptosis and thus has prognostic value in HCC ( 30 ). miR-335-3p, sometimes referred to as miR-335*, is generated concurrently with miR-335. Notably, these miRNAs have also been shown to be responsible for inducing the activation of the p53 tumor suppressor pathway to prevent cellular proliferation and neoplastic cell transformation ( 31 ). Emerging evidence has depicted the role of miR-335 in the majority of oncogenic signaling pathways responsible for cell growth and survival. It has been discovered that the downregulation of miR-335 in lung cancer enhances cell proliferation by activating the AKT/mTOR signaling pathway, which is one of the most commonly dysregulated signaling pathways in human malignancies ( 32 ). This characteristic of miR-335 could be exploited in therapeutic applications for restricting HCC progression and controlling metastasis.

miR-335-5p reportedly targets downstream genes to modulate the biological activity of cancer cells. For example, miR-335-5p overexpression has been shown to suppress the proliferation, migration and invasion of non-small cell lung cancer by targeting CPNE1 ( 33 ). In the case of HCC, miR-335-5p has been reported to significantly attenuate the development of this type of cancer. For example, a previous study demonstrated how the circ_0009910/miR-335-5p/Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) axis is crucial to the onset and development of HCC ( 34 ). In that study, miR-335-5p inhibited HCC cell proliferation, migration and invasion by targeting ROCK1. By enhancing the inhibitory effects of miR-335-5p on the expression of ROCK1 in HCC, circ_0009910 knockdown exhibited anticancer properties. In addition to that, it was found that the invasiveness of cancer cells was positively associated with ROCK1 ( 34 ). In a similar context, Liu et al ( 35 ) demonstrated that miR-335 was involved in suppressing HCC cell proliferation, migration and invasion by downregulating ROCK1 expression. Their research investigating molecular mechanisms revealed that ROCK1, which is associated with cell movement and invasion in various cancer types, was a target gene for miR-335 for modulating the proliferation and metastasis of HCC cells ( 35 ). Reportedly, ROCK1 functions as an oncogene in HCC and promotes the development of HCC ( 36 ).

The significance of miR-335 was further validated by another study which demonstrated that miR-335 restoration inhibited hepatic stellate cell (HSC) migration ( 37 ). The findings of that study concluded that miR-335 considerably decreased during HSC activation. Restoring miR-335 expression markedly decreased collagen type I and α-smooth muscle actin levels and prevented cell migration, at least in part through the downregulation of tenascin-C, an extracellular matrix glycoprotein involved in cell migration. The overexpression of miR-335 in HSC may thus provide a novel strategy for the treatment of hepatic fibrosis ( 37 ). Another recent study by Yang et al ( 38 ) revealed that a newly characterized circular RNA, circ_0005075, promoted HCC progression by suppressing the function of miR-335. Circ_0005075 was discovered to be upregulated in HCC tissues where it was found that the downregulation of circ_0005075 inhibited HCC progression ( 38 ). As per their study mitogen-activated protein kinase 1 (MAPK1) was shown to be regulated by miR-335, divulging it as one of the downstream regulatory targets of circ_0005075. It was also shown that the upregulation of circ_0005075 may be responsible for the elevated level of MAPK1 ( 38 ). As a crucial member of the MAPK family, the main function of MAPK1 involves cell proliferation, gene expression, differentiation, mitosis, cell survival and apoptosis ( 39 ). Likewise, their role is also imperative in tumorigenesis. With the overexpression of MAPK1 in multiple types of cancer, including breast cancer ( 40 ), they could be the likely candidates to be used for the prognosis of patients with HCC ( 38 ). To sum up, Yang et al ( 38 ) demonstrated that miR-335 may target and inhibit MAPK1 ( 38 ). Moreover, Ji et al ( 41 ) identified octamer-binding transcription factor 4 (OCT4) as another target gene of miR-335-5p. miR-335-5p was demonstrated to prevent OCT4 gene expression to restrict the downstream activation of the Akt signaling pathway, which was demonstrated to be a canonical regulator in the development of liver cancer ( 41 ).

Notably, a number of scientific investigations have also discovered a pertinent link between miR-335 expression and the survival of cancer patients ( 26 , 42 ). Furthermore, several findings have indicated that miR-335 influences the chemotherapeutic response in patients receiving standard treatment ( 43 , 44 ). For example, the study by Cui et al ( 45 ) demonstrated that serum miR-335 levels can be utilized as a marker to identify the status of disease progression, apart from clinical outcome in patients receiving TACE therapy. TACE therapy is the standard of care treatment for patients with large or multinodular HCC whose treatment response varies and still lacks any prognostic marker. However, the study by Cui et al ( 45 ) indicated that low levels of miR-335 in patient serum were associated with a low survival rate with a poor treatment response. Chen and Xia ( 46 ) demonstrated the role of miR-335 as a biomarker for HCC treatment and demonstrated its function responsible for regulating sensitivity to sorafenib in HCC. They depicted the fact that miR-335 regulates sorafenib sensitivity in HCC cells by inhibiting the AKT pathway ia targeting C-MET, which is a tyrosine kinase protein involved in the development of cancer ( 46 ).

Dohi et al ( 47 ) first reported that miR-335, which is located within the intron of its protein-coding host gene, MEST, was downregulated due to aberrant promoter hypermethylation. Primary HCC tissues exhibited considerably higher levels of miR-335/MEST methylation and miR-335 expression was much lower in tumors compared to the non-tumor tissue counterparts ( 47 ). Their finding suggested that aberrant DNA methylation in primary HCC was the cause of the decreased miR-335 expression. Furthermore, their findings suggested that a decreased expression of miR-335 may be linked to distant metastases in HCC ( 47 ). Similar findings were also provided on the expression of miR-335 in tumor tissues, which was reported to be much lower than in non-tumor tissues compare to HCC patient samples ( 48 ). The recent study by Nie et al ( 49 ) demonstrated ROCK1 as a target gene of miR-335-5p, where circ_0064288 enhanced ROCK1 expression by competitively binding with miR-335-5p. They suggested that circ_0064288, which is highly expressed in HCC, functions as an oncogene by inhibiting miR-335-5p expression and promoting ROCK1 expression, which is responsible for regulating cell motility. A list of various studies investigating the clinical significance of miR-335 in HCC and their molecular mechanisms is presented in Table I . Thus, potential diagnostic and treatment options for HCC may be provided via the modulation of miR-335.

3. Significance of TRAIL in HCC treatment

TRAIL is a pro-apoptotic ligand that has received increasing attention owing to its property of inducing apoptosis in multiple types of cancer cells without affecting normal cells ( 50 ). This unique feature of TRAIL has allowed several researchers to investigate the development of TRAIL-receptor agonists as a form of anticancer therapy ( 51 , 52 ). Additionally, TRAIL-based therapeutics are independent of p53 in tumor cells, unlike other chemotherapeutic drugs, which renders them unique in terms of the cell death pathway ( 53 , 54 ).

TRAIL is a transmembrane protein reported to be found on natural killer (NK) cells and cytotoxic T-cell surfaces with a predominant expression in tissues, including the prostate, lungs and spleen ( 55 ). TRAIL can be secreted in the soluble form, which is non-toxic to normal cells, while healthy adult plasma contains a trace quantity of endogenous TRAIL (100 pg/ml) ( 56 ). Multiple types of cancer cells overexpress the death receptors (DRs), DR4 and DR5 ( 57 ). Upon secretion from NK cells, TRAIL binds to DR4 and DR5 ( 58 ) and upon binding, it leads to the recruitment of caspase-8 to the Fas-associated death domain adaptor protein. Following activation, it culminates in apoptotic signaling via caspase-3 activation, ultimately leading to cell death ( Fig. 3 ) ( 59 ).

The ability of TRAIL to induce tumor-specific cell death renders it a promising candidate for antitumor therapy. In numerous in vitro studies, TRAIL protein in its soluble recombinant form has been widely explored as an anticancer agent ( 57 , 60 - 62 ). A number of previous publications with the aim of developing antitumor therapy have centered their findings on the in vitro and in vivo tumoricidal activity of TRAIL protein; they demonstrated a peculiar feature of apoptosis induction by recombinant, soluble TRAIL protein in a broad range of cancer cell lines, although they demonstrated no activity against normal cells ( 63 - 65 ). The safe use of TRAIL as a ligand for therapeutic purposes, displaying no noticeable cytotoxicity to normal tissues, has also been validated in mouse models ( 57 ) and in humans ( 66 ).

The study by Grisendi et al ( 67 ) on adipose-derived (AD)-mesenchymal stem cells (MSCs) producing TRAIL revealed that when AD-MSCs loaded with TRAIL were injected into mice, they localized to the tumor site and induced apoptosis without causing any significant toxicity to normal tissues. Those authors also proposed that using stably genetically modified AD-MSCs to deliver TRAIL alone or in conjunction with sensitizing drugs may provide new treatment options for malignancies that remain incurable ( 67 ). Furthermore, El-Shemi et al ( 61 ) revealed TRAIL and inhibitor of growth 4 (ING4) as potent apoptosis-inducing genes in an orthotopic mouse model of human HCC bearing utilizing oncolytic adenoviruses as a gene delivery agent. Their study found that the combination of these drugs significantly reduced tumor-driven angiogenesis and neovascularization, and also triggered apoptosis and immune responses, without exhibiting any overlapping toxicities ( 61 ). Another study by Liu et al ( 65 ) revealed that TRAIL plasmid DNA delivered via HCC-targeted lipid/calcium/phosphate/protamine nanoparticles in conjunction with traditional sorafenib therapy decreased HCC development, as well as liver fibrosis in a mouse model of HCC. Overall, these findings provide a promising treatment strategy for cancer based on TRAIL that may be applied in clinical settings.

Another study recently demonstrated the underlying mechanisms of action of TRAIL, which involved causing substantial cytotoxicity to tumor cells only, but seldom affecting non-transformed cells ( 68 ). That research revealed an interaction between TRAIL and immediate early response gene (IER3), which is expressed in a number of human tissues, and appears to be downregulated in cancer cells, and its overexpression can stimulate the apoptosis of cancer cells and enhance their sensitivity to chemotherapeutic drugs ( 68 ). It was demonstrated that these two proteins may be responsible for directing HCC cells to undergo apoptosis and interrupt with their capacity to proliferate and migrate. These findings demonstrate that TRAIL can partly influence the pathogenesis of HCC by interacting with IER3 to reduce Wnt/-catenin signaling ( 68 ). A number of TRAIL-based therapeutics for the treatment of HCC are currently undergoing or have undergone clinical trials, as demonstrated in Table II .

Challenges with TRAIL therapy

Unfortunately, despite being such a prominent feature of being specific to tumor cells, TRAIL-based therapy still has a long way to go for successful clinical translation. TRAIL-based therapy, including recombinant or agonistic monoclonal antibodies against DR4/DR5 ( 69 , 70 ), has exhibited limited efficacy in a number of clinical trials of different stages due to the short plasma half-life of recombinant TRAIL, limited bioavailability and undesirable systemic toxicity ( 69 ). Moreover, TRAIL-based therapy is associated with several challenges, including the development of TRAIL-mediated cell death resistance that leads to TRAIL-induced apoptosis being ineffective in HCC. The root cause of the development of therapeutic resistance may be intrinsic resistance in some highly malignant tumors and acquired resistance post-frequent exposure to TRAIL ( 71 ). Other factors may include the activation of anti-apoptotic molecules and multiple receptors of various signaling pathways ( 71 - 73 ).

Developing agonist monoclonal antibodies (mAbs) targeting DR4 or DR5 receptors has been the most prevailing approach due to their long serum half-lives in vivo . A number of clinical trials, have been conducted on several agonists, such as mapatumumab ( NCT01258608 ), lexatumumab ( NCT00428272 ) and tigatuzumab ( NCT01307891 ); however, none of them displayed any antitumor response rates in cancer patients ( 69 ). Similarly, in preclinical tumor xenograft mouse models, mapatumumab and lexatumumab have failed to eradicate tumors ( 74 , 75 ). The dimeric structure of the antibody is most likely to blame for the failure of all agonist mAb clinical trials. Since the binding of ligand on DR4 and DR5 induces receptor trimerization following the activation of the extrinsic pathway, maintaining its trimeric structure will be required in the future to enable its functional mechanism of apoptosis induction.

The strategy employing recombinant human native TRAIL appears to be more feasible as it allows the preservation of the original trimeric structure with full functional efficacy. Dulanermin, which is Amgen's version of TRAIL, was not found to be effective against cancer in human clinical trials, even though it was effective in preclinical tumor xenograft models ( 76 ). The probable reason for the inefficacy of dulanermin may be related to its poor pharmacokinetic profile due to its very short half-life in mammals. Given its low molecular weight and its non-covalently linked trimeric structure instability, which may cause rapid renal elimination. All these facts illustrate the requirement of a novel approach to tackle this challenge.

To obtain such biologically active TRAIL, many expression systems, such as His, Flag tag or the incorporation of trimerization domains, such as leucine zipper or isoleucine zipper and the stabilization of trimers with cations, as zinc were identified. It has been validated that to assemble and maintain a functionally folded ligand trimer of TRAIL, zinc chelation is critical ( 77 , 78 ). However, it appears to have its own set of drawbacks. For example, both leucine zipper-fused TRAIL and an N-terminus His-tagged TRAIL stabilized by insertion mutation are likely to be immunogenic in humans ( 57 ). In addition, particularly the His-tagged version, has been linked to hepatotoxicity not observed with native TRAIL ( 79 , 80 ). Other strategies for prolonging the half-life of TRAIL, which include albumin-conjugated TRAIL nanoparticles or liposome conjugated TRAIL, present the issue of production limitation ( 81 , 82 ).

The recent study by Naval et al ( 83 ) demonstrated the significance of the oligomerization of TRAIL receptors in TRAIL-induced apoptosis. They stated that TRAIL, as a transmembrane protein, exhibited more potent pro-apoptotic properties than its soluble form ( 83 ). In immune system cells, TRAIL is expressed as a type II membrane protein in the plasma membrane ( 84 , 85 ) or is enclosed within microvesicles ( 86 , 87 ). DR5 is solely triggered by the membrane-bound form of TRAIL, whereas DR4 can be activated by both the soluble and membrane-bound forms of the ligand ( 88 ). Given the property of TRAIL of being naturally secreted as a membrane protein in exosomes, a number of nanocarriers, such as liposomes, whose lipid composition replicates natural exosomes with surface-bound TRAIL, have been investigated in several pre-clinical studies on its anticancer properties. Various in vitro and in vivo studies have demonstrated that this membrane-bound form of TRAIL has greater antitumor activity than the soluble form against hematological and solid tumors ( 89 - 91 ). In comparison to soluble TRAIL, this liposomal formulation with TRAIL attached to the liposome surface produces improved DR5 clustering and increased DISC recruitment, resulting in a greater apoptotic signal ( 90 ). As TRAIL produces high-order TRAIL oligomers on the lipid nanoparticle surface, improved DR5 clustering and higher DISC recruitment is accomplished ( 92 ). Furthermore, TRAIL-encapsulated liposomes and nanoparticles face hurdles with agent release from carriers ( 93 , 94 ). In summary, the therapeutic benefits of TRAIL therapy have been limited, possibly due to the resistance displayed by HCC cells and poor pharmacokinetics. Thus, an effective delivery agent is required for the delivery of TRAIL, which can increase its circulation time in the human body with optimal encapsulation.

4. Exosomes: A novel approach for drug delivery

Conventional anticancer drugs display limited efficacy owing to their short half-life, poor solubility and inefficacious delivery, resulting in the development of drug resistance and substantial systemic toxicity ( 95 ). Apart from this, poor drug delivery is also a major contributor to treatment failure. After entering the blood circulation by injection, the drug faces a variety of hurdles before reaching and acting on the target site ( 96 ). To improve the efficacy of HCC chemotherapeutics, a drug delivery system with active targeting and local, controlled, and continuous drug release is urgently required.

Extracellular vesicles (EV)-based therapeutics are a promising drug delivery system, since they can penetrate tissues and even cross the blood-brain barrier as natural nanoscale agents ( 97 , 98 ). Exosomes in particular, offer numerous benefits as drug delivery vehicles, which include a small size, low cytotoxicity, long half-life in the circulation, and the ability to load various cargoes with high a biocompatibility ( Fig. 4 ) ( 99 , 100 ). Conventional drug delivery strategies frequently fall short of the intended results for several reasons, including the rapid in vivo degradation of miRNAs, the loss of native structure in proteins and the potential for severe toxicity in normal cells. However, these issues may be resolved by using exosomes as carriers and thus, by deploying exosomes to deliver drugs to tumor sites, an effective and promising approach may be made available for targeted cancer therapy.

Exosomes are endosomal-derived EVs with a size of 30-200 nm that have been reported to be released by a multitude of cell types. They have the characteristic cargo-loading capacity of carrying heterogenous biomolecules, such as DNA, RNA, proteins and lipids, and transferring them to recipient cells, thus acting as intercellular messengers. Exosomes are formed by the intraluminal budding of multivesicular bodies (MVBs) of an intact cell and are released into the extracellular environment when these MVBs fuse with the plasma membrane of the recipient cell ( 101 ).

Exosomes have the potential to mirror the intricacy of the parental cell with the innate capacity to regulate multitude of roles in crucial biological activities ( 102 ). Consequently, this characteristic led to exosome-based applications in cancer treatment and diagnoses being more feasible. Exosomes contain molecules with a wide range of functions, but lack the complexity of cells and organs; as a result, exosomes are regarded as excellent tools for use in the treatment of a variety of disorders, including cancer. Exosomes also have a number of advantages in terms of biocompatibility, stability, cellular uptake mechanism, biodistribution, pharmacokinetics and immunogenicity, rendering them promising anticancer candidates. These characteristics can raise the therapeutic index of exosome-based cancer treatments by preferentially targeting tumor cells, while reducing undesirable side-effects. Below is a brief illustration of key facts of the therapeutic potential of exosomes over present drug delivery platforms.

Source and safety

MSCs have been reported to be the most favorable selection for the commercial production of exosomes due to the ease of availability and are reported to produce an excessive number of exosomes (EVs) with consistent sustainability and reproducibility compared to other cell lines ( 103 ). Most importantly, applying MSC-exosomes as an agent to deliver drugs has been reported to have no safety concerns, including the chances of inducing tumorigenicity ( 104 , 105 ). MSC-derived EVs exhibit significant flexibility for in vitro and in vivo modifications ( 106 ), as well as a high stability in human plasma and at storage at 20°C ( 107 , 108 ).

Furthermore, MSC-derived EVs have been demonstrated to be well-tolerated in a variety of animal models, aside from possessing therapeutic benefits as proven in the treatment of myocardial infarction, chronic kidney disease, wound healing and liver injury in mouse models ( 109 , 110 ). Previous studies have employed EVs as an efficient systemic natural gene carrier for transporting anticancer miRNAs and proteins ( 111 , 112 ). A number of phase I clinical trials have validated the safety of EV administration; no reports of grade II toxicity were reported with the determination of the maximal tolerated dose ( 113 - 115 ).

Drug delivery and cellular uptake

As aforementioned, exosomes, being a natural cellular messenger, provide the benefit of a heterogeneous cargo-loading capacity and specificity. Exosomes are known to possess homing properties that can deliver cargo even to distant targets and in between cells with suitable biocompatibility, and to regulate the functions of targeted cells transiently ( 116 ). Reportedly, the interaction of exosomes with target cells involves multiple mechanisms, as exosomes can directly bind to membrane receptors of the recipient cell for content internalization, or they can transport bioactive cargo by fusing with the plasma membrane of the target cell.

Presently, a number of drugs face the issue of not being able to cross the blood-brain barrier, limiting the efficacy of several therapies, including cancer therapies. However, exosomes can cross the blood-brain barrier to increase intracranial drug concentration ( 117 ). For example, exosomes have been shown to carry medicines or siRNA to the brains of mice with Alzheimer's disease ( 111 , 118 ). Unlike the traditional approach of drug administration, delivery using exosomes does not have the drawback of drug toxicity, intracranial infection and imprecise absorption ( 118 ).

Cargo protection and improved durability

The ideal delivery agent does not only perform the site-specific transportation of enclosed therapeutics, but should also be capable of protecting the enclosed material and avoiding premature degradation by the body's immune system. Exosomes have a lipid bilayer structure that not only aids in transport efficiency and supports the load of hydrophobic or hydrophilic drugs, but also protects the encapsulated material ( 119 ). Furthermore, being able to have a reduced clearance rate can sustain the drugs in the body's circulation. Exosomes, being natural products of the body, do not invoke any immune response and possess a longer circulation half-life, which can prevent therapeutic cargo from degrading too rapidly ( 118 ).

Stability

Exosomes are well-known for their stability, as they preserve the identity of their parental cells, while maintaining their long-term innate integrity ( 107 ). Multiple freeze-thaw cycles have been shown to have no effect on their size, indicating that freezing has no effect on the quality of exosomes stored ( 107 ). Kalra et al ( 108 ) demonstrated the stability of colon cancer-derived exosomes and noted that the majority of samples retained their integrity even without protease inhibitors for 3 months, and that the highest stability was found at 80°C. This property of being stable for a long period of time in storage at 80°C suggests another advantage of exosomes over existing anticancer drugs ( 120 ).

Furthermore, a previous study found that therapeutic exosomes maintained antitumor activity even after being frozen for at least 5 months ( 121 ). Exosomes can shield therapeutic nucleic acids and proteins from RNases and proteinase degradation as they contain fragile bioactive molecules within a lipid bilayer membrane ( 122 ). In addition, under both physiological and pathological conditions, exosomes appear to display an enhanced stability in the blood, that facilitates long-distance travel within the body. Thus, exosome stability covers not just the human body, but also storage in the field. In addition to this, they have also been shown to have improved stability in the blood, allowing them to cover long distances throughout the body under both normal and pathological conditions ( 123 ).

Therapeutic significance and drug resistance

It has been demonstrated that drugs encapsulated with exosomes lead to an enhanced chemotherapeutic efficacy ( 124 ). Exosomes appear to have a much higher potential (>10-fold) in targeting cancer cells compared to liposomes of similar size ( 125 ). Furthermore, currently, the major barrier to an effective therapy or complete cure is multi-drug resistance (MDR). This resistance is commonly shown by all cancer patients undergoing long-term chemotherapy. The exosome is one such natural nanocarrier which has proven to be efficient in overcoming the issue of MDR in tumors. Kim et al ( 126 ) confirmed this by integrating paclitaxel (PTX) into exosomes released from macrophages for the treatment of MDR cancer. They determined that exosomes augmented cytotoxicity in drug-resistant cells by >50-fold when compared to exosome-free drugs ( 126 ). Furthermore, when doxorubicin (DOX)-loaded exosomes were administered intranasally to animals with pulmonary metastasis, confocal fluorescence microscopy revealed an almost perfect co-localization with cancer cells. These findings suggest that PTX-loaded exosomes inhibit MDR tumors and pulmonary metastasis growth more effectively ( 126 ).

5. Challenges, synergism, and strategies against liver cancer

Several drug delivery strategies have been developed over the years for the treatment of cancer; however, only a few of these have obtained clinical approval. The likelihood of an efficacious drug delivery to cancer tissues following in vivo administration is <0.7% ( 127 ). The expected accomplishments in the case of HCC drug development may not be as satisfactory as in the case of other types of cancer. Furthermore, based upon the outcome of recent clinical trials, a single drug therapy appears to be inadequate in the case of treatment for advanced-stage HCC ( 128 ). Thus, combination therapy is a major area of research for the treatment of advanced-stage HCC.

Surprisingly, formulations containing recombinant TRAIL have encountered considerable difficulties in being evaluated for human application due to its undesirable systemic toxicity, the short plasma half-life of recombinant TRAIL, or the activation of anti-apoptotic proteins ( 62 , 69 , 94 , 129 ). However, the exosome-based encapsulation of TRAIL protein can convey better pharmacokinetic characteristics, greater bioavailability and the ability to cross target tissues to the enclosed protein/drug ( 130 ). For example, in a previous study, TRAIL was transduced into leukemia K562 cells with human membrane TRAIL and produced TRAIL secreted exosomes, which were shown to trigger the apoptosis of melanoma and lymphoma cell lines in vitro ( 131 ). These findings reveal that cells that have been genetically engineered to express TRAIL can secrete exosomes that contain the pro-apoptotic ligand in an active form in their membranes. Although therapeutic success varied in the different tumor models studied, TRAIL exosomes exhibited potent killing activity in vitro and in vivo , in both local and systemic therapy modalities ( 131 ).

Another study by Yuan et al ( 132 ) demonstrated that TRAIL-loaded exosomes were more efficient in inducing cell death than recombinant soluble TRAIL. It was shown that the fluidic nature of the lipid bilayer membrane in exosomes harboring TRAIL may allow higher order TRAIL oligomerization and, as a result, the stronger clustering of its receptors, which is a crucial signal for effective extrinsic death pathway activation ( 132 ). It was demonstrated that the limited bioavailability of TRAIL, the low activity and cell resistance to TRAIL ligand can be overcome by TRAIL-expressing EVs derived from MSCs, thereby improving the clinical efficacy of TRAIL. This EV-loaded TRAIL effectively induced apoptosis in a variety of cancer cell lines, including lung (A549, NCI-H460 and NCI-H727), neuroblastoma (SHEP-TET), breast (M231), kidney (RCC10) and malignant pleural mesothelioma lines (H2795). While there was no toxicity to control healthy cells, TRAIL + exosomes were capable of triggering apoptosis in TRAIL-resistant cancer cells ( 132 ).

The TRAIL receptor binding to target cells, which activates the caspase cascade and results in death, was suggested as the therapeutic mechanism of TRAIL-MSC-EVs. Furthermore, TRAIL-MSC-EVs have exhibited therapeutic efficacy in TRAIL-resistant cancer cell lines, which is noteworthy ( 132 ). The study by Shamili et al ( 133 ) also demonstrated the anti-tumor activity of TRAIL-transfected MSC-derived exosomes in a mouse model of melanoma. Their findings suggested that when TRAIL-expressing exosomes were injected into mice, they delayed the appearance of tumors and attenuated tumor growth ( 133 ). Of note, they proposed that a combination of TRAIL-exosomes with another chemotherapeutic may be explored as a promising therapeutic tool ( 133 ). The diagrammatic representation of how TRAIL-expressing exosomes would lead to cell death in recipient HCC cells is presented in Fig. 3 . A list of studies demonstrating the exosomal delivery by TRAIL and its clinical significance is presented Table III .

Several studies have found a synergistic effect between TRAIL and sorafenib, suggesting that combined treatment with these agents may lead to the development of effective therapy for overcoming TRAIL resistance in cancer cells. For example, a previous study demonstrated the synergistic effects of sorafenib and TRAIL, where Sorafenib considerably increased the cytotoxicity TRAIL to HCC cells ( 134 ). The enhancement in cytotoxicity may be obtained from the downregulation of anti-apoptotic proteins by sorafenib. Similarly, another study by Chen et al ( 135 ) revealed that sorafenib sensitized TRAIL-resistant HCC cells to TRAIL-induced apoptosis by inhibiting STAT3 ( 135 ).

Moreover, the application of Sorafenib enclosed within exosomes into the target site offers a number of benefits as opposed to oral administration. As depicted by a previous study, exosome-encapsulated DOX delivery increased the therapeutic index in breast and ovarian cancer mouse models compared to exosome-free DOX ( 124 ). It was also demonstrated by in vitro and in vivo experiments that exosomes loaded with DOX limited heart toxicity by partially reducing the passage of DOX through cardiac endothelial cells ( 124 ).

The major issue with the safe, specific and efficient delivery of miRNAs is their property of being easily degradable before reaching the target organ. The lipid bilayer membrane of EVs protects the enclosed miRNA, preventing it from degradation and facilitating its effective delivery to the target site ( 136 ). Previous research has demonstrated that exosomes can safely enclose and carry miRNA to target cells of multiple types of cancer. For example, Almanza et al ( 137 ) demonstrated that EVs containing miR-335 effectively and long-lastingly restored the endogenous miR-335 pool in human triple-negative breast cancer cells, suppressing the expression of the miR-335 target gene SOX4 transcription factor, and significantly reducing tumor development in vivo .

Previously, another group ( 138 ) reported utilizing EVs as a delivery agent for miR-335 both in vivo and in vitro . They were successful in demonstrating that the safe administration of fibroblast-derived EVs that were loaded with miR-195 may concentrate inside the tumor, reduce the size of tumors, and increase the longevity of treated rats in a rat model of cholangiocarcinoma ( 138 ). Wang et al ( 139 ) demonstrated that miR335-5p could be successfully supplied to hepatoma cells by utilizing exosomes as a delivery agent both in vivo and in vitro . They observed the progression of HCC cell development when stellate cells were co-cultured with HCC cells due to exosomal transfer and noted the downregulated expression of miR-3355p in both cells and exosomes ( 139 ). The target genes identified in terms of HCC are CDC42, NRG1, EIF5, CDK2, EIF2C2, LIMK1, PLK2, RGS19, THBS1, YBX1 and TCF3. However, upregulating the expression of miR-335-5p in stellate cell-derived exosomes has been shown to restrict HCC cell proliferation and invasion in vitro , and cause tumor shrinkage in mouse models ( 139 ). A schematic diagram of the exosomal delivery of miR-335 into recipient HCC cells and the mode of action based on the afore-mentioned investigations is presented in Fig. 5 .

It is also noteworthy that miR-335 modulates the sensitivity of sorafenib against HCC cells. As shown in the study by Kim et al ( 43 ), Siah E3 ubiquitin protein ligase 2 (SIAH2) is the target of miR-335, where miR-335 contributes to the sensitizing effect of anticancer drugs via the expression enhancement of histone deacetylase 3 (HDAC3). SIAH2 overexpression was shown to result in anticancer drug resistance due to its effect on HDAC3 expression and ubiquitination ( 43 ). These findings suggest that miR-335 may be a promising anticancer agent. When combined with sorafenib, encapsulating it in exosomes increases its sensitivity to the drug, thus enhancing the therapeutic efficacy and preventing degradation.

6. Development of exosome-based TRAIL + miR-335 therapy

Although exosome-based cancer therapy displays exceptional therapeutic potential, there are still a number of substantial challenges that need to be resolved in order for its use to be feasible in clinical applications. The technology of exosome mass production is not yet standardized. Although small-scale GMP exosome production has been shown to be viable, there are still numerous obstacles in large-scale production ( 121 , 140 ). Numerous companies are still struggling to produce exosome on mass scale level. However, few companies, such as CK Exogene, a Korean biotechnology firm, have managed to overcome the issue of low exosome yield and have acquired the patented technology (10-2020-0062365) for exosome mass production ( 141 ) and this company is currently developing exosome-based anticancer drug for patients with liver cancer using the aforementioned candidates i.e., TRAIL and miR-335. A schematic overview of anticancer candidates, including dorafenib encapsulated in exosomed as a delivery agent is presented in Fig. 6 ).

To the best of our knowledge, the present review is the first of its kind, exploring and combining the cutting-edge feature of exosome with novel anticancer candidates (TRAIL and miR-335). The aim of the present review article was to present the compiled investigations of TRAIL and miR-335, both of which have been extensively explored in the past, along with the added benefit of utilizing exosomes as a carrier. Exosomes encapsulating TRAIL have the potential to overcome the challenging issue of resistance among HCC cells towards TRAIL-induced apoptosis, thus rendering TRAIL more effective in killing cancer cells. Such a strategy not only provides long-term and effective anticancer treatment for patients with HCC, but it has also been reported to overcome the issue of drug resistance, which is the major challenge to current drug therapy for liver cancer.

Additionally, to receive the successful outcome of any drug treatment, the accessibility of the drug to the target organ is imperative and necessitates the requirement of an effective delivery route. Even though drugs targeted against HCC comprise various delivery routes, including the direct injection into the liver, intra-arterial drug delivery is an effective technique for targeting the tumor site with multiple agents. Compared to intravenous delivery, intra-arterial drug administration expedites the systemic clearance and enhances the intra-tumor drug concentration ( 142 ). Owing to such high magnitude of benefits conferred by exosome-based TRAIL-miR-335 delivery from preventing metastasis to inducing cytotoxicity in cancer cells specifically, this approach has the prospects to be provided to patients with all stages of liver cancer from stages 0 to 4.

7. Conclusions

Exosomes offer the versatile characteristics of an efficient delivery system for both TRAIL and miR-335 as anticancer candidates. The incorporation of the benefits of exosomes, with them being a natural cellular carrier and the combination of these novel candidates with a standard drug, such as sorafenib would have a sensitizing effect and has been proven to yield a synergistic anti-cytotoxicity effect on TRAIL-resistant cancer cells. Moreover, this strategy also has the potential to overcome resistance to sorafenib, the most prevalent issue of the current drug treatment program among patients with HCC. The most prominent significance of exosome-based technology lies in the fact that this approach can be applied to all types of cancer and encompasses the benefit of overcoming drug resistance, which is the most prevalent issue in current drug treatment regimen. The present review thus provides an insight into the development of exosome-based therapy and the possibility of its bench-to-bed translation for providing an exceptional anticancer treatment.

Acknowledgments

Not applicable.

Availability of data and materials

Data sharing is not applicable to this article, as no data sets were generated or analyzed during the current study.

Authors' contributions

All authors (NT, YJC, KHY, TBW, DK, DC and JK) were involved in the drafting and revision of the manuscript, and in critically revising the manuscript for important intellectual content. All authors have read and approved the final manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The purification strategy for the mass production of highly purified and concentrated exosomes is subject to Korean patent application no. 10-2020-0062365, associated with CK-Exogene, Inc. JK and NT are employees of CK-Exogene, Inc. The other authors (YJC, KHY, TBW, DK and DC) are not associated with CK-Exogene, Inc. and declare that they have no competing interests.

References

1 Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J Clin 68 394 424 2018 10.3322/caac.21492 30207593

2 Singal AG El-Serag HB Hepatocellular carcinoma from epidemiology to prevention: Translating knowledge into practice Clin Gastroenterol Hepatol 13 2140 2151 2015 10.1016/j.cgh.2015.08.014 26284591 PMC4618036

3 Dasgupta P Henshaw C Youlden DR Clark PJ Aitken JF Baade PD Global trends in incidence rates of primary adult liver cancers: A systematic review and meta-analysis Front Oncol 10 171 2020 10.3389/fonc.2020.00171 32185125 PMC7058661

4 Gomes MA Priolli DG Tralhão JG Botelho MF Hepatocellular carcinoma: Epidemiology, biology, diagnosis, and therapies Rev Assoc Med Bras (1992) 59 514 524 2013 In English, Portuguese 10.1016/j.ramb.2013.03.005 24041910

5 Center MM Jemal A International trends in liver cancer incidence rates Cancer Epidemiol Biomarkers Prev 20 2362 2368 2011 10.1158/1055-9965.EPI-11-0643 21921256

6 Farinati F Sergio A Baldan A Giacomin A Di Nolfo MA Del Poggio P Benvegnu L Rapaccini G Zoli M Borzio F Early and very early hepatocellular carcinoma: When and how much do staging and choice of treatment really matter? A multi-center study BMC Cancer 9 33 2009 10.1186/1471-2407-9-33 19171074 PMC2640412

7 Kakushadze Z Raghubanshi R Yu W Estimating cost savings from early cancer diagnosis Data 2 30 2017 10.3390/data2030030

8 Finn RS Emerging targeted strategies in advanced hepatocellular carcinoma Semin Liver Dis 33 Suppl 1 S11 S19 2013 10.1055/s-0033-1333632 23457035

9 WHO's International Agency for Research on Cancer (IARC) World Cancer Report 2014 Stewart BW Kleihues P IARC Press Lyon 2014

10 Dimitroulis D Damaskos C Valsami S Davakis S Garmpis N Spartalis E Athanasiou A Moris D Sakellariou S Kykalos S From diagnosis to treatment of hepatocellular carcinoma: An epidemic problem for both developed and developing world World J Gastroenterol 23 5282 5294 2017 10.3748/wjg.v23.i29.5282 28839428 PMC5550777

11 Arruebo M Vilaboa N Saez-Gutierrez B Lambea J Tres A Valladares M González-Fernández A Assessment of the evolution of cancer treatment therapies Cancers (Basel) 3 3279 3330 2011 10.3390/cancers3033279 24212956 PMC3759197

12 El-Serag HB Marrero JA Rudolph L Reddy KR Diagnosis and treatment of hepatocellular carcinoma Gastroenterology 134 1752 1763 2008 10.1053/j.gastro.2008.02.090 18471552

13 Llovet JM Ricci S Mazzaferro V Hilgard P Gane E Blanc JF de Oliveira AC Santoro A Raoul JL Forner A Sorafenib in advanced hepatocellular carcinoma N Engl J Med 359 378 390 2008 10.1056/NEJMoa0708857 18650514

14 Kane RC Farrell AT Madabushi R Booth B Chattopadhyay S Sridhara R Justice R Pazdur R Sorafenib for the treatment of unresectable hepatocellular carcinoma Oncologist 14 95 100 2009 10.1634/theoncologist.2008-0185 19144678

15 Liu L Cao Y Chen C Zhang X McNabola A Wilkie D Wilhelm S Lynch M Carter C Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5 Cancer Res 66 11851 11858 2006 10.1158/0008-5472.CAN-06-1377 17178882

16 Kudo M Finn RS Qin S Han KH Ikeda K Piscaglia F Baron A Park JW Han G Jassem J Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: A randomised phase 3 non-inferiority trial Lancet 391 1163 1173 2018 10.1016/S0140-6736(18)30207-1 29433850

17 Finn RS Qin S Ikeda M Galle PR Ducreux M Kim TY Kudo M Breder V Merle P Kaseb AO Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma N Engl J Med 382 1894 1905 2020 10.1056/NEJMoa1915745 32402160

18 Ikeda K Kudo M Kawazoe S Osaki Y Ikeda M Okusaka T Tamai T Suzuki T Hisai T Hayato S Phase 2 study of lenvatinib in patients with advanced hepatocellular carcinoma J Gastroenterol 52 512 519 2017 10.1007/s00535-016-1263-4 27704266 PMC5357473

19 Calin GA Croce CM MicroRNA signatures in human cancers Nat Rev Cancer 6 857 866 2006 10.1038/nrc1997 17060945

20 Meng F Henson R Wehbe-Janek H Ghoshal K Jacob ST Patel T MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer Gastroenterology 133 647 658 2007 10.1053/j.gastro.2007.05.022 17681183 PMC4285346

21 Jin C Wang A Liu L Wang G Li G Han Z miR-145-5p inhibits tumor occurrence and metastasis through the NF-κB signaling pathway by targeting TLR4 in malignant melanoma J Cell Biochem Jan 30 2019 Epub ahead of print 10.1002/jcb.28388 30701576

22 Su Z Yang Z Xu Y Chen Y Yu Q MicroRNAs in apoptosis, autophagy and necroptosis Oncotarget 6 8474 8490 2015 10.18632/oncotarget.3523 25893379 PMC4496162

23 Hydbring P Wang Y Fassl A Li X Matia V Otto T Choi YJ Sweeney KE Suski JM Yin H Cell-cycle-targeting MicroRNAs as therapeutic tools against refractory cancers Cancer Cell 31 576 590.e8 2017 10.1016/j.ccell.2017.03.004 28399412 PMC5425285

24 Nagy Á Lánczky A Menyhárt O Győrffy B Validation of miRNA prognostic power in hepatocellular carcinoma using expression data of independent datasets Sci Rep 8 9227 2018 10.1038/s41598-018-27521-y 29907753 PMC6003936

25 Wang L Zhao Y Xu M Zhou F Yan J Serum miR-1301-3p, miR-335-5p, miR-28-5p and their target B7-H3 may serve as novel biomarkers for colorectal cancer J BUON 24 1120 1127 2019 31424670

26 Du W Tang H Lei Z Zhu J Zeng Y Liu Z Huang JA miR-335-5p inhibits TGF-β1-induced epithelial-mesenchymal transition in non-small cell lung cancer via ROCK1 Respir Res 20 225 2019 10.1186/s12931-019-1184-x 31638991 PMC6805547

27 Xu X Tao Y Shan L Chen R Jiang H Qian Z Cai F Ma L Yu Y The role of MicroRNAs in hepatocellular carcinoma J Cancer 9 3557 3569 2018 10.7150/jca.26350 30310513 PMC6171016

28 Wang G Dong F Xu Z Sharma S Hu X Chen D Zhang L Zhang J Dong Q MicroRNA profile in HBV-induced infection and hepatocellular carcinoma BMC Cancer 17 805 2017 10.1186/s12885-017-3816-1 29191172 PMC5709924

29 Gougelet A Exosomal microRNAs as a potential therapeutic strategy in hepatocellular carcinoma World J Hepatol 10 785 789 2018 10.4254/wjh.v10.i11.785 30533178 PMC6280159

30 Ye L Wang F Wu H Yang H Yang Y Ma Y Xue A Zhu J Chen M Wang J Zhang QA Functions and targets of miR-335 in cancer Onco Targets Ther 14 3335 3349 2021 10.2147/OTT.S305098 34045870 PMC8144171

31 Scarola M Schoeftner S Schneider C Benetti R miR-335 directly targets Rb1 (pRb/p105) in a proximal connection to p53-dependent stress response Cancer Res 70 6925 6933 2010 10.1158/0008-5472.CAN-10-0141 20713524

32 Liu J Bian T Feng J Qian L Zhang J Jiang D Zhang Q Li X Liu Y Shi J miR-335 inhibited cell proliferation of lung cancer cells by target Tra2β Cancer Sci 109 289 296 2018 10.1111/cas.13452 29161765 PMC5797811

33 Tang H Zhu J Du W Liu S Zeng Y Ding Z Zhang Y Wang X Liu Z Huang J CPNE1 is a target of miR-335-5p and plays an important role in the pathogenesis of non-small cell lung cancer J Exp Clin Cancer Res 37 131 2018 10.1186/s13046-018-0811-6 29970127 PMC6029376

34 Li HW Liu J Circ_0009910 promotes proliferation and metastasis of hepatocellular carcinoma cells through miR-335-5p/ROCK1 axis Eur Rev Med Pharmacol Sci 24 1725 1735 2020 32141540 10.26355/eurrev_202002_20349

35 Liu H Li W Chen C Pei Y Long X MiR-335 acts as a potential tumor suppressor miRNA via downregulating ROCK1 expression in hepatocellular carcinoma Tumour Biol 36 6313 6319 2015 10.1007/s13277-015-3317-2 25804796

36 Chen K Zhang L LINC00339 regulates ROCK1 by miR-152 to promote cell proliferation and migration in hepatocellular carcinoma J Cell Biochem 120 14431 14443 2019 10.1002/jcb.28701 31081143

37 Chen C Wu CQ Zhang ZQ Yao DK Zhu L Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation Exp Cell Res 317 1714 1725 2011 10.1016/j.yexcr.2011.05.001 21586285

38 Yang X Song H Zi Z Kou J Chen S Dai Y Wang J Yuan L Gao K Circ_0005075 promotes hepatocellular carcinoma progression by suppression of microRNA-335 J Cell Physiol 234 21937 21946 2019 10.1002/jcp.28757 31054187

39 Pearson G Robinson F Beers Gibson T Xu BE Karandikar M Berman K Cobb MH Mitogen-activated protein (MAP) kinase pathways: Regulation and physiological functions Endocr Rev 22 153 183 2001 11294822 10.1210/edrv.22.2.0428

40 Zhang D Li X Yao Z Wei C Ning N Li J GABAergic signaling facilitates breast cancer metastasis by promoting ERK1/2-dependent phosphorylation Cancer Lett 348 100 108 2014 10.1016/j.canlet.2014.03.006 24657659

41 Ji YY Song Y Wang AN MiR-335-5p inhibits proliferation of Huh-7 liver cancer cells via targeting the Oct4/Akt pathway Eur Rev Med Pharmacol Sci 25 1853 1860 2021 33660795 10.26355/eurrev_202102_25080

42 Zhang BJ Gong HY Zheng F Liu DJ Liu HX Up-regulation of miR-335 predicts a favorable prognosis in esophageal squamous cell carcinoma Int J Clin Exp Pathol 7 6213 6218 2014 25337272 PMC4203243

43 Kim Y Kim H Park D Jeoung D miR-335 targets SIAH2 and confers sensitivity to anti-cancer drugs by increasing the expression of HDAC3 Mol Cells 38 562 572 2015 10.14348/molcells.2015.0051 25997740 PMC4469914

44 Cheng Y Shen P miR-335 acts as a tumor suppressor and enhances ionizing radiation-induced tumor regression by targeting ROCK1 Front Oncol 10 278 2020 10.3389/fonc.2020.00278 32219065 PMC7078682

45 Cui L Hu Y Bai B Zhang S Serum miR-335 level is associated with the treatment response to trans-arterial chemoembolization and prognosis in patients with hepatocellular carcinoma Cell Physiol Biochem 37 276 283 2015 10.1159/000430352 26305026

46 Chen S Xia X Long noncoding RNA NEAT1 suppresses sorafenib sensitivity of hepatocellular carcinoma cells via regulating miR-335-c-Met J Cell Physiol Apr 1 2019 Epub ahead of print 10.1002/jcp.27567 30937906

47 Dohi O Yasui K Gen Y Takada H Endo M Tsuji K Konishi C Yamada N Mitsuyoshi H Yagi N Epigenetic silencing of miR-335 and its host gene MEST in hepatocellular carcinoma Int J Oncol 42 411 418 2013 10.3892/ijo.2012.1724 23229728 PMC3583616

48 Shang X Li G Liu H Li T Liu J Zhao Q Wang C Comprehensive circular RNA profiling reveals that hsa_ circ_0005075, a new circular RNA biomarker, is involved in hepatocellular crcinoma development Medicine (Baltimore) 95 e3811 2016 10.1097/MD.0000000000003811 27258521 PMC4900729

49 Nie Y Zhu X Bu N Jiang Y Su Y Pan K Li S Circ_0064288 acts as an oncogene of hepatocellular carcinoma cells by inhibiting miR-335-5p expression and promoting ROCK1 expression BMC Cancer 22 265 2022 10.1186/s12885-022-09323-8 35287604 PMC8919637

50 Ashkenazi A Dixit VM Apoptosis control by death and decoy receptors Curr Opin Cell Biol 11 255 260 1999 10.1016/S0955-0674(99)80034-9 10209153

51 Wajant H Molecular mode of action of TRAIL receptor agonists-common principles and their translational exploitation Cancers (Basel) 11 954 2019 10.3390/cancers11070954 31284696 PMC6678900

52 Amarante-Mendes GP Griffith TS Therapeutic applications of TRAIL receptor agonists in cancer and beyond Pharmacol Ther 155 117 131 2015 10.1016/j.pharmthera.2015.09.001 26343199 PMC4955572

53 Willms A Schittek H Rahn S Sosna J Mert U Adam D Trauzold A Impact of p53 status on TRAIL-mediated apoptotic and non-apoptotic signaling in cancer cells PLoS One 14 e0214847 2019 10.1371/journal.pone.0214847 30947287 PMC6448923

54 Micheau O Shirley S Dufour F Death receptors as targets in cancer Br J Pharmacol 169 1723 1744 2013 10.1111/bph.12238 23638798 PMC3753832

55 Lim B Allen JE Prabhu VV Talekar MK Finnberg NK El-Deiry WS Targeting TRAIL in the treatment of cancer: New developments Expert Opin Ther Targets 19 1171 1185 2015 10.1517/14728222.2015.1049838 26004811

56 Graves JD Kordich JJ Huang TH Piasecki J Bush TL Sullivan T Foltz IN Chang W Douangpanya H Dang T Apo2L/TRAIL and the death receptor 5 agonist antibody AMG 655 cooperate to promote receptor clustering and antitumor activity Cancer Cell 26 177 189 2014 10.1016/j.ccr.2014.04.028 25043603

57 Walczak H Miller RE Ariail K Gliniak B Griffith TS Kubin M Chin W Jones J Woodward A Le T Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo Nat Med 5 157 163 1999 10.1038/5517 9930862

58 Zamai L Ahmad M Bennett IM Azzoni L Alnemri ES Perussia B Natural killer (NK) cell-mediated cytotoxicity: Differential use of TRAIL and Fas ligand by immature and mature primary human NK cells J Exp Med 188 2375 2380 1998 10.1084/jem.188.12.2375 9858524 PMC2212426

59 Huang Y Yang X Xu T Kong Q Zhang Y Shen Y Wei Y Wang G Chang KJ Overcoming resistance to TRAIL-induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs Int J Oncol 49 153 163 2016 10.3892/ijo.2016.3525 27210546 PMC4902065

60 Ashkenazi A Pai RC Fong S Leung S Lawrence DA Marsters SA Blackie C Chang L McMurtrey AE Hebert A Safety and antitumor activity of recombinant soluble Apo2 ligand J Clin Invest 104 155 162 1999 10.1172/JCI6926 10411544 PMC408479

61 Galal El-Shemi A Mohammed Ashshi A Oh E Jung BK Basalamah M Alsaegh A Yun CO Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: First evidence in preclinical hepatocellular carcinoma Gene Ther 25 54 65 2018 10.1038/gt.2017.86 28925992 PMC5817393

62 Herbst RS Eckhardt SG Kurzrock R Ebbinghaus S O'Dwyer PJ Gordon MS Novotny W Goldwasser MA Tohnya TM Lum BL Phase I dose-escalation study of recombinant human Apo2L/TRAIL, a dual proapoptotic receptor agonist, in patients with advanced cancer J Clin Oncol 28 2839 2846 2010 10.1200/JCO.2009.25.1991 20458040

63 Wiley SR Schooley K Smolak PJ Din WS Huang CP Nicholl JK Sutherland GR Smith TD Rauch C Smith CA Identification and characterization of a new member of the TNF family that induces apoptosis Immunity 3 673 682 1995 10.1016/1074-7613(95)90057-8 8777713

64 Pitti RM Marsters SA Ruppert S Donahue CJ Moore A Ashkenazi A Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family J Biol Chem 271 12687 12690 1996 10.1074/jbc.271.22.12687 8663110

65 Liu CH Chern GJ Hsu FF Huang KW Sung YC Huang HC Qiu JT Wang SK Lin CC Wu CH A multifunctional nanocarrier for efficient TRAIL-based gene therapy against hepatocellular carcinoma with desmoplasia in mice Hepatology 67 899 913 2018 10.1002/hep.29513 28885731

66 Kim CY Jeong M Mushiake H Kim BM Kim WB Ko JP Kim MH Kim M Kim TH Robbins PD Cancer gene therapy using a novel secretable trimeric TRAIL Gene Ther 13 330 338 2006 10.1038/sj.gt.3302658 16195699

67 Grisendi G Bussolari R Cafarelli L Petak I Rasini V Veronesi E De Santis G Spano C Tagliazzucchi M Barti-Juhasz H Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy Cancer Res 70 3718 3729 2010 10.1158/0008-5472.CAN-09-1865 20388793

68 Liu S Qiu J He G He W Liu C Cai D Pan H TRAIL promotes hepatocellular carcinoma apoptosis and inhibits proliferation and migration via interacting with IER3 Cancer Cell Int 21 63 2021 10.1186/s12935-020-01724-8 33472635 PMC7816514

69 Lemke J von Karstedt S Zinngrebe J Walczak H Getting TRAIL back on track for cancer therapy Cell Death Differ 21 1350 1364 2014 10.1038/cdd.2014.81 24948009 PMC4131183

70 Holland PM Death receptor agonist therapies for cancer, which is the right TRAIL? Cytokine Growth Factor Rev 25 185 193 2014 10.1016/j.cytogfr.2013.12.009 24418173

71 Zhang L Gu J Lin T Huang X Roth JA Fang B Mechanisms involved in development of resistance to adenovirus-mediated proapoptotic gene therapy in DLD1 human colon cancer cell line Gene Ther 9 1262 1270 2002 10.1038/sj.gt.3301797 12215894

72 Hinz S Trauzold A Boenicke L Sandberg C Beckmann S Bayer E Walczak H Kalthoff H Ungefroren H Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis Oncogene 19 5477 5486 2000 10.1038/sj.onc.1203936 11114725

73 Eggert A Grotzer MA Zuzak TJ Wiewrodt BR Ho R Ikegaki N Brodeur GM Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression Cancer Res 61 1314 1319 2001 11245427

74 Marini P Denzinger S Schiller D Kauder S Welz S Humphreys R Daniel PT Jendrossek V Budach W Belka C Combined treatment of colorectal tumours with agonistic TRAIL receptor antibodies HGS-ETR1 and HGS-ETR2 and radiotherapy: Enhanced effects in vitro and dose-dependent growth delay in vivo Oncogene 25 5145 5154 2006 10.1038/sj.onc.1209516 16636678

75 Pukac L Kanakaraj P Humphreys R Alderson R Bloom M Sung C Riccobene T Johnson R Fiscella M Mahoney A HGS-ETR1, a fully human TRAIL-receptor 1 monoclonal antibody, induces cell death in multiple tumour types in vitro and in vivo Br J Cancer 92 1430 1441 2005 10.1038/sj.bjc.6602487 15846298 PMC2361994

76 Kelley SK Harris LA Xie D Deforge L Totpal K Bussiere J Fox JA Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efcacy, pharmacokinetics, and safety J Pharmacol Exp Ther 299 31 38 2001 11561060

77 Hymowitz SG O'Connell MP Ultsch MH Hurst A Totpal K Ashkenazi A de Vos AM Kelley RF A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL Biochemistry 39 633 640 2000 10.1021/bi992242l 10651627

78 Mérino D Lalaoui N Morizot A Solary E Micheau O TRAIL in cancer therapy: Present and future challenges Expert Opin Ther Targets 11 1299 1314 2007 10.1517/14728222.11.10.1299 17907960 PMC2976473

79 Lawrence D Shahrokh Z Marsters S Achilles K Shih D Mounho B Hillan K Totpal K DeForge L Schow P Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions Nat Med 7 383 385 2001 10.1038/86397 11283636

80 Ashley DM Riffkin CD Lovric MM Mikeska T Dobrovic A Maxwell JA Friedman HS Drummond KJ Kaye AH Gan HK In vitro sensitivity testing of minimally passaged and uncultured gliomas with TRAIL and/or chemotherapy drugs Br J Cancer 99 294 304 2008 10.1038/sj.bjc.6604459 18594532 PMC2480982

81 Bae S Ma K Kim TH Lee ES Oh KT Park ES Lee KC Youn YS Doxorubicin-loaded human serum albumin nanoparticles surface-modified with TNF-related apoptosis-inducing ligand and transferrin for targeting multiple tumor types Biomaterials 33 1536 1546 2012 10.1016/j.biomaterials.2011.10.050 22118776

82 Mitchell MJ Wayne E Rana K Schaffer CB King MR TRAIL-coated leukocytes that kill cancer cells in the circulation Proc Natl Acad Sci USA 111 930 935 2014 10.1073/pnas.1316312111 24395803 PMC3903223

83 Naval J de Miguel D Gallego-Lleyda A Anel A Martinez-Lostao L Importance of TRAIL molecular anatomy in receptor oligomerization and signaling Implications for cancer therapy Cancers (Basel) 11 444 2019 10.3390/cancers11040444 30934872 PMC6521207

84 Griffith T Wiley SR Kubin MZ Sedger LM Maliszewski CR Fanger NA Monocyte-mediated tumoricidal activity via the tumor necrosis factor-related cytokine, TRAIL J Exp Med 189 1343 1354 1999 10.1084/jem.189.8.1343 10209050 PMC2193036

85 Kayagaki N Yamaguchi N Nakayama M Kawasaki A Akiba H Okumura K Yagita H Involvement of TNF-related apoptosis-inducing ligand in human CD4+ T cell-mediated cytotoxicity J Immunol 162 2639 2647 1999 10072506

86 Monleón I Martínez-Lorenzo MJ Anel A Lasierra P Larrad L Pineiro A Naval J Alava MA CD59 cross-linking induces secretion of APO2 ligand in overactivated human T cells Eur J Immunol 30 1078 1087 2000 10.1002/(SICI)1521-4141(200004)30:4<1078::AID-IMMU1078>3.0.CO;2-Q 10760796

87 Monleón I Martinez-Lorenzo MJ Monteagudo L Lasierra P Taulés M Iturralde M Piñeiro A Larrad L Alava MA Naval J Anel A Differential secretion of Fas ligand- or APO2 ligand/TNF-related apoptosis-inducing ligand-carrying microvesicles during activation-induced death of human T cells J Immunol 167 6736 6744 2001 10.4049/jimmunol.167.12.6736 11739488

88 Wajant H Moosmayer D Wüest T Bartke T Gerlach E Schönherr U Peters N Scheurich P Pfizenmaier K Differential activation of TRAIL-R1 and -2 by soluble and membrane TRAIL allows selective surface antigen-directed activation of TRAIL-R2 by a soluble TRAIL derivative Oncogene 20 4101 4106 2001 10.1038/sj.onc.1204558 11494138

89 De Miguel D Basáñez G Sánchez D Malo PG Marzo I Larrad L Naval J Pardo J Anel A Martinez-Lostao L Liposomes decorated with Apo2L/TRAIL overcome chemoresistance of human hematologic tumor cells Mol Pharm 10 893 904 2013 10.1021/mp300258c 23331277

90 De Miguel D Gallego-Lleyda A Anel A Martinez-Lostao L Liposome-bound TRAIL induces superior DR5 clustering and enhanced DISC recruitment in histiocytic lymphoma U937 cells Leuk Res 39 657 666 2015 10.1016/j.leukres.2015.03.019 25882551

91 De Miguel D Gallego-Lleyda A Ayuso JM Erviti-Ardanaz S Pazo-Cid R del Agua C Fernández LJ Ochoa I Anel A Martinez-Lostao L TRAIL-coated lipid-nanoparticles overcome resistance to soluble recombinant TRAIL in non-small cell lung cancer cells Nanotechnology 27 185101 2016 10.1088/0957-4484/27/18/185101 27001952

92 De Miguel D Gallego-Lleyda A Ayuso JM Pejenaute-Ochoa D Jarauta V Marzo I Fernández LJ Ochoa I Conde B Anel A Martinez-Lostao L High-order TRAIL oligomer formation in TRAIL-coated lipid nanoparticles enhances DR5 cross-linking and increases antitumour effect against colon cancer Cancer Lett 383 250 260 2016 10.1016/j.canlet.2016.10.005 27725224

93 Kim TH Jiang HH Park CW Youn YS Lee S Chen X Lee KC PEGylated TNF-related apoptosis-inducing ligand (TRAIL)-loaded sustained release PLGA microspheres for enhanced stability and antitumor activity J Control Release 150 63 69 2011 10.1016/j.jconrel.2010.10.037 21062635

94 Wang S El-Deiry WS TRAIL and apoptosis induction by TNF-family death receptors Oncogene 22 8628 8633 2003 10.1038/sj.onc.1207232 14634624

95 Chinnappan M Srivastava A Amreddy N Razaq M Pareek V Ahmed R Mehta M Peterson JE Munshi A Ramesh R Exosomes as drug delivery vehicle and contributor of resistance to anticancer drugs Cancer Lett 486 18 28 2020 10.1016/j.canlet.2020.05.004 32439419 PMC7327711

96 Wang J Yeung BZ Cui M Peer CJ Lu Z Figg WD Guillaume Wientjes M Woo S Au JL Exosome is a mechanism of inter-cellular drug transfer: Application of quantitative pharmacology J Control Release 268 147 158 2017 10.1016/j.jconrel.2017.10.020 29054369 PMC5722714

97 Lee Y El Andaloussi S Wood MJ Exosomes and microvesicles: Extracellular vesicles for genetic information transfer and gene therapy Hum Mol Genet 21 R125 R134 2012 10.1093/hmg/dds317 22872698

98 Lee JK Park SR Jung BK Jeon YK Lee YS Kim MK Kim YG Jang JY Kim CW Exosomes derived from mesenchymal stem cells suppress angiogenesis by down-regulating VEGF expression in breast cancer cells PLoS One 8 e84256 2013 10.1371/journal.pone.0084256 24391924 PMC3877259

99 Syn NL Wang L Chow EK Lim CT Goh BC Exosomes in cancer nanomedicine and immunotherapy: Prospects and challenges Trends Biotechnol 35 665 676 2017 10.1016/j.tibtech.2017.03.004 28365132

100 Ha D Yang N Nadithe V Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: Current perspectives and future challenges Acta Pharm Sin B 6 287 296 2016 10.1016/j.apsb.2016.02.001 27471669 PMC4951582

101 Farooqi AA Desai NN Qureshi MZ Librelotto DRN Gasparri ML Bishayee A Nabavi SM Curti V Daglia M Exosome biogenesis, bioactivities and functions as new delivery systems of natural compounds Biotechnol Adv 36 328 334 2018 10.1016/j.biotechadv.2017.12.010 29248680

102 Maia J Caja S Strano Moraes MC Couto N Costa-Silva B Exosome-based cell-cell communication in the tumor microenvironment Front Cell Dev Biol 6 18 2018 10.3389/fcell.2018.00018 29515996 PMC5826063

103 Yeo RW Lai RC Zhang B Tan SS Yin Y The BJ Lim SK Mesenchymal stem cell: An efficient mass producer of exosomes for drug delivery Adv Drug Deliv Rev 65 336 341 2013 10.1016/j.addr.2012.07.001 22780955

104 Lou G Chen Z Zheng M Liu Y Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases Exp Mol Med 49 e346 2017 10.1038/emm.2017.63 28620221 PMC5519012

105 Cho BS Kim JO Ha DH Yi YW Exosomes derived from human adipose tissue-derived mesenchymal stem cells alleviate atopic dermatitis Stem Cell Res Ther 9 187 2018 10.1186/s13287-018-0939-5 29996938 PMC6042362

106 El-Andaloussi S Lee Y Lakhal-Littleton S Li J Seow Y Gardiner C Alvarez-Erviti L Sargent IL Wood MJ Exosome-mediated delivery of siRNA in vitro and in vivo Nat Protoc 7 2112 2126 2012 10.1038/nprot.2012.131 23154783

107 Sokolova V Ludwig AK Hornung S Rotan O Horn PA Epple M Giebel B Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy Colloids Surf B Biointerfaces 87 146 150 2011 10.1016/j.colsurfb.2011.05.013 21640565

108 Kalra H Adda CG Liem M Ang CS Mechler A Simpson RJ Hulett MD Mathivanan S Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma Proteomics 13 3354 3364 2013 10.1002/pmic.201300282 24115447

109 Rani S Ryan AE Griffin MD Ritter T Mesenchymal stem cell-derived extracellular vesicles: Toward cell-free therapeutic applications Mol Ther 23 812 823 2015 10.1038/mt.2015.44 25868399 PMC4427881

110 Sun L Xu R Sun X Duan Y Han Y Zhao Y Qian H Zhu W Xu W Safety evaluation of exosomes derived from human umbilical cord mesenchymal stromal cell Cytotherapy 18 413 422 2016 10.1016/j.jcyt.2015.11.018 26857231

111 Alvarez-Erviti L Seow Y Yin H Betts C Lakhal S Wood MJ Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes Nat Biotechnol 29 341 345 2011 10.1038/nbt.1807 21423189

112 El Andaloussi S Lakhal S Mäger I Wood MJ Exosomes for targeted siRNA delivery across biological barriers Adv Drug Deliv Rev 65 391 397 2013 10.1016/j.addr.2012.08.008 22921840

113 Escudier B Dorval T Chaput N André F Caby MP Novault S Flament C Leboulaire C Borg C Amigorena S Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: Results of thefirst phase I clinical trial J Transl Med 3 10 2005 10.1186/1479-5876-3-10 15740633 PMC554765

114 Morse MA Garst J Osada T Khan S Hobeika A Clay TM Valente N Shreeniwas R Sutton MA Delcayre A A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer J Transl Med 3 9 2005 10.1186/1479-5876-3-9 15723705 PMC551593

115 Dai S Wei D Wu Z Zhou X Wei X Huang H Li G Phase I clinical trial of autologous ascites-derived exosomes combined with GM-CSF for colorectal cancer Mol Ther 16 782 790 2008 10.1038/mt.2008.1 18362931 PMC7106337

116 Iranifar E Seresht BM Momeni F Fadaei E Mehr MH Ebrahimi Z Rahmati M Kharazinejad E Mirzaei H Exosomes and microRNAs: New potential therapeutic candidates in Alzheimer disease therapy J Cell Physiol 234 2296 2305 2019 10.1002/jcp.27214 30191975

117 Qu M Lin Q Huang L Fu Y Wang L He S Fu Y Yang S Zhang Z Zhang L Sun X Dopamine-loaded blood exosomes targeted to brain for better treatment of Parkinson's disease J Control Release 287 156 166 2018 10.1016/j.jconrel.2018.08.035 30165139

118 Wang H Sui H Zheng Y Jiang Y Shi Y Liang J Zhao L Curcumin-primed exosomes potently ameliorate cognitive function in AD mice by inhibiting hyperphosphorylation of the Tau protein through the AKT/GSK-3β pathway Nanoscale 11 7481 7496 2019 10.1039/C9NR01255A 30942233

119 Kim MS Haney MJ Zhao Y Yuan D Deygen I Klyachko NL Kabanov AV Batrakova EV Engineering macro-phage-derived exosomes for targeted paclitaxel delivery to pulmonary metastases: In vitro and in vivo evaluations Nanomedicine 14 195 204 2018 10.1016/j.nano.2017.09.011 28982587

120 Azmi AS Bao B Sarkar FH Exosomes in cancer development, metastasis, and drug resistance: A comprehensive review Cancer Metastasis Rev 32 623 642 2013 10.1007/s10555-013-9441-9 23709120 PMC3843988

121 Mendt M Kamerkar S Sugimoto H McAndrews KM Wu CC Gagea M Yang S Blanko EVR Peng Q Ma X Generation and testing of clinical-grade exosomes for pancreatic cancer JCI Insight 3 e99263 2018 10.1172/jci.insight.99263 29669940 PMC5931131

122 Wang Y Balaji V Kaniyappan S Krüger L Irsen S Tepper K Chandupatla R Maetzler W Schneider A Mandelkow E Mandelkow EM The release and trans-synaptic transmission of Tau via exosomes Mol Neurodegener 12 5 2017 10.1186/s13024-016-0143-y 28086931 PMC5237256

123 Jiang XC Gao JQ Exosomes as novel bio-carriers for gene and drug delivery Int J Pharm 521 167 175 2017 10.1016/j.ijpharm.2017.02.038 28216464

124 Hadla M Palazzolo S Corona G Caligiuri I Canzonieri V Toffoli G Rizzolio F Exosomes increase the therapeutic index of doxorubicin in breast and ovarian cancer mouse models Nanomedicine (Lond) 11 2431 2441 2016 10.2217/nnm-2016-0154 27558906

125 Smyth TJ Redzic JS Graner MW Anchordoquy TJ Examination of the specificity of tumor cell derived exosomes with tumor cells in vitro Biochim Biophys Acta 1838 2954 2965 2014 10.1016/j.bbamem.2014.07.026 25102470 PMC5657189

126 Kim MS Haney MJ Zhao Y Mahajan V Deygen I Klyachko NL Inskoe E Piroyan A Sokolsky M Okolie O Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells Nanomedicine 12 655 664 2016 10.1016/j.nano.2015.10.012 26586551 PMC4809755

127 Wilhelm S Tavares AJ Dai Q Ohta S Audet J Dvorak HF Chan WCW Analysis of nanoparticle delivery to tumours Nat Rev Mater 1 16014 2016 10.1038/natrevmats.2016.14

128 Faivre S Rimassa L Finn RS Molecular therapies for HCC: Looking outside the box J Hepatol 72 342 352 2020 10.1016/j.jhep.2019.09.010 31954496

129 Trivedi R Mishra DP Trailing TRAIL resistance: Novel targets for TRAIL sensitization in cancer cells Front Oncol 5 69 2015 10.3389/fonc.2015.00069 25883904 PMC4382980

130 Sterzenbach U Putz U Low LH Silke J Tan SS Howitt J Engineered exosomes as vehicles for biologically active proteins Mol Ther 25 1269 1278 2017 10.1016/j.ymthe.2017.03.030 28412169 PMC5474961

131 Rivoltini L Chiodoni C Squarcina P Tortoreto M Villa A Vergani B Bürdek M Botti L Arioli I Cova A TNF-related apoptosis-inducing ligand (TRAIL)-armed exosomes deliver proapoptotic signals to tumor site Clin Cancer Res 22 3499 3512 2016 10.1158/1078-0432.CCR-15-2170 26944067

132 Yuan Z Kolluri KK Gowers KH Janes SM TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy J Extracell Vesicles 6 1265291 2017 10.1080/20013078.2017.1265291 28326166 PMC5328331

133 Shamili FH Bayegi HR Salmasi Z Sadri K Mahmoudi M Kalantari M Ramezani M Abnous K Exosomes derived from TRAIL-engineered mesenchymal stem cells with effective anti-tumor activity in a mouse melanoma model Int J Pharm 549 218 229 2018 10.1016/j.ijpharm.2018.07.067 30075248

134 Nojiri K Sugimoto K Shiraki K Tameda M Inagaki Y Ogura S Kasai C Kusagawa S Yoneda M Yamamoto N Sorafenib and TRAIL have synergistic effect on hepatocellular carcinoma Int J Oncol 42 101 108 2013 10.3892/ijo.2012.1676 23123700

135 Chen KF Tai WT Liu TH Huang HP Lin YC Shiau CW Li PK Chen PJ Cheng AL Sorafenib overcomes TRAIL resistance of hepatocellular carcinoma cells through the inhibition of STAT3 Clin Cancer Res 16 5189 5199 2010 10.1158/1078-0432.CCR-09-3389 20884624

136 Munir J Yoon JK Ryu S Therapeutic miRNA-enriched extracellular vesicles: Current approaches and future prospects Cells 9 2271 2020 10.3390/cells9102271 33050562 PMC7601381

137 Almanza G Rodvold JJ Tsui B Jepsen K Carter H Zanetti M Extracellular vesicles produced in B cells deliver tumor suppressor miR-335 to breast cancer cells disrupting oncogenic programming in vitro and in vivo Sci Rep 8 17581 2018 10.1038/s41598-018-35968-2 30514916 PMC6279829

138 Li L Piontek K Ishida M Fausther M Dranoff JA Fu R Mezey E Gould SJ Fordjour FK Meltzer SJ Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model Hepatology 65 501 514 2017 10.1002/hep.28735 27474881 PMC5258762

139 Wang F Li L Piontek K Sakaguchi M Selaru FM Exosome miR-335 as a novel therapeutic strategy in hepatocellular carcinoma Hepatology 67 940 954 2018 10.1002/hep.29586 29023935 PMC5826829

140 Andriolo G Provasi E Lo Cicero V Brambilla A Soncin S Torre T Milano G Biemmi V Vassalli G Turchetto L Exosomes from human cardiac progenitor cells for therapeutic applications: Development of a GMP-grade manufacturing method Front Physiol 9 1169 2018 10.3389/fphys.2018.01169 30197601 PMC6117231

141 Yoo KH Thapa N Kim BJ Lee JO Jang YN Chwae YJ Kim J Possibility of exosome-based coronavirus disease 2019 vaccine (review) Mol Med Rep 25 26 2022 10.3892/mmr.2021.12542 34821373 PMC8630821

142 Cooke JN Ellis JA Hossain S Nguyen J Bruce JN Joshi S Computational pharmacokinetic rationale for intra-arterial delivery to the brain Drug Deliv Transl Res 6 622 629 2016 10.1007/s13346-016-0319-6 27431401

143 Ciuleanu T Bazin I Lungulescu D Miron L Bondarenko I Deptala A Rodriguez-Torres M Giantonio B Fox NL Wissel P A randomized, double-blind, placebo-controlled phase II study to assess the efficacy and safety of mapatumumab with sorafenib in patients with advanced hepatocellular carcinoma Ann Oncol 27 680 687 2016 10.1093/annonc/mdw004 26802147

144 von Pawel J Harvey JH Spigel DR Dediu M Reck M Cebotaru CL Humphreys RC Gribbin MJ Fox NL Camidge DR Phase II trial of mapatumumab, a fully human agonist monoclonal anti-body to tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1), in combination with paclitaxel and carboplatin in patients with advanced non-small-cell lung cancer Clin Lung Cancer 15 188 196.e2 2014 10.1016/j.cllc.2013.12.005 24560012

145 Davies A Sage B Kolluri K Alrifai D Graham R Weil B Rego R Bain O Patrick PS Champion K TACTICAL: A phase I/II trial to assess the safety and efficacy of MSCTRAIL in the treatment of metastatic lung adenocarcinoma J Clin Oncol 37 TPS9116 2019 10.1200/JCO.2019.37.15_suppl.TPS9116

146 Forero A Bendell JC Kumar P Janisch L Rosen M Wang Q Copigneaux C Desai M Senaldi G Maitland ML First-in-human study of the antibody DR5 agonist DS-8273a in patients with advanced solid tumors Invest New Drugs 35 298 306 2017 10.1007/s10637-016-0420-1 28050790

147 Xie Y Bai O Zhang H Yuan J Zong S Chibbar R Slattery K Qureshi M Wei Y Deng Y Xiang J Membrane-bound HSP70-engineered myeloma cell-derived exosomes stimulate more efficient CD8(+) CTL- and NK-mediated antitumour immunity than exosomes released from heat-shocked tumour cells expressing cytoplasmic HSP70 J Cell Mol Med 14 2655 2666 2010 10.1111/j.1582-4934.2009.00851.x 19627400 PMC4373481

148 Ke C Hou H Li J Su K Huang C Lin Y Lu Z Du Z Tan W Yuan Z Extracellular vesicle delivery of TRAIL eradicates resistant tumor growth in combination with CDK inhibition by dinaciclib Cancers (Basel) 12 1157 2020 10.3390/cancers12051157 32375399 PMC7281120

149 Yuan Q Su K Li S Long X Liu L Yang M Yuan X Sun J Hu J Li Q Pulmonary delivery of extracellular vesicle-encapsulated dinaciclib as an effective lung cancer therapy Cancers (Basel) 14 3550 2022 10.3390/cancers14143550 35884614 PMC9318050

150 Jiang L Gu Y Du Y Tang X Wu X Liu J Engineering exosomes endowed with targeted delivery of triptolide for malignant melanoma therapy ACS Appl Mater Interfaces 13 42411 42428 2021 10.1021/acsami.1c10325 34464081