The Role of Exosomes in the Female Reproductive System and Breast Cancers.

Exosomes have been used to deliver biomolecules and chemotherapeutic drugs for tumor treatment. Han et al successfully isolated exogenous hormones derived from natural killer cells (NK-Exos) by ultra-high speed centrifugation, and prepared a PTX-NK-Exos drug delivery system using NK-Exos as castor oil-based PTX carrier by electroporation. It was found that PTX-NK-Exos played an anti-tumor role in BC cells by inducing the upregulation of Bax and Caspase-3 in the apoptosis signaling pathway of tumor cells. This finding indicated that exosomes loaded with drugs could effectively inhibit the proliferation and induce tumor cell apoptosis, thus playing an anti-tumor role in BC cells. 120 Exosomes are the perfect choice for gene targeting on account of their innate non-toxic, non-immunogenic, 121 biodegradable and targetable characteristics. Limoni et al adopted the Her2 -specific anchor protein repeat protein (DARPins) 122 developed by Plueckthun to produce Her2 -targeted exosomes, which were used to deliver siRNA to Her2 -overexpressing BC cells. Studies have shown that targeted exosomes can be successfully loaded with high levels of siRNA and used in Her2 -positive BC gene therapy. This approach offers a variety of options for gene therapy and drug delivery. 123 Exosomes derived from human mesenchymal stroma/stem-like cells (MSCs) have shown significant biocompatibility and reduced innate immunogenicity, thus representing valuable vectors for drug delivery in oncology therapies. Catharina et al found that drug-loaded MSC-derived exosomes showed excellent in vitro cytotoxicity by effectively targeting primary and metastatic tumors and reducing side-effects in BC cell populations and in vivo, offering promising treatment prospects for BC. 124 Kobayashi et al demonstrated that therapy with miR199a-3p-loaded-exosomes (miR-199a-3p-Exo) greatly enhanced miR199a-3p expression in OC cells, and that miR-199a-3p-Exo suppressed the expression of C-Met , the specific target of miR199a-3p, hence inhibiting cell propagation and aggression. These results suggested that exosomes derived from OC patients could be used as a novel drug delivery system (DDS) for prospective targeted molecular therapies. 125 Liu et al constructed a triptolide-loaded exosome delivery system (TP-Exos) and observed its effect on the propagation and apoptosis of OC cells in vitro and vivo. The results showed that TP-Exos have the normal features of exosomes, as well as exhibiting advanced drug encapsulation efficiency, suggesting that TP-Exos may be a promising treatment strategy for OC. 126 Kim et al discovered that tumor cell-derived exosomes act as natural vectors for effective delivery of the CRISPR/Cas9 plasmid to cancer cells. Exosomes loaded with CRISPR/Cas9 inhibited the expression of poly (ADP-ribose) polymerase-1 ( PARP-1 ), leading to the induction of OC cell apoptosis. These results suggested the promise of tumor-derived exosomes for drug delivery in tumor therapy. 127 Zhang et al studied the clinical significance and biological function of miR-320a encapsulated in EVs released by CAFs in EC. The results showed that the miR-320a encapsulated in exosomes secreted by CAF was directly transferred to EC cells, thereby inhibiting their proliferation. This effect which was achieved by miR-320a-induced downregulation of HIF1α , leading to decreased expression of VEGFA in vitro. These results suggested that CAF-derived EVs overexpressing miR-320a offer a new direction for EC treatment strategies. 128 These studies suggest that tumor-derived exosomes can facilitate drug delivery, which could have broad prospects for the therapy of the female reproductive system cancers and BC.

Cancers of the female reproductive system, including cervical cancer (CC), ovarian cancer (OC) and endometrial cancer (EC), as well as breast cancer (BC) are significant causes of death among women, and their incidence continues to increase. Despite advances in chemotherapy, radiation therapy and surgery in recent years, there is still a lack of methods for early diagnosis and effective treatment. A better comprehension of the potential molecular mechanisms of carcinogenesis and developments in particular biological markers are therefore needed. The tumor environment is composed of tumor cells and non-stationary cells with diverse types of endocellular communication mechanisms. 1 In recent years, more and more research has been conducted on exosomes derived from diverse cancer and non-cancer cells. 2 Exosomes play a crucial role in establishing intercellular communication and maintaining tumor cell homeostasis. These vesicles shuttle miscellaneous biomolecules to target cells and are released from tumor cells. Therefore, analysis of tumor-derived exosomes may offer useful markers for precise monitoring of cancers. 3 Tumor-derived exosomes also mediate immune responses and drug resistance in tumors, and can transfer endogenous and exogenous compounds making them good candidates for the delivery of nanotech drugs. Here, we review the progress in our understanding of the use of exosomes for the diagnosis and treatment of female reproductive system cancers and BC.

Several clinical trials have been reported in recent years that further clarify the role of exosomes in the treatment of female reproductive system and breast cancers. Human mesenchymal stem cells (MSCs) have a well-established tumor homing capability, highlighting their potential as a delivery vehicle for targeting tumors. 138 Makiko et al co-cultured BC cells with bone marrow mesenchymal stem cells (BM-MSCs) isolated from human donors. An increase in miR-23b was observed in BM-MSC-derived exosomes, and the miR-23b overexpression induced a dormant phenotype by inhibiting the target gene MARCKS . These findings indicated that the transfer of miRNAs from the bone marrow-derived exosomes of BC patients may promote the dormancy of breast cancer cells in the metastatic niche to achieve the effective treatment of BC. 139 Senthilkumar et al isolated exosome simulators (EM) from BM-MSCs in breast cancer patients, mixed the cells with paclitaxel (PTX), and isolated PTX-loaded EM (PTX-MSC-EM), which were found to significantly inhibit the growth of BC. 140 O’Brien et al found that miR-379 expression was significantly reduced in lymph node metastasis compared with BC tumor tissues from the same patients. MSC-379 secreted by MSC exosomes encapsulates COX-2 as an effective tumor suppressor in BC, showing exciting potential for innovative therapies for metastatic BC. 138 Musa et al found that conditioned medium (CM) derived from human fat MSC (hAMSC) inhibited OC cells by blocking the cell cycle and activating mitochondria-mediated apoptosis signal transduction. Exosomes derived from hAMSC-CM induced apoptotic signals by upregulating different pro-apoptotic signaling molecules (such as BAX , CASP9 and CASP3 ) and downregulating the anti-apoptotic protein BCL2 . Moreover, exosomal miRNAs are important participants in the inhibitory effect of hAMSC-CM on OC cells. These results will provide new advances in the research and treatment of OC. 141 Li et al found that the presence of tumor-specific antigens on exosomes isolated from malignant ascites of ovarian cancer patients could be presented by DC from unrelated cord blood sources, thus inducing tumor-specific cytotoxicity, which may represent a new immunotherapy for OC. 142 Niko et al also isolated exosomes from the malignant ascites of patients with OC, and showed that the secretion of body in mononuclear precursor cells may also trigger in other immune cells dependence toll-like receptor ( TLR ) signaling pathway. This process reveals that ovarian cancer and inflammatory diseases are induced through an immunosuppressive mechanism, and that immune therapy of OC is crucial. 143 These studies suggest that exosomes isolated from MSCs and malignant ascites from cancer patients may have specific inhibitory effects on female reproductive system and breast cancers, and these results will provide new advances in the research and treatment of female reproductive system and breast cancers ( Table 4 ). Table 4 Overview of Clinical Trials of Using Exosomes to Treat Female Reproductive System and Breast Cancers Cancer Exosome Donor Cell Recipient Cell Pathway Function Reference Breast CA miR-23b mesenchymal stem cells(MSC) Breast CA cells inhibiting the target gene MARCKS therapy [ 139 ] Breast CA Exosome mimetics (EMs) MSC Breast CA cells As drug delivery vehicles Therapy [ 140 ] Breast CA miR-379 MSC Breast CA cells As drug delivery vehicles Therapy [ 138 ] Ovarian CA Exosome mimetics (EMs) Human fat MSC (hAMSC) Ovarian CA cells blocking cell cycle and activating mitochondria-mediated apoptosis signal transduction. Therapy [ 141 ] Ovarian CA Exosomal proteins Malignant ascites of ovarian cancer patients Ovarian CA cells Presenting tumor-specific antigens Therapy [ 142 ] Ovarian CA Exosomal proteins Malignant ascites of ovarian cancer patients Ovarian CA cells The knockdown of Toll-like receptor 2 ( TLR2 ) and TLR4 blocked NFκB and STAT3 activation Therapy [ 143 ] Overview of Clinical Trials of Using Exosomes to Treat Female Reproductive System and Breast Cancers

Exosome-mediated drug resistance is a crucial challenge in cancer therapy. Mounting evidence suggests that tumor-derived exosomes may transform the extracellular substrate by secreting or activating matrix metalloproteinases ( MMPs ). 106 Sadegh–Nejadi et al found that circulating exosomes in the plasma of obese women promoted the propagation, migration and aggression of BC cells as well as the activation of MMP2 and MMP9 . Circulating plasma exosomes in obese women may also lead to tamoxifen resistance of BC cells. 107 Han et al found that the lncRNA actin filament-associated protein 1 antisense RNA 1 ( AFAP1-AS1 ) is related to trastuzumab resistance. Receptor tyrosine-protein kinase erbB-2 ( ERBB2 ) is one of the most studied oncogenes and is considered a biomarker of BC. It was found that exosomal AFAP1-AS1 induced trastuzumab resistance by binding to AU binding factor 1 ( AUF1 ) and facilitating ERBB2 translation. Consequently, exosomal AFAP1-AS1 levels can be used to predict trastuzumab resistance and treatment efficacy in BC. 108 Pan et al suggested that miR-221-3p in drug-resistant BC cell-derived exosomes targets PIK3R1 via the PI3K / AKT both in vivo and vitro, thereby enhancing BC cells resistance to adriamycin (ADR). 109 Santos et al demonstrated miR-155 induction in exosomes isolated from cancer stem cells (CSCs) and drug-resistant cells, and also showed that exosomes in BC cells may mediate resistance and migration to sensitive cells in part through the migration of exosomes containing miR-155. This finding confirmed the importance of exosome-mediated miR-155 resistance in BC cells. 110 Dong et al demonstrated that exosomal regulated diversion of lncRNA- SNHG14 induced trastuzumab resistance in BC cells, and that exosome lncRNA- SNHG14 in human serum could be considered as an underlying diagnostic biological marker for BC to improve trastuzumab treatment efficacy. 111 Alharbi et al studied the role of platinum in heterogeneous populations of OC cells and their derived exosomes, and found that miR-21-3p, miR-21-5p and miR-891-5p were enriched in exosomes. These exosomal miRNAs could play a role of chemotherapy resistance in OC by upregulating detoxification metabolic pathways and DNA repair mechanisms. 112 Asare–Werehene et al demonstrated increased expression and secretion of plasma gelsolin (pGSN) in chemotherapy-resistant OC cells compared with the chemically sensitive counterparts. pGSN is secreted and carried by exosomes (ex-pGSN) to upregulate HIF1α -induced pGSN expression via an autocrine pathway in chemotherapy-resistant OC cells and induces cisplatin resistance in other chemosensitive OC cells. 113 Zhu et al found that exosomes derived from anoxic macrophagocytes strengthened the tumorigenic phenotype of EOC cells. Furthermore, under hypoxic conditions, macrophage-derived exosomes rich in miR-223 promoted chemoresistance of EOC cells via PTEN - PI3K / AKT . 114 Li et al observed that urothelial carcinoma-associated 1 ( UCA1 ) was upregulated in tissues and cell lines of cisplatin-resistant patients, and inhibition of UCA1 promoted miR-143 expression and regulated the expression of FOSL2 in OC to promote cisplatin resistance. 115 Cao et al demonstrated that DNMT1 counterparts were highly concentrated in exosomes of OC cells, and co-incubation with exosomes promoted endogenetic expression and made host cells resistant to cisplatin cytotoxicity. These results elucidated new mechanisms of cisplatin resistance in OC foreign secrete DNMT1 and indicated the potential of exosome inhibitors combined with cisplatin in drug-resistant patients. 116 Kanlikilicer et al found that miR-1246 expression in paclitaxel-tolerant OC exosomes was significantly higher than in the sensitive counterparts, while Cav1 gene was the specific target of miR-1246 and participated in the process of exosome transfer. Cav1 overexpression and anti-miR-1246 therapy markedly sensitized OC cells to paclitaxel. This research provided a novel therapy to overcome chemical resistance in OC patients with exosomal miR-1246. 117 Guo et al showed that CDKN1A was highly expressed in cisplatin sensitive OC cells, and exosomal miR-98-5p targeted CDKN1A to restrain CDKN1A expression. These results suggested that CAF-derived exosomes transferred the overexpressed miR-98-5p to facilitate cisplatin resistance in OC by downregulating CDKN1A . 118 Luo et al found that exosomal lncRNA HNF1A-AS1 was upregulated in DDP-resistant (HeLa/DDP) cells, and HNF1A-AS1 acted as a competitive endogenous RNA (ceRNA) of miR-34b, promoting the expression of TUFT1 and consequently promoting DDP resistance in CC cells. 119 These studies revealed a new mechanism underlying exosome-mediated tumor chemoresistance, and also demonstrated the potential of exosome inhibitors for the treatment of drug-resistant female reproductive system cancers and BC ( Figure 4 ). Figure 4 The proposed function of exosomes in the regulation of tumor chemotherapy resistance. The proposed function of exosomes in the regulation of tumor chemotherapy resistance.

Exosomes are the most widely studied of the three major subunits (exosomes, microvesicles, and apoptotic vesicles (ApoEVs) of extracellular vesicles (EVs) liberated from mammiferous cells. 4 , 5 In addition to these three primary hypotypes, other EVs include membrane particles, exosome-like particles, EVs derived from neutrophils, 6 EVs from the prostate, 7 , 8 migrasomes, 9 large liposomes 10 and others. 11 There are various names for exosomes in the literature: exosomes, oncosomes, dexosomes, exosome-like particles, and membrane blebs. Exosomes originate from the multivesicular body (MVB) and are cup-shaped when viewed under an electron microscope, with a diameter of 50–150 nm. 12 ApoEVs comprise nucleoprotein histones and DNA, with a diameter of 1000–5000 nm. 13 Exosomes are produced by all normal and pathological cells and coexist in all body fluids, including plasma, urine, saliva, amniotic fluid, ascites, and cerebrospinal fluid. 4 Each exosome carries the imprint of the contents of its parent cell, including nucleic acids, proteins, enzymes, lipids, cytokines, and other soluble factors, depending on the cell of origin, environmental conditions, stage of development, epigenetic variations, and biogenesis. 13 , 14 Messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and long non-coding RNA (lncRNA) 15 have also been identified in exosomes, providing insights into the epigenetic modifications of cells and changes in their biological activity and function. The biogenesis of exosomes differs from that of other EVs. 16 , 17 Studies of the maturation of sheep reticular cells in the 1980s revealed that exosomes were generated by intracellular budding ( Figure 1 ) through the plasmalemma to shape endocellular endosomes and achieve cell surface protein expression. 16 More invasion of endocellular endosomes results in the production of MVB, which contain vesicles with diameters ranging from 40–150nm. Subsequently, the MVB fuses with lysosomes to degrade inclusions, or release their inclusions into the extracellular space accompanied by serosomes, 17 a process known as exosome biogenesis. Figure 1 Biogenesis and content of exosomes. Notes: Internal germination of the plasmalemma results in the construction of primitive endosomes that bind panniculus proteins. The entrapment of the endosome and encapsulation of the selected cargo (such as nucleic acids and proteins) then leads to the production of MVBs through ESCRT-dependent or ESCRT-independent mechanisms. These MVBs then fuse with the plasmalemma, releasing exosomes to the environment outside the cell. Exosomes transfer the cargos (proteins, mRNAs, miRNAs, lncRNAs, circRNAs, and DNAs) to recipient cells through mechanisms that include a) direct fusion, b) surface protein binding, and c) endocytosis. Biogenesis and content of exosomes. The mechanisms of exosomal biogenesis are highly regulated via respective pathways, 18 including endosomal sorting complexes required for transport ESCRT-dependent and ESCRT-independent pathways. In the mechanism of ESCRT panniculus rupture, exosome biogenesis requires four multiprotein subcomplexes (ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III). Inchoate ESCRT composites (ESCRT-0, ESCRT-I, and ESCRT-III) discern the ubiquitinated cargo through their ubiquitin-bonding subgroups to form steady protein composites in the protoplasm. ESCRT-III is then assembled instantly on the nucleosome and undergoes vesicle division. 19 Recently, it has been shown that a number of assistant elements, such as ATPase, vacuolar protein sort-associated protein (VPS4) and ALG-2 interacting protein X (ALIX), are involved in the mechanism of ESCRT panniculus rupture. 19 By comparison, the liberation of exosomes via the ESCRT-independent pathway is mediated by lipids, like spinolamide, 20 spinol-1-phosphate, and Rab family proteins, including Rab27a and Rab27b. 21 Exosomes export many proteins that are promoters or inhibitors of tumors. 22 For example, the widespread presence of heat shock proteins, p53, phosphatase, and tension protein homologues in exosomes is closely associated with tumor development. 23–26 MiRNAs in exosomes account for the majority of circulating miRNAs and have been studied as biomarkers of different cancers. 27 These discoveries suggest that exosomes are vital in the development of tumors.

Our knowledge of exosomes has grown dramatically in recent years. Exosomes have been shown to be important modulators in tumor biology, and tumor-derived exosomes containing tumor-specific antigens and nucleic acids can be used as potential diagnostic and predictive biomarkers for noninvasive assessment. Exosomes are also used to identify patients who may develop metastatic disease, and the production of exosomes may provide new targets for cancer treatment. The use of exosomes as a carrier of cellular information is a promising strategy in the field of targeted drug delivery in the treatment of cancer, and improved functional exosome mimics have greatly improved drug acceptability in this DDS. However, more efficient and widespread use of exosomes is still problematic. For example, drug delivery systems require nucleic acid drugs to be effectively transfected into exosomes, and host cells suitable for exosome injection pose a challenge for future clinical applications. Appropriate cell selection can also determine the natural population of exosome surface proteins, which ensures ideal ligand-receptor interactions with the proposed target cell. Optimization of the producer-target cell combination is critical to the production of exosomes for therapeutic use. Despite these potential drawbacks and reservations, this area of research is highly dynamic and promises to provide novel approaches to the diagnosis and treatment of cancer patients. In addition, the accurate isolation, identification and high-throughput clinical application of EVs face great challenges. The further development of cancer exosome proteomics and the improvement of microfluidics technology to detect exosomes will improve their application in cancer diagnosis.

The current research on exosomes has reshaped our understanding of exosomes and provided new targets for cancer diagnosis and treatment. In this review, we describe the biogenesis of exosomes and the main mechanisms of exosome-mediated immunity, chemoresistance, and drug delivery. Regarding clinical applications, our review helps to understand the role of exosomes as biomarkers, nanotherapy in female reproductive system cancer and breast cancers, and clinical trials of the use of exosomes. However, it must be pointed out that the key components of exosomes have not been fully clarified, and there is still a long way to go to fully understand the role of exosomes in female reproductive system cancer and breast cancers. More extensive and in-depth research is still needed to fully understand the role of exosomes and to develop exosome-based clinical programs for the diagnosis, prognosis and treatment of female reproductive system cancer and breast cancers in the future.

In recent years, the application of exosomes in tumor treatment has been extensively studied, and the nanotherapy of exosomes has better research value. Exosomes are endogenous nanoparticles secreted by a variety of cells and have been explored as drug delivery nanocarriers. 129 , 130 Zhao et al developed exosome membrane-coated nanoparticles that can protect siRNA from degradation and have excellent biocompatibility. Further studies in vivo showed that exosomal membrane-coated nanoparticles had higher affinity, which significantly inhibited the growth of malignant BC cells. 131 Tran et al loaded aspirin into exosomes as an anticancer agent, and converted crystalline aspirin into the nano-amorphous form in exosomes with a nano-matrix structure, thereby improving the efficiency of drug encapsulation of exosomes and the dissolution and cytotoxicity of aspirin in BC. Thus, in this study, a novel nano-amorphous exosome delivery system consisting of nanorods was created that can transform anti-inflammatory drugs into effective cancer drugs. 132 By in vivo adoptive transfer of bone marrow MSCs in CC mice, Naseri et al demonstrated that MSCs-Exo permeate tumor sites and act as an appropriate nanocarriers for the delivery of inhibitory oligonucleotides into neoplastic tissues to downregulate the expression levels of miR-142-3p and miR-150. 133 Aqil et al found that exosomes containing Anthos exhibited marked anti-proliferative activity against OC cell development and restrained tumor development more effectively compared with the effects of Anthos from berries and carrier controls alone. Anthos has been shown to be effective against OC, and milk exosomes are excellent nanocortors that enhance oral bioavailability of drugs for the treatment of OC. 134 Aqil et al further demonstrated that exosomal curcumin (ExoCUR) showed stronger anti-proliferative and anti-inflammatory activity in BC and CC cell lines (as measured by NF-κB activation) compared with free curcumin, suggesting that exosomes are suitable for development as potential nanocarriers to deliver curcumin to improve tissue bioavailability. 135 Reprogrammed exosomes can be used as nanocontrollers of cellular immunity. Cheng et al found that endogenous exosomes can be used as artificial cellular immune controllers to redirect immune effector cells and regulate their immune reactivity in BC cells. This exosome-based nano-agent provides unique and enhanced pharmacological properties that can be used to develop new therapies for BC. 136 Endogenous exosome levels may also affect the efficiency of nanoparticles for targeted drug delivery. Wang et al found that pre-treatment with peripheral blood-derived exosomes reduced the deposition grapefruit-derived nanovector (GNV) in the liver and improved the treatment efficiency of GNV-carrying drugs in BC mice. 137 These studies demonstrate the role of exosomes in nanotherapy and open a new chapter in the treatment of female reproductive system cancers and BC.