Exosomes as crucial emerging tools for intercellular communication with therapeutic potential in ovarian cancer

Metastasis is a primary reason for cancer-related deaths [ 79 ] and is induced by the cancer cells undergoing EMT [ 80 ]. EMT is a process by which epithelial cells lose cell polarity and adhesive properties and attain migratory and invasive characteristics, promoting malignancy [ 81 ]. The exosomes originating from tumor cells carry EMT-inducing molecules such as the developmental transcription factors (SNAIL/SLUG, TWIST1/TWIST2, and ZEB1/ZEB2 family) [ 82 ], TGF-β, miRNAs, β-catenin, or hypoxia-inducible factors. These factors can confer mesenchymal characteristics to epithelial cells, promoting transformation and metastasis [ 58 ]. Recently, EMT was described to progress through a spectrum of stable hybrid states, namely intermediate EMT, partial EMT (pEMT), hybrid epithelial/mesenchymal, and EMT-like states [ 83–85 ]. pEMT is characterized by the partial loss of epithelial markers and gain of mesenchymal markers and has been revealed to modulate the hallmarks of cancer [ 86 ]. In addition to paracrine signaling via the stromal cells in the TME, genetic, epigenetic, and post-translational modifications in the primary tumor cells are responsible for inducing the pEMT cell state [ 83 , 87 , 88 ]. In OC, pEMT cell states confer more malignancy than mesenchymal phenotype and may promote ascites formation and peritoneal metastasis [ 89 ]. Exosomes are known to regulate the phenotypic pEMT by altering the transcription factors to promote invasiveness and chemoresistance [ 90 ]. The reversal of mesenchymal phenotype to epithelial state is termed mesenchymal-epithelial transition (MET), which commonly occurs during cellular development and cancer metastasis [ 91 ]. In OC, miRNA-1294 promoted MET by suppressing insulin-like growth factor 1 receptor ( IGF1R ), an anti-apoptotic gene, thereby inhibiting AKT, mTOR, and ErbB [ 92 ]. Similarly, in liver cancer, Phosphatase of regenerating liver-3 ( PRL-3 ) promotes MET via the activation of the epidermal growth factor receptor (EGFR) signaling pathway [ 93 ]. Cancer cells have the ability to activate the oncogenic pathways that promote cell proliferation in the local niches, followed by the recruitment of EVs, in particular the exosomes, to communicate with cells at distant sites. The aberrations in the pathways within the cells receiving these EVs can result in cancer progression [ 94 ]. Interestingly, the exosomal contents promote EMT by regulating EMT-associated signaling pathways such as the Hippo pathway, β-catenin signaling pathway, and ERK pathway [ 81 ]. In breast cancer cells, exosomes obtained from mesenchymal stem cell differentiated adipocytes induced EMT via the activation of YAP and TAZ, the downstream effectors of the Hippo pathway [ 95 ]. In prostate cancer, miR-301a promoted EMT, via the Wnt/β-catenin pathway, by inhibiting p63 thereby releasing ZEB1/2 which suppresses E-cadherin [ 96 ]. Similarly, miR-1260b derived from lung cancer exosome promoted EMT via the Wnt/β-catenin pathway by suppressing SMAD4 and sFRP1 [ 97 ]. In non-small lung cell cancer (NSLCC), miR-92a promoted EMT through the PI3K/AKT pathway by inhibiting PTEN expression [ 98 ]. In hepatocellular carcinoma, exosomes induced EMT by activating the TGF- β/Smad signaling pathway, thereby promoting migration and invasion by down-regulating E-cadherin expression and up-regulating Vimentin expression [ 99 ]. Furthermore, melanoma cell-derived exosomes induced EMT via the upregulation of Let7i , let7a , miR-191 , SNAIL2 , ZEB2 , and vimentin and downregulation of E cadherin [ 100 ].

Exosomes contain several proteins such as cytosolic, common membrane proteins, and origin-specific sub-set of proteins that reflect cell function [ 120 ]. Numerous studies have shown the potential role of exosomal proteins in serving as a biomarker for various diseases, including cancer [ 120 ]. In chronic myeloid leukemia patients, the exosomes derived from the tumor cells carried cytokine TGF-β, which promoted tumorigenesis by activating the AKT, ERK, and anti-apoptotic pathways [ 148 ]. In another study, CD63, a member of the tetraspanin family and an exosomal marker, was significantly elevated in melanoma patients compared with healthy individuals [ 149 ]. In glioblastoma patients, glioblastoma-specific epidermal growth factor VIII was detected in serum exosomes, serving as a diagnostic tool for glioblastoma detection [ 150 ]. A previous study reported the metastatic potential of the exosomes secreted by the glioma cells containing EGFRvIII factor, which promoted the growth of the recipient cells by activating transforming signaling pathways such as mitogen-activated protein kinase and Akt pathway [ 151 ]. In breast cancer patients, nephronectin, a protein inducing anchorage-independent growth, was highly expressed in exosomes derived from tumor tissues [ 152 ]. Similarly, in the early stage of breast cancer patients, survivin levels were higher in the serum exosomes, suggesting its potential use as a diagnostic and prognostic marker [ 153 ]. In EOC, exosomal proteins play a role in tumor growth and metastasis [ 28 ]. A previous finding on the content of IGROV-1 and OVCAR-3-derived exosomes showed the presence of proteins involved in signal transduction, adhesion, immune response regulation, and membrane transport and fusion. Liang et al. showed the enrichment of cancer-associated signaling pathway proteins within the exosomes such as epithelial cell surface antigen (EpCAM), epidermal growth factor receptor (EGFR), apolipoprotein E (APOE), proliferation cell nuclear antigen (PCNA), with a potential role as diagnostic markers and therapeutic targets for OC treatment [ 154 ]. In the study of Marta Szajnik et al. , exosomal protein content was higher in advanced stages than in the early stages of OC. Furthermore, TGF-β and MAGE3/6 could be used as markers to distinguish patients with benign tumors from OC patients [ 155 ]. Hence, exosomal proteins could reflect the stage of the tumor and serve as a prognostic marker to determine the response to OC treatment [ 28 ].

The therapeutic potential of exosomes in-vivo has been characterized by emerging techniques such as liquid biopsy, thereby unraveling their clinical importance [ 156 ]. Furthermore, exosomes serve as efficient drug delivery vehicles due to their characteristic properties such as low toxicity, low immunogenicity, stability, biocompatibility, permeability, and ability to cross the blood-brain barrier [ 157 , 158 ]. Understanding the constituents, the mechanism of biogenesis and specific cell targeting of exosomes will shed light on their physiological activity. Also, identifying the process regulating the release of the exosomes and their fate in the recipient cells has the potential to unravel novel ways involved in cell–cell communication and inhibition of cancer progression [ 51 ]. miRNAs and lncRNAs play a key role in human pathologies such as cancer. Profiling exosomal miRNAs serve as a promising tool for cancer and may be used for monitoring disease recurrence and responses to therapies [ 78 ]. Recently, exosomes were found to inhibit tumor angiogenesis when loaded with tumor suppressor miRNA, which acts against pro angiogenetic mRNAs [ 29 ]. Similarly, exosomes produced from self-derived dendritic cells were loaded with siRNA and injected into mice, which, in turn, delivered the siRNA to neurons, oligodendrocytes, and microglia in the brain. This resulted in the knockdown of a gene involved in Alzheimer's disease, demonstrating nanovesicle-mediated siRNA delivery's therapeutic use [ 159 ]. Furthermore, exosomes enriched with miRNA-155 inhibitor were successfully delivered to the mouse hepatocytes resulting in the downregulation of TNF in the recipient cells, demonstrating its application in nanomedicine [ 160 ]. Several reports have identified the ability of exosomes to precisely deliver drugs to the tumor cells, thereby enhancing the efficacy of cancer treatment. Kim et al. demonstrated 50-times higher cytotoxicity in drug-resistant cancer cells upon treatment with paclitaxel-loaded exosomes compared with paclitaxel alone [ 161 ]. Similarly, exosomes derived from siGRP78 transfected bone marrow mesenchymal stem cells in hepatocellular cancer inhibited sorafenib resistance, growth, and metastasis in-vivo [ 162 ]. In triple-negative breast cancer, exosomes engineered with surface peptides against the mesenchymal-epithelial transition factor gene displayed efficient cellular uptake and increased tumor targeting effects of doxorubicin [ 163 ]. Similarly, exosomal enclosed curcumin promoted apoptosis in brain cells upon intranasal administration [ 164 ].

Intercellular communication is crucial for maintaining physiological homeostasis and pathological processes [ 50 ]. The development and progression of cancer require intercellular communication within the primary tumor microenvironment (TME) and at distant sites [ 51 ]. Hence, TME has a crucial function in influencing the hallmarks of cancer [ 52 , 53 ]. TME is composed of nonmalignant cells, blood vessels, stromal cells (fibroblasts, mesenchymal stem cells (MSCs), endothelial cells, adipocytes, and lymphatic network), immune cells (natural killer cells, B and T lymphocytes, and tumor-associated macrophages), exosomes, tumor cells and extracellular matrix [ 54–56 ]. Exosomes serve as signaling molecules by transferring bioactive molecules between the cancer cells and the surrounding cells [ 57 ], thereby modifying the TME to affect the normal cells. Hence, exosomes are crucial for linking healthy cells to cancer cells. Furthermore, exosomes derived from tumor play a key role in the crosstalk between the tumor microenvironment and cancer cells by facilitating epithelial-to-mesenchymal transition (EMT), migration, invasion, angiogenesis, immune regulation, and stromal reprogramming to a cancer-associated phenotype within the TME [ 58 , 59 ]. Therefore, exosomes have a profound impact on the TME [ 60 ]. Furthermore, EVs derived from tumor cells were found to influence the stromal cells, such as fibroblasts and endothelial cells, within the tumor microenvironment [ 50 ], thereby engaging in crosstalk for sustaining the signaling networks for cancer progression [ 61 ]. In order to promote metastasis, stromal cells such as a tumor associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and MSCs secrete cytokines and exosomes that induce cell proliferation, EMT, and the extracellular matrix (ECM) remodeling [ 62 ]. In addition, CAFs play a key role in initiating an inflammatory process and suppressing the activity of cytotoxic T lymphocytes [ 63 ].

Exosomes are a subset of extracellular vesicles (EV) composed of a lipid bilayer ranging from 30–100 nm in diameter [ 29 ]. It is released by several cell types, such as red blood cells, lymphocytes, dendritic cells, platelets, and tumor cells [ 30 ]. It has been detected in various biological fluids, including blood plasma, saliva, nasal secretion, cerebrospinal fluid, urine [ 30 ], and ascites [ 31 ], suggesting its role in modulating physiological responses [ 32 ]. Exosomes were earlier identified as a tool to eliminate cellular waste [ 33 , 34 ]. However, with advancements in current research, exosomes have been revealed to serve as carriers of a complex mixture of relevant biological molecules, including lipids, proteins, microRNAs (miRNAs), and long noncoding RNAs (lncRNAs), some of which have the potential to serve as a biomarker [ 35 ]. Furthermore, exosomes transport their cargo to the encountered recipient cells, with the potential to reprogram these target cells [ 36 ].

The molecular contents enriched within the exosomes strongly reflect the parental cell [ 41 ], which vary according to the cellular origin, developmental stages, environmental conditions, and epigenetic changes [ 32 ]. In addition to messenger RNA (mRNA) and DNA, several other RNA species such as miRNAs, ribosomal RNA (rRNAs), transfer RNAs (tRNAs), or lncRNAs are enriched within the exosomes. Lipids such as cholesterol, phospholipids, sphingolipids, and ceramide form the bilayer membrane structure of the exosomes [ 41 , 48 ]. Interestingly, exosomes carry distinct proteins such as ESCRT-I associated protein (TSG101), MVB associated protein (Alix), tetraspanins (CD9, CD63, CD81, and CD82) exclusively associated with the biogenesis of the exosomes and are used as markers for their authentication (see Figure 1 ) [ 46 ]. Furthermore, Ras-related proteins (Rabs), membrane-binding proteins (annexins), and major histocompatibility molecules are found in the exosomes [ 49 ]. Created with BioRender.com.

One of the reasons for the failure of EOC treatment is the detection of the disease at advanced stages in addition to either ineffective drug delivery to the tumor site or acquired resistance to chemotherapy. Therefore, biological markers with the potential to detect EOC at early stages would help improve therapies and overall survival. Exosomes can potentially serve as an effective noninvasive tool as they are carriers of nucleic acids and proteins, which are known to be altered according to the pathological conditions of the patients. Liquid biopsy, a minimally invasive blood test, is increasingly used for the early detection and prognosis of EOC patients, and circulating exosomes are a great source of liquid biopsy analysis. The circulating exosomal ncRNAs are gaining wide attention as they represent a crucial source of liquid biopsy markers in cancer diagnosis, prognosis, and response to treatment. In addition, exosomes could be utilized in real-time cancer detection, identifying tumor stages and therapeutic responses [ 165 ]. Furthermore, exosomes have a promising role in nanomedicine due to their pathophysiological and biological features and functioning as therapeutic targets, anticancer drug delivery vehicles, and diagnostic markers. Despite the strides in elucidating exosome-associated molecular mechanisms of tumor progression, further efforts are required to utilize the therapeutic potential of exosomes for clinical practice. In addition, further studies on the enormous sample size are required to attain knowledge of its diagnostic value as well as a standardized protocol should be devised for the isolation and characterization of exosomes. Hence, a deeper understanding of the exosome heterogeneity and its use as a drug delivery tool can open new avenues for combating EOC. Exosomes are nanovesicles released in the extracellular milieu by diverse cell types and serve as carriers of proteins, and nucleic acids, with the potential to modulate the recipient cells. Understanding the functions of exosomes as messengers between cancer and healthy cells unfolds new avenues in intercellular signaling mechanisms. Functional characterization of exosomal contents through different techniques opens a new approach to discovering novel EOC biomarkers with the potential to discriminate patients at early stages. Several findings have highlighted the involvement of exosome encapsulated genes in EOC angiogenesis, proliferation, migration, and tumor growth, thereby serving as diagnostic and prognostic factors in EOC progression and metastasis. Exosomes could represent a powerful tool for therapeutic interventions due to their ability to function as drug carriers or vaccines posing a promising option in curing EOC.

The remodeling of ECM is a crucial step in facilitating the invasiveness of the tumor [ 50 ]. Recent studies have shown the role of cancer cell-derived exosomes in organizing the ECM. The exosomes released by the cancer cells are enriched with proteases, which can modify the ECM by degrading collagen, laminin, and fibronectin resulting in tumor and host cell adhesion, metastasis, and invasiveness [ 64 ]. In a previous finding, Rab27B regulated the release of HSP90 enriched exosomes from metastatic breast cancer cells to activate matrix metalloproteinase 2 (MMP2), thereby degrading the components of ECM and facilitating the secretion of growth factors leading to invasion [ 65 ]. The interactions between exosomes from EOC cells and TME has a pivotal role in cancer (see Table 1 ) [ 66 ]. In EOC, lymphatic dissemination is preceded by peritoneal dissemination, a specific form of metastasis. Peritoneal dissemination is a major obstacle in treating OC. A better understanding of the functions of exosomes in facilitating peritoneal dissemination could result in the better management of EOC. Exosomes facilitate peritoneal dissemination through cross-talks between the cancer cells and the tumor microenvironment [ 73–75 ]. Malignant ascites are common in OC patients and are characterized by high levels of tumor-enhancing factors such as IL-6, IL-8, IL-10, TGFβ, and vascular endothelial growth factor (VEGF). Furthermore, malignant ascites promote OC cell proliferation, anti-apoptosis, invasion, and chemoresistance via these factors [ 76–78 ].

One of the main obstacles to the treatment of cancer patients is chemoresistance, with the highest chemoresistance rates observed in OC patients. Recent studies have revealed the role of exosomal miRNA in developing chemoresistance in OC cells [ 23 ]. Exosomal transfer of miR-21 to OC cells promoted resistance to paclitaxel by targeting the APAF1 gene [ 145 ]. Similarly, miR-433 was observed to cause the downregulation of cyclin-dependent kinase 6 to promote paclitaxel resistance in OC cells [ 146 ]. In OC patients, exosomal miR-223 obtained from hypoxic macrophages induced chemoresistance of OC cells by regulating the PTEN-PI3K/AKT signaling pathway [ 147 ]. Similarly, long noncoding RNA UCA1  was elevated in serum exosomes derived from cisplatin-resistant OC tissues and cell lines compared with normal ovarian samples. Moreover, inverse relation was observed between UCA1 and miR-143 in serum exosomes obtained from OC samples. Hence, UCA1 induced cisplatin resistance in OC by sponging miR-143 , which resulted in the upregulation of FOSL2 [ 139 ].