Exosomes in Ovarian Cancer: Promoters, Biomarkers, and Therapeutic Targets

Abstract

Ovarian cancer is an aggressive malignancy treated primarily with surgery and platinum-based chemotherapy. The high recurrence rate and platinum resistance are the primary reasons for poor prognosis in ovarian cancer. Early screening can improve patient survival, but there are currently no high-precision biomarkers available. Exosomes are nanoscale vesicles that mediate intercellular communication by transferring bioactive molecules, and their composition reflects pathological states. Late diagnosis is the primary cause of poor prognosis in patients with ovarian cancer. Owing to the high stability conferred by their unique structure, exosomes can serve as an efficient, non-invasive approach for early screening. In the context of drug delivery, engineered exosomes using novel advanced technologies can enhance the specificity of clinical pharmacotherapy and reduce adverse toxic reactions. This review summarizes the latest research findings on ovarian cancer-related exosomes and introduces their important roles in exploring the mechanisms of ovarian cancer progression, metastasis, and chemoresistance, as well as their potential as prognostic biomarkers and therapeutic targets.

Introduction

Ovarian cancer (OC) is one of the common malignant tumors in the female reproductive system, and its disease burden continues to increase globally. Epidemiological data indicate that OC ranks eighth among female malignancies in terms of both mortality and incidence, with more than 207,000 women dying from ovarian cancer worldwide each year.¹ In 2020, there were over 310,000 new cases and approximately 200,000 deaths.² Metastasis of primary OC can occur through three pathways: peritoneal implantation, lymphatic metastasis, and hematogenous metastasis,³ among which peritoneal implantation is the most common. 90% of patients with advanced ovarian cancer develop peritoneal carcinomatosis and excessive intra-abdominal fluid known as malignant ascites.⁴

Nowadays, with the continuous advancement of medicine, the treatment of ovarian cancer is no longer limited to simple surgical resection of lesions, but has gradually developed into more precise application of refined surgery combined with comprehensive approaches such as platinum-based chemotherapy, immunotherapy, and targeted therapy.⁵ Although modern treatment methods such as bevacizumab and poly (ADP-ribose) polymerase (PARP) inhibitors have significantly improved patient survival,⁶ OC still has the highest mortality rate among all gynecological malignancies due to the high recurrence rate after resection and widespread chemoresistance.⁵ Since ovarian cancer has no obvious symptoms in the early stage, most patients are already in the advanced stage (stage III or IV) when they seek medical treatment, with a 5-year survival rate of less than 40%.⁷ Early diagnosis helps to improve patient survival. Therefore, exploring new therapeutic directions and biomarkers is crucial for improving the prognosis of OC patients.

Cancer nanomedicine employs nanotechnology to deliver therapeutic agents—including nucleic acids, proteins, and small-molecule drugs—to tumor tissues. In cancer immunotherapy, nanoscale drug delivery systems offer superior safety and efficacy compared with conventional antineoplastic therapies.⁸ Conventional nanoparticles are generally classified into organic and inorganic categories, such as liposomes, lipid nanoparticles (LNPs), metallic nanoparticles, silica nanoparticles, and carbon nanotubes (CNTs). Organic nanoparticles typically exhibit low toxicity but are often compromised by poor stability and suboptimal biodistribution, resulting in unsatisfactory drug delivery efficiency.⁹ Inorganic nanoparticles, while possessing greater stability, are frequently more toxic and associated with a higher incidence of adverse reactions.¹⁰ Neither class of conventional nanoparticles achieves ideal performance in drug delivery applications. In contrast, exosomes are fully biodegradable and biocompatible, as they constitute essential components of normal physiological processes.¹¹ Their low immunogenicity, absence of toxicity, and lack of adverse reactions translate to minimal safety concerns.¹²

Functional biomolecules delivered by exosomes to recipient cells can effectively alter the biological state of recipient cells and activate signaling pathways within them.¹³ This exosome-mediated response may either promote or inhibit disease progression, providing new targets for disease treatment. Such vesicles can penetrate biological barriers including the blood–brain barrier, a property that further underscores their application potential in therapeutic fields.¹⁴ In addition, exosomes can serve as biomarkers for liquid biopsy because they are present in most body fluids, such as blood, urine, saliva, pleural and peritoneal effusions, and breast milk,¹⁵ and have the potential for multi-component analysis.¹⁶ Exosomes are widely present in various body fluids and enable the targeted delivery of bioactive molecules. Accordingly, they hold great promise as noninvasive biomarkers for cancer diagnosis, therapeutic monitoring, and prognosis assessment, with extensive application prospects.¹⁷ Nevertheless, exosome detection remains technically challenging, and existing isolation methods suffer from certain limitations. Exosome enrichment strategies based on advanced nanotechnology are expected to improve the accuracy of liquid biopsies.

Exosome-targeting strategies represent a transformative and cutting-edge frontier in tumor nanomedicine, providing innovative insights for the development of more precise and personalized therapies. Engineered exosomes exhibit great potential in overcoming tumor drug resistance and mitigating drug-induced toxicity.¹⁸ Conventional nanoparticles accumulate at tumor sites via the enhanced permeability and retention (EPR) effect. However, recent studies have demonstrated that endogenous targeting relying solely on the EPR effect is extremely inefficient, with less than 1% of nanoparticles actually reaching tumor tissues.^19,20 Engineered exosomes can improve therapeutic efficacy by enhancing the targeting capability of nanoparticles. For instance, engineered exosomes derived from activated immune cells, following antibody conjugation through copper-free click chemistry, can significantly suppress tumor growth and constitute a highly efficient and feasible novel strategy for cancer immunotherapy.²¹

This article briefly introduces the new roles of exosomes in OC and reviews their significant effects in promoting tumor growth and metastasis, chemoresistance, and serving as prognostic biomarkers and potential therapeutic targets. Exosomes hold promising prospects for clinical application, yet numerous challenges remain to be addressed, including ensuring the quality and safety of medicinal products, the lack of standardized isolation methods for clinical detection, and associated ethical issues. These challenges are also discussed in this review.

Introduction to Exosomes

Contents and Biogenesis of Exosomes

In 1946, experiments on human plasma by Chargaff and West demonstrated that the plasma fraction remaining after removing precipitates via high-speed centrifugation could inhibit plasma coagulation.²² A few years later, Peter Wolf identified these coagulation inhibitors as platelet-derived vesicles with a diameter of 20–50 nanometers.²³ This marked the beginning of recognition and subsequent research on extracellular vesicles.

Extracellular vesicles are mainly classified into two categories: exosomes and ectosomes. Ectosomes are vesicles formed by direct budding from the plasma membrane, which can develop into microvesicles, microparticles, and large vesicles with diameters ranging from approximately 50 nanometers to 1 micrometer.²⁴ In contrast, exosomes are extracellular vesicles with a diameter of 50 to 150 nanometers (averaging 100 nanometers). They are composed of a lipid bilayer and contain various functional biomolecules, including proteins, DNA, lipids, as well as coding and non-coding RNAs (ncRNAs).²⁵ NcRNAs consist of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Current studies have identified exosomes as having a diverse composition, including approximately 4400 proteins, 194 lipids, 1639 messenger RNAs (mRNAs), and 764 miRNAs.²⁶

Under physiological conditions, exosomes generally function to clear harmful and excess intracellular components, including various drugs, thereby maintaining intracellular homeostasis. Exosomes also exhibit significant advantages in crossing biological barriers²⁷ and are involved in both short-distance and long-distance intercellular communication.²⁸ When exosomes are taken up by other cells, the substances they carry are transferred, influencing the phenotype of the recipient cells. Additionally, exosomes are highly heterogeneous, which can reflect the phenotypic state of the cells that produce them.²⁹

The tumor microenvironment (TME) constitutes a dynamic ecosystem shaped by reciprocal interactions between tumor cells and host stromal components. It consists of malignant cells, endothelial cells, microvessels, immune cells, cancer-associated fibroblasts, extracellular matrix, and diverse bioactive signaling molecules.³⁰ Beyond cellular compartments, the TME also incorporates non-cellular constituents. As pivotal mediators within the TME, exosomes modulate metabolic reprogramming, activate oncogenic signaling pathways, and facilitate immune evasion, thereby driving the remodeling of the tumor microenvironment.³¹ Exosomes derived from ovarian cancer cells mediate the intercellular transfer of biological materials between cancer cells and multiple recipient cell types, thereby reconstructing the TME and ultimately facilitating tumor invasion and metastasis.³² As depicted in Figure 1, the present review elaborates on the critical functions of exosomes in multiple pathological processes, including TME remodeling, angiogenesis promotion, and tumor chemoresistance (Figure 1).

Figure 1.

Multiple roles of exosomes in TME. Exosomes mediate intercellular communication by transporting bioactive molecules, including miRNAs, circRNAs, lncRNAs, and proteins, between tumor cells and various stromal cells. This schematic illustrates how ovarian cancer cell-derived exosomes regulate multiple protumorigenic processes, including promoting macrophage polarization, angiogenesis, EMT, and therapeutic resistance. Key regulatory molecules are highlighted, such as miR-21, LAMA5, circNFIX, and ANXA2.

The biogenesis of exosomes involves multiple steps:^33–35

1. Formation of early endosomes: Through endocytosis of the plasma membrane, cells cause invagination of the plasma membrane near active endocytic sites, forming early endosomes that provide an endocytic space for lipids, proteins, and other molecules.

2. Formation of multivesicular bodies: Early endosomes gradually transform into late endosomes. The membrane of late endosomes buds inward, forming multivesicular bodies (MVBs) containing intraluminal vesicles. During this process, components such as proteins and nucleic acids are packaged into these MVBs.

3. Release or degradation of exosomes: If MVBs fuse with the plasma membrane, exosomes are released into the extracellular space; if MVBs fuse with lysosomes, they are degraded.

The mechanism by which exosomes enter recipient cells may involve multiple pathways simultaneously:^36–38

1. Endocytosis: Including clathrin-mediated endocytosis, caveolin-mediated endocytosis, lipid raft-mediated endocytosis, macropinocytosis, and phagocytosis.

2. Membrane fusion: Direct fusion of the exosomal membrane with the recipient cell membrane, which can directly release the contents of exosomes, such as proteins, nucleic acids, and lipids, into the cytoplasm of the recipient cell, thereby achieving information transmission and functional regulation.

3. Receptor-based binding: Corresponding receptors on the surface of recipient cells can recognize and bind to various proteins present on the surface of exosomes, triggering intracellular signaling cascades and promoting the uptake of exosomes (Figure 2).

Figure 2.

The release of exosomes into the extracellular matrix involves three steps: the formation of early endosomes, the formation of multivesicular bodies, and the release or degradation of exosomes. Cytoplasmic contents of donor cells, such as nucleic acids, proteins, and lipids, are sorted into exosomes. Subsequently, exosomes are internalized by recipient cells through three modes: endocytosis, membrane fusion, and receptor-based binding.

Methods for Isolation and Enrichment of Exosomes

Exosome characterization and identification are critical steps for assessing sample quality and supporting subsequent research, with the core principle of employing orthogonal methods to avoid biased conclusions resulting from incomplete characterization. MISEV2023 proposes a framework of five categories of protein markers.³⁹ Among them, transmembrane anchored proteins and cytosolic proteins are classical positive markers of EVs, which can directly confirm the presence of vesicular structures; however, the exclusive detection of these markers has limitations, as they are not absolutely specific to EVs and may also be carried by some non-vesicular particles, nor can they reflect sample purity. Therefore, the detection of co-isolated contaminant markers such as apolipoproteins, albumin, and immunoglobulins is equally essential, as they objectively reflect the level of residual impurities during isolation. In addition, organelle markers are used to indicate cellular debris contamination, while secreted proteins are associated with the functional properties of exosomes and their interactions with the microenvironment. No single detection method can fully define exosomes.³⁹ Only by combining morphological, physicochemical, and molecular marker analyses can the true state of the sample be comprehensively and objectively characterized, laying a solid foundation for the reliability of subsequent research.

Currently, common traditional methods for the isolation and detection of exosomal proteins include ultracentrifugation, precipitation techniques, and molecular characterization via enzyme-linked immunosorbent assay (ELISA). Among these, ultracentrifugation is the most widely used method.⁴⁰ It first uses low-speed centrifugation to remove cell debris, then medium-speed centrifugation to eliminate other large vesicles, and finally high-speed centrifugation to pellet exosomes. This method remains the foundational choice in most studies due to its requirement for no complex media and capability to process large-volume samples. However, it is highly sensitive to parameters such as rotor type, centrifugation speed, and duration, resulting in poor reproducibility. Precipitation techniques utilize highly hydrophilic polymers to compete for water molecules around exosomes, thereby precipitating them. Polyethylene glycol (PEG) is the most commonly used polymer,⁴¹ and many commercial kits based on this technology, such as ExoQuick™, have been developed.⁴² Although PEG precipitation is simple to perform and low-cost, it suffers from severe co-isolation of impurities, resulting in insufficient purity of the isolated product; thus, it is particularly not recommended for complex biological fluids such as serum and plasma. While commercial kits simplify the workflow, their underlying principles and formulations are often undisclosed, lacking standardized quality control measures, which may compromise the reliability of results.

Due to the difficulty in exosome detection and the challenge of achieving continuous tracking and monitoring, these traditional methods have certain drawbacks. Some methods are too costly, while others are prone to contaminating exosomes during operation, with each method having its own limitations. In recent years, with the development of science and technology, new breakthroughs have been made in exosome isolation methods, such as chemiluminescence,⁴³ microfluidic chip-based optical detection,⁴⁴ and surface-enhanced Raman scattering (SERS).^39,45

Microfluidic technology enables efficient and low-damage isolation of exosomes by virtue of its miniaturization, automation, and high-throughput characteristics. By integrating principles such as size sieving, immunoaffinity capture, and hydrodynamic sorting, microfluidic chips can process samples in minute volumes, significantly reducing isolation time and minimizing exosome loss and structural damage, making them particularly suitable for the analysis of trace clinical samples.⁴⁶ Some chips can even integrate multiple functional modules, offering potential for rapid clinical detection.⁴⁷ However, microfluidic technology also has notable drawbacks: high chip fabrication costs and low throughput make it difficult to meet the demands of large-scale preparation with high sample volumes; additionally, substantial variations in chip design and operational parameters, along with the lack of unified standardized protocols, compromise the reproducibility and comparability of results.

Integration of SERS technology with microfluidics or nanomaterials provides novel strategies for simultaneous isolation, enrichment, and detection of exosomes.⁴⁸ Based on the specific interaction between exosomal surface markers and targeted probes, SERS signals enable capture and highly sensitive identification of target exosomes. This approach combines both separation and preliminary characterization functions, with sensitivity far exceeding conventional methods.⁴⁹ However, the application of SERS is limited by probe specificity and substrate stability; nonspecific adsorption may cause signal interference and compromise isolation purity. Moreover, matrix effects in complex biological fluids can attenuate detection signals, restricting its widespread clinical application. These new methods have shown significant effectiveness in exosome detection, but their role in practical applications of liquid biopsy is still limited. Therefore, exploring more accurate exosome detection methods has become a hot topic in current research.

Electrochemical detection is a method that detects analytes by measuring the electrochemical potential or current of a sample, and it has the advantages of high sensitivity and a wide measurement range.⁵⁰ Jeong S et al developed an integrated magnetic electrochemical sensor (iMEX) technology for exosome analysis, which combines magnetic separation and electrochemical detection. Compared with ELISA, iMEX has higher correlation, faster detection speed (only 1 hour), and smaller sample usage (only 10 μL) when analyzing exosomal transmembrane proteins. In biological fluid detection, iMEX has a detection limit of 3×10⁴ exosomes and a dynamic range spanning four orders of magnitude, with performance superior to ELISA.⁵¹

Microfluidic chips are chip platforms that achieve precise control and manipulation of fluids at the micrometer scale using micro-electromechanical processing technology.⁵² Studies by Zhao et al have shown that a microfluidic chip composed of a Y-shaped injector, a serpentine fluid mixer, and a microcavity with a replaceable magnet can enrich tumor-derived circulating exosomes from plasma and perform in-situ multi-label detection. Compared with ultracentrifugation, the exosomes isolated by this chip have a higher proportion of those smaller than 150 nm, indicating strong separation specificity.⁵³ In addition, Zhang et al developed a microfluidic chip with 3D nano-patterns, using a multi-scale integrated design self-assembly (MINDS) strategy, which applies microfluidic engineered colloid self-assembly (CSA) to construct 3D nanostructured functional microelements, such as nano-herringbone (nano-HB) mixers. The nano-HB chip can effectively promote microscale mass transport, increase surface area and probe density, while reducing near-surface hydrodynamic resistance. Its detection limit is as low as 10 exosomes/μL, and its detection performance is superior to traditional planar channel microfluidic chips.⁵³

Fluorospectrophotometry in fluorescence detection is a method that identifies substances and determines their content based on the positivity and intensity of fluorescence spectral lines.⁵⁰ Carney et al used multi-spectral optical tweezers (MS-OTs) technology, combining a laser trapping Raman spectroscopy (LTRS) system with a fluorescence imaging system, to construct a new device, realizing molecular fingerprint analysis of single exosomes.⁵⁴ This device focuses on the CD9-positive exosome subpopulation, which helps to more clearly define exosome subpopulations and thus promote the application of exosomes in clinical treatment and diagnosis.

These modern technologies are not complete replacements for conventional methods, but rather important complements. They exhibit unique advantages in scenarios involving trace samples, rapid detection, and high-sensitivity analysis. However, they still present limitations in large-scale preparation, standardization, and cost control. The future direction should focus on promoting complementary integration between these advanced technologies and traditional approaches, while establishing unified quality control standards. Only in this way can the clinical translation and widespread application of exosome isolation and enrichment technologies be truly realized.

Exosomes Promote the Growth and Metastasis of Ovarian Cancer

The role of exosomes in tumor development has been extensively studied. Substances contained in exosomes, such as RNA, miRNA, proteins, DNA, and even metabolites, can alter the fate of recipient cells through autocrine and paracrine signals.³⁸ Based on various secretory compounds including exosomes, cancer cells and stromal cells have established a bidirectional communication network, which is involved in the process of cancer metastasis.⁵⁵ These tumor-derived exosomes have become new carriers of intercellular communication networks and new components of the tumor microenvironment that support the localization, proliferation, and survival of tumor cells.⁵⁶ Due to their ability to transport and transfer bioactive molecules, exosomes’ capacity to regulate tumor metastasis has attracted much attention recently.⁵⁷

Exosomes Promote Angiogenesis

The proliferation of tumor cells requires sufficient nutrients and oxygen, so they gather near blood vessels to obtain the necessary nutrients. Based on this, Judah Folkman proposed that tumor angiogenesis is one of the necessary conditions for tumor progression.⁵⁸ When the tumor grows to a certain extent, due to the lack of nutrients, a microenvironment characterized by hypoxia, ischemia, acidosis, and high interstitial pressure is gradually formed in the tissue. This environment releases a large number of growth factors and cytokines, stimulating angiogenesis and lymphangiogenesis to meet the needs of tumor growth and metabolism.⁵⁹ Tumor progression is usually accompanied by inward growth of blood vessels, which is consistent with the fact that malignant cells need to enter the circulatory system to thrive.

MiRNAs secreted by OC cells can be packaged into exosomes and participate in tumor angiogenesis. The upregulation of exosomal miR-205 is positively correlated with high microvessel density in OC patients, and high levels of miR-205 in circulating exosomes are associated with OC metastasis. Studies have found that it induces angiogenesis through the PTEN-AKT pathway and accelerates angiogenesis and tumor growth in mouse models.⁶⁰ Importantly, miR-205 is significantly enriched in the serum of OC patients, suggesting that exosomal miR-205 is a potential therapeutic target for OC.

Similarly, exosome-packaged proteins also affect angiogenesis. Exosomal prokineticin receptor 1 (PKR1) protein enhances the migration of vascular endothelial cells and promotes tube formation of vascular endothelial cells through signal transducer and activator of transcription 3 (STAT3) phosphorylation.⁶¹ In addition, the homeobox protein homeobox D11 (HOXD11) in exosomes derived from cancer-associated fibroblasts (CAFs) can bind to the promoter of fibronectin 1 (FN1), upregulate the expression of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (PECAM-1) proteins, thereby promoting angiogenesis and tumor growth.⁶² Proteomic studies have also found that proteins such as activating transcription factor 2 (ATF2), Metastasis-associated protein 1 (MTA1), and rho-associated coiled-coil containing protein kinase 1/2 (ROCK1/2) may be involved in the angiogenic function of exosomes.⁶³

Human umbilical vein endothelial cells (HUVECs) refer to the single layer of squamous epithelial cells located on the inner surface of umbilical veins, and they are a type of vascular endothelial cells. Multiple studies have shown that exosomes can affect HUVECs through various signaling pathways, thereby promoting angiogenesis as well as the growth and metastasis of OC. OC-related exosomal circNFIX regulates the JAK/STAT1 pathway in HUVECs through the miR-518a-3p/TRIM44 axis, thereby promoting angiogenesis.⁶⁴ MiR-141-3p in exosomes reduces the expression level of suppressor of cytokine signaling 5 (SOCS-5), leading to the upregulation of the JAK-STAT3 pathway in endothelial cells.⁶⁵ A variety of lncRNA can also exert stimulatory effects on HUVECs, lncRNA activated by TGF-β (ATB) promotes the viability and migration of HUVECs by regulating the miR-204-3p/TGFβR2 axis.⁶⁶ OC cells transfer lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) to recipient HUVECs through exosomes, and finally promote angiogenesis by stimulating the expression of vascular endothelial growth factor-A (VEGF-A) and vascular endothelial growth factor-D (VEGF-D).⁶⁷

Exosomes Promote Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a cellular program crucial for embryogenesis, wound healing, and malignant progression of tumors. In early-stage cancer, tumor cells exhibit an epithelial-like state, while as the tumor progresses, they gradually acquire more mesenchymal characteristics.⁶⁸ During EMT, epithelial cells lose intercellular junction structures such as tight junctions and desmosomes, and simultaneously lose cell polarity. This enables tumor cells to detach from the epithelial layer, thereby gaining migratory and invasive capabilities, creating conditions for tumor cells to break through the basement membrane and infiltrate surrounding tissues.⁶⁹ In the course of cancer development, EMT promotes tumor metastasis and spread, thereby driving tumor progression.⁷⁰

Exosomes can alter the levels of EMT markers such as fibronectin, α-smooth muscle actin (α-SMA), vimentin, and E-cadherin. For example, exosomes derived from ovarian cancer ascites can transfer miR-6780b-5p to ovarian cancer cells, reducing the expression of E-cadherin while increasing the expression of N-cadherin and vimentin. Under this effect, cell morphology gradually becomes elongated, scattered, and more spindle-like, thereby promoting the EMT process and ultimately facilitating ovarian cancer metastasis.⁷¹

Peritoneal mesothelial cells originally act as a barrier to prevent ovarian cancer from breaking through the peritoneum and metastasizing to deeper layers, but exosomes can alter their physiological state. In moderately and poorly differentiated ovarian cancer tissues, exosomal circWHSC1 is highly expressed, which can upregulate downstream targets mucin 1 (MUC1) and human telomerase reverse transcriptase (HTERT) and act on peritoneal mesothelial cells.⁷² This process provides favorable conditions for tumor metastasis, assisting tumor spread within the abdominal cavity. Annexin A2 (ANXA2) produced by ovarian cancer cells can also be transferred via exosomes, regulating morphological changes in peritoneal mesothelial cells and inducing fibrosis, promoting the EMT process and degradation of the extracellular matrix, and ultimately affecting the pre-metastatic microenvironment of ovarian cancer.⁷³

Tumor cells that have undergone EMT can interact with other cells in the tumor microenvironment, such as CAFs, endothelial cells, and immune cells. Recent studies have shown that the interaction between tumor cells and CAFs can activate CAFs, enabling them to secrete more growth factors and extracellular matrix components, providing support for the growth and migration of tumor cells.⁷⁴ MiR-141 is an exosomal miRNA highly secreted by ovarian cancer cells, which can reprogram stromal fibroblasts into CAFs, thereby promoting the metastatic colonization of tumor cells.⁷⁵ MiR-124 secreted by human ovarian surface epithelial cells can be transferred to CAFs via exosomes, leading to reduced expression of α-SMA and fibroblast activation protein (FAP) and weakened cell motility, while downregulation of miR-124 expression promotes the transformation of normal fibroblasts into CAFs.⁷⁶

Exosomes Induce Macrophage Polarization

Macrophages are a diverse group of infiltrating immune-related stromal cells present in all parts of the human body.⁷⁷ Under different stimuli, they can polarize into classically activated M1-type macrophages or alternatively activated M2-type macrophages.⁷⁸ Tumor-associated macrophages (TAMs), considered similar to M2-type macrophages, exist in the TME. They influence the progression and metastasis of various cancers through interactions with cancer cells and other cell types.⁷⁹ Colorectal cancer cells can secrete substances that induce M2 macrophage polarization, thereby regulating crosstalk between cancer cells and TAMs and further inducing colorectal cancer liver metastasis.⁸⁰ Similarly, gastric cancer cells can promote the polarization of peritoneal TAMs into the M2 phenotype.⁸¹ These pieces of evidence indicate that the mechanism of macrophage polarization toward M2 has become a hotspot in cancer research and a potential therapeutic target.

Exosomes derived from OC cells can reshape macrophages into TAMs with a tumor-promoting phenotype. Recent studies have emphasized the important role of miRNAs in this process. One study found that a set of miRNAs are enriched in hypoxic exosomes. Under hypoxic conditions, these miRNAs can promote macrophage polarization in peripheral blood mononuclear cells (PBMCs) by inducing hypoxia-inducible factor (HIF) expression, and macrophages “trained” by hypoxic exosomes can promote the proliferation and migration of OC cells.⁸² Another study showed that miR-940 is highly expressed in exosomes isolated from the ascites of OC patients, and the pro-tumor function of miR-940 is achieved by inducing TAM polarization.⁸³ In addition to miRNAs, lncRNAs can also promote M2 macrophage polarization. LINC00958 can bind to glioma-associated oncogene homolog 1 (GLI1) and activate the Hedgehog pathway, thereby maintaining the stemness of OC cells and inducing M2 differentiation.⁸⁴

Further research by Haiyang Li et al showed that some special exosomes are also involved in the formation of TAMs. OC cells overexpressing the E26 transformation-specific sequence 1 (ETS1) transcription factor secrete larger exosomes (LV-ETS1Exos), which have higher laminin levels. LV-ETS1Exos can stimulate the polarization of macrophages toward the M2 phenotype and also promote macrophages to produce more chemokines C-X-C motif chemokine ligand 5(CXCL5) and C-C motif chemokine ligand 2(CCL2), thereby promoting the metastasis of ovarian cancer to the omentum⁸⁵(Table 1).

Table 1.

Exosomes That Promote the Growth and Metastasis of Ovarian Cancer

Effect	Exo-resource	Exo-content	Functional Mechanism	Ref
Promote angiogenesis	Patient’s serum	miR-205	Activate the PTEN/AKT pathway	[60]
	A2780 and HO-8910 ovarian cell lines	PKR1	Induces STAT3 phosphorylation and promotes vascular endothelial cell migration	[61]
	Patient’s tumor tissue	HOXD11	Binds to the promoter of FN1 and upregulates the protein expression of VEGF and CD31	[62]
	SKOV3 and CAOV3 ovarian cell lines	ATF2, MTA1, ROCK1/2	Upregulate VEGF and stabilize MTA1	[63]
	Patient’s tumor tissue	circNFIX	Activate the JAK/STAT1 pathway	[64]
	Patient’s serum	lncRNA ATB	Regulates the miR-204-3p/TGFβR2 axis	[66]
	Patient’s serum	MALAT1	Upregulate VEGF-A, VEGF-D, ENA-78, PlGF, IL-8	[67]
	SKOV-3 ovarian cell line	miR-141-3p	Activate the JAK/STAT3 pathway	[65]
Promote EMT	OVCA433 and OVKATE ovarian cell lines	miR-141	Regulates the Hippo/YAP1/GROα/CXCR1/2 axis	[75]
	Patient’s ascites	miR-6780b-5p	Activate Notch pathway and MAPK pathway	[71]
	Patient’s tumor tissue	circWHSC1	Initiate miR-145 and miR-1182, and upregulate downstream targets MUC1 and HTERT	[72]
	CAOV3, OVCAR3 and SKOV3 ovarian cell lines	ANXA2	Activate the PI3K/AKT/mTOR pathway	[73]
	Patient’s tumor tissue and normal ovarian tissue	miR-124	Downregulate α-SMA and FAP	[76]
Induce macrophage polarization	SKOV3 ovarian cell line	miR-21-3p, miR-125b-5p和miR-181d-5p	Regulates the SOCS4/5/STAT3 axis	[82]
	CAOV3 and OVCAR-3 ovarian cell lines	lncRNA LINC00958	Binds to GLI1, activates the Hedgehog pathway, and maintains the stemness of OC cells	[84]
	Patient’s omental tissue	LAMA5, LAMB1 and LAMC1	Regulates the integrin αvβ5/AKT/Sp1 axis	[85]
	Patient’s omental tissue and ascites	microRNA-940	Upregulate CD163 and CD206	[83]

Exosomes Promote Chemoresistance in Ovarian Cancer

The treatment of ovarian cancer is usually based on surgery, supplemented by chemotherapy, molecular targeted therapy and other methods. However, chemoresistance is a major challenge in current ovarian cancer treatment. The mechanisms of chemoresistance in ovarian cancer are complex and diverse. Abnormal transmembrane transport, changes in DNA damage repair, dysregulation of cancer-related signaling pathways, and epigenetic modifications can all cause chemoresistance.⁸⁶ Platinum or paclitaxel derivatives combined with surgical debulking is the first-line treatment strategy for ovarian cancer,⁸⁷ which can induce DNA damage in rapidly proliferating cells, trigger apoptosis and lead to massive cell death. Most patients initially respond to platinum-based chemotherapy, but about 70–80% of patients will experience tumor recurrence and develop resistance to chemotherapy.⁸⁸ Tumor cells have various mechanisms to develop resistance to cisplatin, such as reduced drug uptake, enhanced efflux pump activity, decreased active drug components in cancer cells, subsequent changes in drug molecular targets, improved DNA damage repair ability, reduced pro-apoptotic factors or upregulated anti-apoptotic genes, etc.⁸⁹ It is worth noting that the number of exosomes secreted by cisplatin-resistant ovarian cancer cells is 2.6 times that of drug-sensitive ovarian cancer cells,⁹⁰ and the more invasive the ovarian cancer cells, the more exosomes they secrete.⁹¹ Thus, exosomes are closely related to chemoresistance in ovarian cancer.

The omentum is a fat cell-rich organ in the peritoneum and a common site of ovarian cancer invasion. Omental adipocytes are associated with disease progression, metastasis, and chemoresistance. Exosomal miR-21 derived from the omentum of OC patients can drive EMT and reduce the response to paclitaxel treatment.⁹² Ovarian cancer cells engineered to exogenously express mothers against decapentaplegic homolog 4 (SMAD4) mutations showed upregulated EMT markers after carboplatin treatment, increased resistance to carboplatin, and also released exosomal miR-21, which increased the resistance of initial recipient cells by approximately 1.7-fold compared with controls.⁹³ This study demonstrated that the exchange of tumor-derived exosomes perpetuates the EMT phenotype, leading to the development of platinum-resistant cell subpopulations.

Hypoxia is a ubiquitous hallmark of cancer, characterized by insufficient oxygen tension within tumor tissues. It modulates therapeutic resistance and accelerates tumor progression in cancer cells.^94,95 A hypoxic microenvironment drives cellular adaptive alterations that further promote tumor growth and induce treatment resistance. Exosomal miR-223 derived from hypoxic macrophages can promote chemoresistance in OC cells through the PTEN-PI3K/AKT pathway both in vivo and in vitro. Meanwhile, the level of circulating exosomal miR-223 is closely associated with OC recurrence.⁹⁶ The expression of miR-1246 in exosomes from paclitaxel-resistant OC cells is significantly higher than that in exosomes from drug-sensitive cells. When resistant OC cells are co-cultured with macrophages, they can transfer miR-1246 to M2-type macrophages via exosomes, thereby enhancing cell resistance to paclitaxel and promoting tumor progression.⁹⁷

Hypoxia also leads to dysregulated metabolic reprogramming in cancer cells. Autocrine is a mode of cell communication in which signaling molecules act on the secreting cells themselves without long-distance transport, with an extremely short acting distance. Plasma gelsolin (pGSN), a secreted isoform of the actin-associated cytoplasmic protein gelsolin (GSN), can upregulate the expression of HIF1α in chemoresistant OC cells in an autocrine manner. HIF1α directly interacts with hypoxia-response elements (HREs) in pGSN, thereby elevating the mRNA expression of pGSN under hypoxia and conferring cisplatin resistance to otherwise chemosensitive OC cells.⁸⁸

Exosomes can not only alter downstream signal transduction through their carried contents, affecting the phenotype and state of recipient cells, but also directly efflux drugs, reducing intracellular drug concentration and leading to drug resistance in OC cells. In studies on cisplatin-resistant ovarian cancer patients, it was found that the concentration of serum exosomes in patients increased significantly after cisplatin treatment, while the size of exosomes did not change. This may be due to the fact that OC cells in a hypoxic environment significantly increase exosome release by upregulating ras-related protein Rab-27a (Rab27a), downregulating ras-related protein Rab-7 (Rab7), lysosome-associated membrane protein 1/2 (LAMP1/2), and neuraminidase-1 (NEU-1), and promoting the production of more secretory lysosome phenotypes. More importantly, high concentrations of cisplatin were detected in these exosomes.⁹⁸ This phenomenon indicates that exosomes have an enhanced ability to efflux chemotherapeutic drugs under hypoxic conditions. In addition, when comparing platinum-sensitive cell lines with samples from resistant patients, it was found that transmembrane protein 205 (TMEM205) and cluster of differentiation 1B (CD1B) are highly expressed in samples from platinum-resistant patients.TMEM205 can alter the exosome secretion pathway by regulating Rab11 expression, thereby increasing platinum efflux in platinum-resistant cells⁹⁹(Table 2).

Table 2.

Exosomes That Promote Chemoresistance in Ovarian Cancer

Exo-resource	Exo-content	Functional Mechanism	Resistant Drug	Ref
Patient’s omental tissue and ascites	miR-21	Downregulate APAF1	Paclitaxel	[92]
A2780 and OVCAR10 ovarian cell lines	miR-21	Activate the TGF-β/SMAD pathway	Cisplatin	[93]
Patient’s serum	miR-223	Regulates the miR-223/PTEN-PI3K/AKT axis	Cisplatin	[96]
A2780 and SKOV-3 ovarian cell lines	pGSN	HIF1α interacts with HREs in pGSN, thereby elevating the mRNA expression of pGSN under hypoxic conditions.	Cisplatin	[88]
HeyA8, Skov3 and A2780 ovarian cell lines	miR-1246	Regulates the Cav1/p-gp/ M2-type macrophages axis	Paclitaxel	[97]
TR127 and TR182 ovarian cell lines	Cisplatin	Promote the secretory lysosome phenotype, thereby facilitating drug efflux	Cisplatin	[98]
TR127 ovarian cell line	Cisplatin	Alter the exosome secretion pathway, thereby promoting drug efflux	Cisplatin	[99]

Exosomes as Biomarkers for Ovarian Cancer Screening

The early clinical manifestations of ovarian cancer are insidious, and most patients are diagnosed at an advanced stage.¹⁰⁰ Late diagnosis is the main cause of poor prognosis in OC patients, with an overall 5-year survival rate of less than 30%.¹⁰¹ In sharp contrast, the 5-year survival rate of OC patients diagnosed at an early stage exceeds 90%, which fully demonstrates the great potential of accurate early diagnosis in improving prognosis. Currently, common screening methods for OC include ultrasound examination, gynecological examination, and detection of serum cancer antigen 125 (CA125) levels. However, conventional examinations have the problem of poor specificity and sensitivity,¹⁰² and there is an urgent need for more effective and non-invasive early screening methods to improve the early diagnosis rate of OC patients.

Exosomes are small vesicles secreted by cells, with a unique lipid bilayer membrane structure that can better protect their contents from degradation.¹⁰³ Therefore, compared with other biomarkers in body fluids, exosomes have higher stability. Exosomes also have many natural advantages: their membrane proteins can be detected without lysis,¹⁰⁴ and they can be detected in almost all biological fluids, making them an ideal tool for minimally invasive liquid biopsy.

RNAs in Exosomes as Biomarkers

Within a single epithelial ovarian tumor, there exist tumor cells with varying invasive abilities, and the exosomes they release also differ significantly. Among them, highly invasive tumor cells release more exosomes, and in these exosomes, the expression of the let-7 family, which inhibits cell proliferation, is significantly higher. In contrast, the miR-200 family, which inhibits EMT, is only expressed in exosomes released by low-invasive cells.¹⁰⁵

Exosomes are abundant in serum, and it has been confirmed that exosomes have diagnostic value in various cancers. Comparing exosomes from patients’ serum with those from healthy individuals’ serum, it was found that the expression level of hsa-miR-6835-3p in patients’ serum exosomes is the highest, and the expression stability of hsa-miR-4468 is the strongest; the combination of the two achieves optimal expression stability.¹⁰⁶ Exosomes not only help in screening OC patients but also provide references for patients’ clinical staging. For example, the levels of miR-200b and miR-200c in serum exosomes of patients with international federation of gynecology and obstetrics (FIGO) stage III–IV are significantly higher than those in early-stage patients. The levels of these two exosomal miRNAs are also associated with elevated CA125 values and shortened overall survival of patients.¹⁰⁷

Some OC patients develop peritoneal effusion. An analysis of lncRNA expression in exosomes derived from peritoneal effusion of patients with high-grade serous carcinoma (HGSC) showed that among patients with peritoneal effusion before chemotherapy, those with higher embryonic stem cell - related gene (ESRG) levels in exosomes had longer overall survival; for patients with peritoneal effusion after chemotherapy, those with higher Link-A levels in exosomes had better overall survival and progression-free survival.¹⁰⁸

Urine, as a non-invasive and easily accessible biological fluid, has a lower protein content than blood samples, which can reduce interference from proteins in RNA extraction and subsequent analysis, making it highly promising in the field of liquid monitoring. Quantitative real-time PCR was used to detect the expression of cell-free urinary microRNAs in OC patients, and it was found that miR-92a was significantly upregulated and miR-106b was significantly downregulated compared with control samples.¹⁰⁹ Another study found that the level of urinary miR-30a-5p in OC patients decreased significantly after surgical resection of the tumor, indicating that urinary miR-30a-5p is likely derived from tumor tissue. In addition, the expression of urinary miR-30a-5p is closely related to the early development and lymph node metastasis of ovarian serous adenocarcinoma. Comparing urine samples from patients with ovarian serous adenocarcinoma with those from patients with other cancers, it was found that urinary miR-30a-5p levels were lower in 20 gastric cancer patients and 20 colon cancer patients, suggesting that the upregulation of urinary miR-30a-5p may be a characteristic manifestation of ovarian serous adenocarcinoma.¹¹⁰

Proteins in Exosomes as Biomarkers

Exosomes contain a wide variety of proteins, which also have the potential to serve as diagnostic biomarkers for OC. Specific marker proteins exist on the exosome membrane, such as CD63, CD9, and CD81, which can promote the recognition and binding of exosomes to target cells. An analysis of the specific marker proteins on the surface of exosomes isolated from ovarian cancer cell lines and their correlation with histological tumor types revealed that the exosomes from primary OC tumors had undetectable CD117 signals and high epithelial cell adhesion molecule (EpCam) signals on their surface; in contrast, the exosomes from recurrent OC tumors showed significant CD117 signals and low EpCam signals.¹¹¹ This finding indicates that the characteristics of these marker proteins on the exosome surface can be used for the diagnosis of tumor recurrence and provide guidance for targeted therapy.

Detection of exosomes derived from patients’ serum found that the zinc finger protein 587B (ZNF587B) protein may be unique to exosomes, and low expression of ZNF587B is associated with poor prognosis of OC.¹¹² To further explore the relationship between the exosome-related gene risk score (ERGRS) and clinical features, immune infiltration, immune checkpoint-related genes, copy number variations, and drug sensitivity, an analysis of patients’ serum exosomes showed that the expression levels of programmed death-ligand 1 (PD-L1), programmed death-1 (PD-1), and indoleamine 2,3-dioxygenase 1 (IDO1) in high-risk patients were lower than those in low-risk patients, and the low-risk group had better prognosis in four immunotherapies.¹¹³ This suggests that the expression levels of anti-ovarian cancer genes in the serum of ovarian cancer patients are closely related to the occurrence and development of ovarian cancer, which can better predict the prognosis of ovarian cancer patients and provide guidance for the selection of immunotherapeutic strategies.

When analyzing exosomes from the ascites of OC patients, full-length transmembrane activated leukocyte cell adhesion molecule (ALCAM) was detected, and the level of ALCAM in ascites was higher than that in serum, indicating that local processing of ALCAM may occur in the peritoneal cavity. Meanwhile, the study also found that the level of ALCAM was significantly higher in type II tumors, even in stage I/II tumors, implying that elevated ALCAM is an early feature of aggressive OC.¹¹⁴ Another study showed that higher levels of tumor suppressor candidate 6 (TSAP6) protein were associated with shorter overall survival in OC patients, while higher levels of Rab27a protein were significantly correlated with longer overall survival¹¹⁵ (Table 3).

Table 3.

Exosomal RNA and Proteins as Biomarkers for Ovarian Cancer Screening

Type	Exo-content	Exo-resource	Theory	Specificity/Sensitivity/AUC	Ref
RNA	let-7 family and miR-200 family	SKOV-3 and OVCAR-3 ovarian cell lines	The let-7 family, which inhibits cell proliferation, is significantly more expressed in exosomes of highly invasive OC cells. The miR-200 family, which inhibits EMT, is only expressed in exosomes of low-invasive OC cells.	let-7 family: AUC=0.80 miR-200 family: AUC=0.82	[105]
	OAZ1 and hsa-miR-6835-3p	Patient’s serum	It is recommended to use the OAZ1/SERF2/MPP1 combination for mRNA analysis and the hsa-miR-6835-3p/hsa-miR-4468 combination for miRNA analysis.	-	[106]
	miR-200b	Patient’s serum	Associated with FIGO stage, lymph node metastasis, CA125 level, and overall survival.	AUC=0.868, sensitivity: 64%, specificity: 86%.	[107]
	ESRG and Link-A	HGSC exudate	Higher levels of ESRG and Link-A are associated with better prognosis.	Link-A was identified as an independent prognostic biomarker (P = 0.045).	[108]
	miR-92a, miR-106b	Patient’s urine	miR-92a is significantly upregulated, and miR-106b is significantly downregulated.	miR-92a: significantly upregulated by 31.97-fold (P = 0.0009) miR-106b: significantly downregulated to 0.27-fold (P = 0.0026)	[109]
	miRNA-30a-5p	Patient’s urine	The expression of miR-30a-5p is closely associated with the early development and lymph node metastasis of serous adenocarcinoma of the ovary.	AUC=0.862	[110]
Protein	CD117 and EPCAM	SKOV-3 and MES-OV ovarian cell lines	The exosomes from recurrent tumors shows significant CD117 signals and low EpCam signals.	-	[111]
	ZNF587B	Patient’s serum	Low expression of ZNF587B is associated with poor prognosis in OC.	Low expression of ZNF587B was significantly associated with shorter overall survival (P=0.009).	[112]
	PD-L1,PD-1 and IDO1	Patient’s serum	The expression levels of PD-L1, PD-1, and IDO1 in high-risk patients are lower than those in low-risk patients.	-	[113]
	ALCAM	Patient’s ascites	Elevated ALCAM is an early feature of aggressive OC.	AUC=0.8067	[114]
	TSAP6 and Rab27a	Patient’s ascites	The higher the level of TSAP6 and the lower the level of Rab27a, the shorter the overall survival of patients.	Higher expression of the TSAP6 protein was significantly associated with shorter overall survival in patients (P=0.01). Higher expression levels of the Rab27a protein were significantly associated with longer overall survival (P=0.025).	[115]

Exosomes and Ovarian Cancer Treatment

Tumor-derived exosomes are regarded as being closely linked to the pathogenesis of cancer and the formation of the tumor microenvironment, as cancer cells produce more exosomes than normal cells.¹¹⁶ Numerous studies have shown that after exosomes deliver their carried substances to target cells, they can induce functional changes in recipient cells and affect the host immune response, thereby promoting or inhibiting tumor proliferation, invasion, and metastasis.¹¹⁷ In addition, exosomes possess many characteristics such as easy uptake by cancer cells, small size, good biocompatibility, convenient acquisition, and high stability,¹¹⁸ which make it possible to use exosomes for drug delivery or engineered exosomes for targeted therapy of ovarian cancer. Therefore, exosomes show great potential and unique advantages in the field of ovarian cancer treatment.

Exosomes Activate Immune Cells

TME refers to the complex environment where tumor cells reside, including the tumor cells themselves, surrounding stromal cells (such as fibroblasts, immune cells, endothelial cells, etc), extracellular matrix, and various signaling molecules and bioactive substances.¹¹⁹ Accumulating evidence indicates that the tumor microenvironment plays a crucial role in the progression, metastasis, and development of drug resistance in ovarian cancer.¹²⁰ Various immune cells exist in the TME, and exploring how to activate these immune cells to generate anti-tumor immune responses has become a new direction in OC treatment.

Exosomes can transfer tumor antigens to autologous dendritic cells, thereby promoting the activation of tumor antigen-specific T cells. These activated T cells can eliminate autologous tumor cells in vitro through a major histocompatibility complex (MHC) class I-restricted manner.¹²¹ Using toll-like receptor 3 (TLR3) agonists to break tumor-induced immune tolerance, while using tumor-derived exosomes as a rich source of ovarian cancer antigens, can stimulate effective and long-lasting tumor antigen-specific T cell immune responses.¹²² Therefore, tumor-derived exosomes can serve as a cell-free vaccine to induce tumor antigen-specific cytotoxic T lymphocytes (CTLs) in vivo, and these CTLs have the ability to kill tumor cells in OC patients.

Cancer cells exhibit an imbalance in redox homeostasis, and elevated levels of reactive oxygen species (ROS) can significantly downregulate the expression of miR-155-5p in OC cell-derived exosomes. Co-culturing macrophages preconditioned with exosomal miR-155-5p with T lymphocytes can increase the percentage of CD8+ T lymphocytes and reduce the apoptosis of CD3+ T cells by downregulating PD-L1, thereby activating cellular immunity and exerting anti-tumor effects.¹²³ Thus, the development of exosome-mimetic liposomal formulations of miR-155-5p may have the potential to bring therapeutic benefits to OC patients.

Exosomes isolated from the human tumor microenvironment express phosphatidylserine (PS) on their surface, which inhibits T cell activity and blocks tumor clearance.¹²⁴ In response to this phenomenon, Bhatta M et al designed and synthesized a new compound called ExoBlock.¹²⁵ ExoBlock is a hexamer engineered to carry six PS-binding sites, with high PS affinity, and its efficacy has been verified in two models: a melanoma-based xenograft (X-BMT) mouse model and an ovarian tumor-based omental tumor xenograft (OTX) model. The results showed that ExoBlock directly activates CD4+ and CD8+ T cells in the models, promotes the generation of Tregs, and upregulates checkpoint molecules such as PD-L1, thereby improving the therapeutic effect of checkpoint blockade therapy.

Exosomes and Chemosensitivity Enhancement in Chemoresistance

Platinum-based chemotherapy is currently the mainstream treatment for ovarian cancer, but the development of chemoresistance in OC cells significantly hinders patient treatment and even reduces their quality of life and survival time. Therefore, reversing chemoresistance in OC cells has become a key research direction in current OC therapy.

Exosomes, with their excellent biocompatibility and ability to precisely target tumor sites for rapid drug release, are expected to serve as ideal drug delivery systems for treating chemoresistance. Triptolide (TP) and miR-497 show promise in overcoming OC resistance. However, TP has strong systemic toxicity and poor water solubility,¹²⁶ while miR-497 has low transcriptional efficiency,¹²⁷ which hinder their application. A study constructed hybrid nanoparticles called miR497/TP-HENPs, which integrate liposomes and exosomes and encapsulate the chemotherapeutic drug TP and miR-497.¹²⁸ These nanoparticles can specifically block the PI3K/AKT/mTOR signaling pathway in OC cells, thereby disrupting the normal cellular microenvironment, inducing tumor cell death, and successfully overcoming OC chemoresistance both in vitro and in vivo.¹²⁸ In another study, exosomes derived from dendritic cells expressing lysosome-associated membrane glycoprotein 2b (Lamp2b) fused with RGD (CRGDKGPDC) could target and deliver miR-484 to induce vascular normalization, thereby sensitizing cancer cells to chemotherapy-induced apoptosis, with efficacy demonstrated in mice.¹²⁹

In addition to acting as drug delivery systems, cell-derived exosomes also play an important role in targeted therapy.MiR-146a from human umbilical cord mesenchymal stem cells (hUCMSCs)-derived exosomes can target laminin γ2, downregulate the PI3K/AKT signaling pathway, and increase the sensitivity of OC cells to docetaxel and taxanes.¹³⁰ Natural killer cell-derived exosomes (eNK-EXO) not only serve as sustained-release carriers for cisplatin but also target and activate NK cells in the immunosuppressive tumor microenvironment, thereby enhancing the killing effect on OC-resistant cells.¹³¹

Engineered Exosomes

Exosomes possess unique biological properties: they can target specific tissues, remain stable during in vivo metabolism, and have membrane permeability, enabling them to cross the cytoplasmic membrane and the blood-brain barrier. The exosome membrane consists of a bilayer structure, which allows for controlled and sustained release of loaded materials.¹³² Based on these characteristics, exosomes have significant advantages in drug delivery: they can enhance drug stability and solubility, and protect drugs from degradation during blood circulation. In summary, exosomes as carriers can overcome the drawbacks of poor bioavailability, and reduce non-target cell toxicity and immunogenicity.¹³³

Recently, natural plant sources have been explored for their possible use in sustainable synthesis of nanoparticles, which can be applied in designing hybrid exosomes as a novel drug delivery system for ovarian cancer treatment. For example, biosynthesis of selenium nanoparticles using potato peels (Solanum tuberosum) has exhibited cytotoxicity on cancer cells along with antimicrobial activity, emphasizing the importance of agricultural waste material utilization in nanotechnology applications.¹³⁴ Likewise, the antimicrobial and anticholinesterase activity of phenolics present in Prunus cerasus fruits is another potential candidate for developing hybrid exosomes as drug delivery systems.

Moreover, identification of phenolic antioxidants present in Myrtus communis fruits has confirmed its strong antioxidant capacity, which can be exploited in designing novel exosomes as drug delivery vehicles to alleviate oxidative stress inside tumors.¹³⁵ On the other hand, the antimicrobial activity of phenolics obtained from Glycyrrhiza glabra roots has provided a natural source for exosome surface modification purposes.¹³⁶ Functional characteristics of different types of Origanum plants, as well as the presence of active compounds in their structure, have also been studied as a possible source of functional foods.¹³⁷ Manipulating phenolic compositions in Echinacea species by altering culture conditions can shed light on enhancing the antioxidant ability of nanovehicles.¹³⁸ Additionally, manipulating the characteristics of carbon dots using phenol-rich agricultural waste can serve as an environmentally friendly approach to utilize the concept of fluorescence labeling for engineered exosomes.¹³⁹ Lastly, bioactivity and antioxidant characteristics of kiwano (Cucumis metuliferus) fruits grown in certain regions highlight the availability of diverse sources of plants to design innovative exosome-based drugs.¹⁴⁰

Phytochemical compounds have potential regulatory effects on OC. Hanan M Alharbi et al designed exosomes loaded with mangiferin (derived from mango) and curcumin (derived from turmeric) based on specific nanotechnology principles.¹⁴¹ The synergistic interaction between the two can target the PI3K/Akt/mTOR pathway, thereby promoting cell apoptosis. Compared with traditional administration methods, this exosome- and liposome-based delivery method significantly improves cellular drug uptake, effectively enhances drug bioavailability, and minimizes drug side effects. Currently, carriers carrying CRISPR/Cas9 have limited in vivo delivery efficiency due to low immunogenicity.¹⁴² Exosomes loaded with cas9 and PARP-1 sgRNA plasmids via electroporation can enhance their tolerance. Moreover, compared with exosomes derived from epithelial cells, exosomes derived from cancer cells can more efficiently inhibit the expression of PARP-1 due to their cell tropism, thereby enhancing the promotion of cell apoptosis.¹⁴³

However, natural exosomes as drug delivery carriers have certain limitations, such as a short half-life in circulation and poor targeting ability. Nevertheless, these defects can be overcome through chemical and protein engineering technologies. Currently, the most promising approach is to engineer exosomes to express custom peptides on their outer surface that can target specific tissues.¹⁴⁴ Enhancing the specificity of engineered exosomes can not only alter their in vivo half-life and distribution but also the fusion of external targeting peptides with exosomal transmembrane proteins not only helps exosomes accurately reach target cells but also reduces uptake by off-target cells, thereby lowering drug toxicity.¹⁴⁵

A study showed that HEK293 cells were used to produce exosomes carrying the ephrin-B2 ligand, which can fuse with the exosomal membrane protein LAMP-2b.¹⁴⁶ In this way, the engineered targeting ligand can be delivered to ephrin-B4 on the surface of ovarian cancer cells. After researchers injected such engineered exosomes into mice, it was found that compared with mice treated with wild-type exosomes, the changes in immune markers in mice treated with engineered exosomes were minimal.

Exosomes engineered with Arg-Gly-Asp peptides derived from SKOV3-92b cells have stronger cell penetration ability than parental exosomes. The overexpressed miR-92b-3p in these exosomes can target SRY-related HMG-box 4 (SOX4) to inhibit tumor growth and tumor-associated angiogenesis.¹⁴⁷ Moreover, whether used alone or in combination with apatinib, these exosomes strongly inhibit tumor growth through in vivo anti-angiogenesis.

Si/TP@Exos, constructed based on peritoneal mesothelial cell-derived exosomes, co-encapsulates TP and siRNA-A4B2.¹⁴⁸ It leverages the homotypic homing capability of exosomes to precisely accumulate in ovarian cancer lesions, enabling targeted drug delivery. On the one hand, siRNA-A4B2 silences ITGA4B2, blocks the formation of the ITGA4B2/AEP ternary complex, inhibits the NF-κB signaling pathway and epithelial-mesenchymal transition, and thereby suppresses peritoneal metastasis. On the other hand, TP exerts potent anti-tumor cytotoxicity and induces cancer cell apoptosis. In vitro and in vivo experiments confirm that this system significantly enhances tumor uptake, promotes cancer cell apoptosis, inhibits tumor growth and peritoneal metastasis, and greatly reduces the systemic toxicity of free TP. It achieves triple functions of targeted efficacy enhancement, toxicity reduction, and anti-metastasis, providing a safe and efficient synergistic therapeutic strategy for ovarian cancer.

In summary, both loading drugs into natural exosomes and engineering exosomes can achieve efficient therapeutic effects on OC. In addition, there are some new approaches that can produce therapeutic effects on OC based on exosomes, such as M-Trap, a tumor cell capture technology based on exosomes that embeds exosomes purified from the ascites of OC patients into 3D-polystyrene/polycaprolactone objects.¹⁴⁹ By simulating the artificial environment of the extracellular matrix and competing with the natural site of peritoneal implantation, M-Trap can effectively disrupt the natural process of peritoneal metastasis, which has been verified in a mouse model of ovarian cancer peritoneal metastasis (Figure 3).

Figure 3.

(a) Activated immune cells: TLR3 agonists reverse tumor-induced immune tolerance. Cell-free vaccines composed of tumor-derived exosomes can elicit robust and durable tumor antigen-specific T cell responses. Exosome-mimicking liposomal formulations carrying miR-155-5p, as well as engineered hexamer ExoBlock with six high-affinity PS-binding sites, can activate CD8⁺ T cells, promote Treg proliferation and upregulate PD-L1, thereby markedly improving the efficacy of checkpoint blockade therapy. (b) Chemoresistance sensitization: miR497/TP-HENPs, hybrid nanoparticles integrating liposomes and exosomes and co-loaded with TP and miR497, specifically block the PI3K/AKT/mTOR pathway and effectively reverse chemoresistance in ovarian cancer. Dendritic cell-derived exosomes expressing RGD-Lamp2b fusion proteins can deliver miR-484 in a targeted manner to normalize tumor vasculature and sensitize cancer cells to chemotherapy. (c) Engineered exosomes: Exosomes loaded with mangiferin and curcumin, as well as exosomes electroporated with plasmids encoding Cas9 and PARP-1 sgRNA, efficiently induce apoptosis in ovarian cancer cells. Exosomes carrying ephrin-B2 ligands bind to LAMP-2b and target ephrin-B4 on ovarian cancer cell surfaces, which greatly reduces systemic toxicities. RGD-modified exosomes possess enhanced cellular penetration ability. Their highly expressed miR-92b-3p targets SOX4 to suppress tumor growth and angiogenesis; combined with apatinib, they exert a potent synergistic anti-tumor effect via anti-angiogenesis. M-Trap is an exosome-based tumor capture technology that immobilizes exosomes purified from ascites of ovarian cancer patients onto 3D polystyrene/polycaprolactone scaffolds. It mimics the extracellular microenvironment to compete for implantation sites and inhibit peritoneal metastasis, which has been validated in relevant models.

These studies demonstrate that exosomes as delivery vehicles in cancer therapy represent a cutting-edge field of nanomedicine. Even so, determining whether additional interactions exist between exosomes and the human body is critical for evaluating their potential risks. Exosomes may trigger allergic reactions and could even modulate the body’s intrinsic immune competence, which might adversely affect therapeutic outcomes and, in the worst-case scenario, promote cancer progression. Therefore, precise identification and monitoring of adverse effects are essential to ensure treatment-related safety and efficacy. Despite their therapeutic promise, regulatory and manufacturing constraints must be carefully addressed when translating exosome-based therapeutics from research to clinical application. To date, most studies are in Phase I, with encouraging positive findings regarding safety, tolerability, and early efficacy, laying a solid foundation for future Phase II trials. With advancing research, exosome-related concepts are expected to be integrated into therapeutic regimens, opening new avenues for cancer treatment.

Conclusions and Future Prospects

This review elaborates on the important roles of exosomes in the clinical management of ovarian cancer, including their mechanisms mediating cancer metastasis and chemoresistance, as well as the main principles underlying their use as biomarkers and potential therapeutic targets. A deep understanding of the molecular mechanisms by which exosomes mediate ovarian cancer metastasis and chemoresistance is conducive to the development of novel therapies targeting exosome-mediated tumorigenesis, metastasis, and chemoresistance.

Exosomes contain a variety of substances, some of which can serve as biomarkers for early detection, diagnosis, prognosis prediction, and evaluation of therapeutic efficacy of cancer. Cancer-derived exosomes carry bioactive molecules that reflect the molecular characteristics of tumors and are secreted into body fluids. By precisely localizing and analyzing molecular transcriptomic markers, the information derived from exosomes enables us to understand cancer progression and develop improved diagnostic and therapeutic strategies. The standardization of exosome isolation and characterization methods is expected to advance cancer research and diagnosis. However, exosome purity and specificity may pose major challenges, and clinical standards for exosome isolation from body fluids remain to be established. The development of standardized protocols is critical to moving research forward. Consistent and reproducible results across different studies are essential to validate the potential of exosomes as reliable biomarkers.^150,151 Current research is hampered by variability in isolation techniques, characterization methods, and analytical approaches.¹⁵¹ Establishing consensus guidelines for these processes will improve data comparability and facilitate the nanomedical translation of exosome-based diagnostic strategies into clinical practice.

Exosome-based therapy represents a breakthrough in nanomedicine, particularly in personalized medicine and cancer treatment. Exosomes are small extracellular vesicles and well-established tools in nanomedicine. Researchers are harnessing these vesicles to develop novel therapeutic approaches, including the targeted delivery of therapeutic agents to cells or tissues and the use of exosome-derived materials for the diagnosis of diseases and symptoms.

However, several issues impede their broad clinical application. One major challenge is the cost and complexity associated with large-scale manufacturing. The clinical translation of exosomes must overcome technical hurdles, such as difficulties in efficient isolation, purification, and storage, as well as the challenge of ensuring consistent quality and functionality during large-scale production. Furthermore, the high cost of manufacturing processes means cost reduction is a top priority.^152,153 Meanwhile, the use of exosomes in research and medicine raises a range of ethical concerns, including how to ensure their legality, confidentiality, and prevention of misuse. Exosomes isolated from biological samples cannot be used directly without donor consent. Donors must be fully informed about how their samples will be used and the associated risks. Since exosomes may carry sensitive biological information, confidentiality is critical and requires strict regulations and data management. To address these issues, clear and genuine informed consent procedures must be followed to ensure donors understand the purpose of their biological materials. Standards must also be established for isolation, purification, and characterization methods to minimize variability and potential biohazards.

Another major limitation is the uncertainty surrounding exosome-based therapies within the regulatory landscape. Insufficient regulatory guidance can delay market adoption and the implementation of clinical trials. Furthermore, exosomes may elicit immune responses or adverse biological effects, and could even promote tumor invasion and metastasis. Accordingly, the safety profile of exosomes must be rigorously evaluated in preclinical studies to ensure patient protection.

Exosomes exhibit a distinct double-edged sword characteristic in the complex pathogenesis of OC. On one hand, exosomes secreted by certain cells can act as tumor promoters and serve as key drivers of malignant progression. Furthermore, exosomes mediate multiple forms of drug resistance, including chemoresistance and targeted therapy resistance, representing a major cause of clinical treatment failure. On the other hand, exosomes also show considerable application potential and represent a ray of hope for ovarian cancer therapy. Exosomes are widely distributed in biofluids such as blood and urine, and carry tumor-specific molecular signatures, making them ideal biomarkers for liquid biopsy. Engineered exosomes possess excellent biocompatibility, tissue penetration capacity, and targeting specificity, qualifying them as promising nanocarriers.

In-depth exploration of the complex signaling networks mediated by exosomes and their bidirectional roles in OC is of great significance. It will not only facilitate a more comprehensive understanding of the disease essence and help overcome clinical challenges such as tumor metastasis and drug resistance, but also lay a solid foundation for the development of exosome-based noninvasive diagnostic techniques, precise prognostic evaluation methods, and targeted therapeutic strategies.

With the continuous deepening of understanding of the heterogeneity of extracellular vesicles, their carried substances, and their functions, the demand for accurate characterization of exosomes is also increasing. Although numerous studies have been devoted to revealing the production and endocytosis processes of exosomes, as well as the molecular mechanisms underlying their biological roles in tumor progression, chemoresistance, diagnosis, and treatment, many issues remain to be further studied in depth. Issues including large-scale production in compliance with good manufacturing practice (GMP), long-term biosafety evaluation, and ethical considerations related to biological origin remain the core barriers impeding their clinical translation. Only by continuously exploring these issues can it be possible to translate research results into clinical practice, optimize the diagnostic procedures, screening methods, and treatment decisions for ovarian cancer, and even achieve personalized treatment.

Funding Statement

Key Research and Development Project of Liaoning Province (2024JH2/102500019); LiaoNing Revitalization Talents Program (XLYC2412037).

Abbreviations

OC, Ovarian cancer; PARP, Poly (ADP-ribose) polymerase; LNPs, Lipid nanoparticles; CNTs, Carbon nanotubes; EPR, Enhanced permeability and retention; ncRNAs, Non-coding RNAs; miRNAs, MicroRNAs; lncRNAs, Long non-coding RNAs; circRNAs, Circular RNAs; mRNAs, Messenger RNAs; TME, Tumor microenvironment; MVBs, Multivesicular bodies; ELISA, Enzyme-linked immunosorbent assay; PEG, Polyethylene glycol; SERS, Surface-enhanced Raman scattering; iMEX, Integrated magnetic electrochemical sensor; MINDS, Multi-scale integrated design self-assembly; CSA, Colloid self-assembly; nano-HB, Nano-herringbone; MS-OTs, Multi-spectral optical tweezers; LTRS, Laser trapping Raman spectroscopy; PKR1, Prokineticin receptor 1; STAT3, Signal transducer and activator of transcription 3; HOXD11, Homeobox protein homeobox D11; CAFs, Cancer-associated fibroblasts; FN1, Fibronectin 1; VEGF, Vascular endothelial growth factor; PECAM-1, Platelet endothelial cell adhesion molecule-1; ATF2, Activating transcription factor 2; MTA1, Metastasis-associated protein 1; ROCK1/2, Rho-associated coiled-coil containing protein kinase 1/2; HUVECs, Human umbilical vein endothelial cells; SOCS-5, Suppressor of cytokine signaling 5; ATB, Activated by TGF-β; MALAT1, Lung adenocarcinoma transcript 1; VEGF-A, Vascular endothelial growth factor-A; VEGF-D, Vascular endothelial growth factor-D; EMT, Epithelial-mesenchymal transition; α-SMA, α-smooth muscle actin; MUC1, Mucin 1; HTERT, Human telomerase reverse transcriptase; ANXA2, Annexin A2; FAP, Fibroblast activation protein; TAMs, Tumor-associated macrophages; PBMCs, Polarization in peripheral blood mononuclear cells; HIF, Hypoxia-inducible factor; GLI1, Glioma-associated oncogene homolog 1; ETS1, E26 transformation-specific sequence 1; CXCL5, C-X-C motif chemokine ligand 5; CCL2, C-C motif chemokine ligand 2; SMAD4, Mothers against decapentaplegic homolog 4; pGSN, Plasma gelsolin; GSN, Gelsolin; HREs, Hypoxia-response elements; Rab27a, Ras-related protein Rab-27a; Rab7, Ras-related protein Rab-7; LAMP1/2, Lysosome-associated membrane protein 1/2; NEU-1, Neuraminidase-1; TMEM205, Transmembrane protein 205; CD1B, Cluster of differentiation 1B; CA125, Cancer antigen 125; FIGO, International federation of gynecology and obstetrics; HGSC, High-grade serous carcinoma; ESRG, Embryonic stem cell - related gene; EpCam, Epithelial cell adhesion molecule; ZNF587B, Zinc finger protein 587B; ERGRS, Exosome-related gene risk score; PD-L1, Programmed death-ligand 1; PD-1, Programmed death-1; IDO1, Indoleamine 2,3-dioxygenase 1; ALCAM, Activated leukocyte cell adhesion molecule; TSAP6, Tumor suppressor candidate 6; MHC, Major histocompatibility complex; TLR3, Toll-like receptor 3; CTLs, Cytotoxic T lymphocytes; ROS, Reactive oxygen species; PS, Phosphatidylserine; X-BMT, Melanoma-based xenograft; OTX, Ovarian tumor-based omental tumor xenograft; TP, Triptolide; Lamp2b, Lysosome-associated membrane glycoprotein 2b; hUCMSCs, Human umbilical cord mesenchymal stem cells; eNK-EXO, Natural killer cell-derived exosomes; SOX4, SRY-related HMG-box 4; GMP, Good manufacturing practice.

Author Contributions

Fengyi Wang drafted this review and designed the figures; Haiyan Dong contributed substantially to the discussion and revision of all the content,Yuli Song checked the manuscript,Yi Zhang provided the design and revision of the manuscript. All authors made substantial, direct and intellectual contribution to the review. All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no competing interests.