Extracellular Vesicles as Immunotherapeutic Mediators in Gastrointestinal Cancers and Diseases: From Mechanisms to Clinical Translation

Abstract

Immunotherapy has transformed cancer treatment, yet its efficacy in gastrointestinal (GI) cancers and inflammatory diseases remains limited, underscoring the need for more effective immunomodulatory strategies. Multiple biomaterial‐based delivery platforms, including liposomes, polymeric nanoparticles, viral vectors, and inorganic nanocarriers, have been employed to enhance immune regulation and therapeutic transport. However, their clinical translation is hindered by intrinsic limitations such as immunogenicity, variable biocompatibility, nonspecific tissue distribution, payload instability, and challenges in scalable and reproducible manufacturing. These constraints highlight the need for safer and more clinically adaptable delivery systems. Extracellular vesicles (EVs), as naturally derived nanoscale carriers, have emerged as promising therapeutic tools due to their unique advantages, such as biocompatibility, low immunogenicity, precise targeting, and multifunctional molecular delivery. This review synthesizes recent advances in EV‐based immunotherapeutic strategies for GI cancers and diseases, emphasizing approaches relevant to clinical pharmacology. Key innovations include EV vaccines, modulation of the immune microenvironment using cell source‐specific EVs, and engineered EVs for the targeted delivery of cytokines, nucleic acids, or immune checkpoint inhibitors. Emerging applications involving EV‐mediated transport of CAR constructs, oncolytic agents, and gene‐editing tools further broaden their therapeutic potential. In conclusion, EV‐based therapies offer transformative approaches through multimodal immune modulation and precision drug delivery to GI diseases. However, standardization, large‐scale manufacturing, targeted delivery, and addressing tumor heterogeneity with personalized EV engineering will be crucial for clinical success in GI diseases. Overcoming these barriers will accelerate EV integration into next‐generation immunotherapy and precision oncology.

OVERVIEW OF EXTRACELLULAR VESICLES

Gastrointestinal (GI) cancers and diseases rank among the most prevalent and lethal malignancies worldwide, characterized by high heterogeneity and a complex immune microenvironment. These features present significant challenges for diagnosis and treatment. While conventional therapies such as surgery, chemotherapy, and radiotherapy remain the cornerstone of clinical management, their efficacy is often limited in advanced and metastatic cases. In recent years, emerging therapeutic strategies, including immunotherapy, nanomedicine, and cell‐based therapies, have shown considerable promise in addressing GI‐related diseases. ¹ , ² However, challenges such as drug resistance, interpatient variability, and insufficient targeting precision continue to hinder their widespread clinical application. ³ , ⁴ Notably, the intricate tumor immune microenvironment further compromises therapeutic efficacy, particularly in immunotherapy, highlighting the urgent need for approaches that enable precise delivery and coordinated immune modulation. The complexity of the tumor immune microenvironment also poses a major barrier to immunotherapy efficacy, necessitating combination approaches and reliable predictive biomarkers. Consequently, the development of novel precision therapies and reliable biomarkers is imperative, not only to refine diagnostic and prognostic approaches but also to advance personalized treatment paradigms and improve patient survival outcomes.

In recent years, extracellular vesicles (EVs) have emerged as a compelling biological platform with unique advantages for both therapeutic intervention and biomarker development in GI cancers. EVs are typically considered in three closely related aspects: their biogenesis, the methods for collection and isolation, and approaches for characterization. In the canonical model, exosomes are generated through the endosomal pathway and released when multivesicular bodies fuse with the plasma membrane, although the origin of some vesicle subtypes remains incompletely defined. ⁵ , ⁶ Current isolation methods, including ultracentrifugation, size‐exclusion chromatography, precipitation‐based approaches, and immunoaffinity capture, possess inherent advantages and limitations in terms of yield, purity, scalability, and reproducibility. ⁷ Increasing evidence therefore supports the use of combined workflows to improve vesicle quality and reduce contamination. ⁸ Likewise, reliable characterization requires complementary methods, including morphological assessment, particle size and concentration analysis, and detection of representative membrane markers. Additional proteomic and RNA‐based analyses can further refine the evaluation of vesicle composition and heterogeneity. Together, these methodological considerations are fundamental for interpreting EV biology and developing clinically relevant EV‐based diagnostics and therapeutics.

Exosomes, a subclass of small EVs ranging from 30 to 150 nm, differ from larger microvesicles (100–1,000 nm) in size, biogenesis, and surface protein composition. ⁵ , ⁹ , ¹⁰ Exosomes originate from the endosomal system via inward budding of multivesicular bodies, whereas microvesicles arise from direct outward budding of the plasma membrane. These compositional differences are functionally significant: exosomal surface proteins, including tetraspanins (CD9, CD63, CD81), integrins, and adhesion ligands, mediate selective interactions with receptors on target cells, facilitating receptor‐mediated endocytosis or membrane fusion and enabling delivery of miRNAs, proteins, or therapeutic cargo. ¹¹ , ¹² Compared with exosomes, microvesicles exhibit broader and less receptor‐specific interactions due to differences in their surface protein repertoire. The intrinsic tropism of exosomes allows them to accumulate in specific tissues, and their nanoscale size, lipid bilayer composition, and membrane protein compatibility enable them to traverse biological barriers, including the blood–brain barrier, via receptor‐mediated transcytosis or endocytosis. ¹³ , ¹⁴ Beyond their natural targeting capacity, engineering strategies can further enhance recognition and delivery efficiency. Surface modification with targeting peptides or antibodies, membrane fusion, morphological optimization (e.g., nanorods or microspheres), and stimulus‐responsive designs (such as pH‐sensitive or externally triggered release) have all been shown to improve uptake by receptor‐expressing cells and therapeutic efficacy in vitro and in vivo. ¹¹ , ¹² , ¹⁵ Together, these intrinsic and engineered features explain how exosomes achieve specific recognition and efficient cargo delivery to recipient cells, distinguishing them from other EV subtypes and supporting their potential as precision nanocarriers for gastrointestinal (GI) and other diseases.

EVs exhibit unique biological properties, enabling them to efficiently transport a diverse array of bioactive molecules, including miRNAs, proteins, lipids, and RNAs, while shielding these cargoes from degradation through their lipid bilayer membrane. ¹⁶ Furthermore, their low immunogenicity and excellent biocompatibility render EVs ideal therapeutic carriers. These attributes have spurred significant interest in their application for treating GI cancers and GI‐related diseases. Importantly, accumulating evidence suggests that the functions of EVs extend beyond passive cargo delivery. Recent studies further indicate that EVs function not merely as passive carriers, but also as active biological modulators capable of reprogramming recipient cells and reshaping the tumor microenvironment (TME). Accumulating preclinical and clinical evidence shows that EV‐based delivery enhances therapeutic specificity, improves pharmacokinetic profiles, and reduces systemic toxicity, thereby reinforcing their translational potential in cancer therapy. ¹⁷ Mechanistically, EVs can exert therapeutic effects through multiple aspects. First, natural EVs exhibit unique advantages in cancer therapy owing to their low immunogenicity, intrinsic bioactivity, and tumor‐homing properties. ¹⁸ , ¹⁹ Beyond their direct biological functions, EVs also play an active role in shaping tumor progression through intercellular communication. Second, therapeutic strategies targeting EVs primarily focus on disrupting their biogenesis, secretion, or uptake to interfere with their TME‐modulating functions. Emerging evidence has identified several key components within EVs—such as miRNAs, long noncoding RNAs (lncRNAs), proteins, and signaling molecules—as potential therapeutic targets. ²⁰ Third, EVs serve as versatile nanocarriers for delivering bioactive molecules, including miRNAs, siRNAs, shRNAs, and chemotherapeutic agents. Finally, engineered EVs hold immense potential in disease treatment. Current engineering strategies include surface modification, drug/gene loading, endogenous modification, hybrid EV construction, and bioproduction optimization. ²¹ , ²²

We have contributed to the translational application of EVs. We previously demonstrated that tumor‐derived exosomal PTPRO exerts antitumor activity by polarizing macrophages and suppressing breast cancer cell invasion and migration. Furthermore, in esophageal cancer, we identified salivary exosomal miRNAs, small RNAs, and chimeric GOLM1‐NAA35 RNA as promising noninvasive biomarkers for early detection and prognostic assessment. Collectively, these findings further support the multifunctional and translational potential of EVs. ²³ , ²⁴ , ²⁵ , ²⁶ Building upon these properties, EVs have gained particular attention in the context of cancer immunotherapy. ²⁷ EV‐based immunotherapeutic strategies encompass a wide range of approaches, including the loading of tumor‐specific antigens onto EVs to elicit immune responses, using immune cell‐derived EVs to remodel the TME, and using tumor cell‐derived EVs to directly activate immune cells or inhibit tumor growth. ²⁸ Moreover, EVs can serve as carriers for small‐molecule drugs or RNAs to modulate immune pathways, while engineered EVs can be designed to amplify immunotherapeutic effects. ²⁹ Notably, EV‐based platforms also provide opportunities to enhance existing immunotherapies. Combining EVs with immune checkpoint inhibitors has further demonstrated synergistic efficacy in enhancing treatment outcomes. ³⁰ These strategies have already yielded promising results in the immunotherapy of GI‐related diseases. Collectively, these findings position EVs at the intersection of precision medicine, immunotherapy, and nanotechnology.

In summary, EVs represent a transformative therapeutic platform with vast potential in the treatment of GI cancers and related diseases. Their multifunctionality, precision targeting, and low toxicity highlight their potential as key components of future therapeutic strategies.

EXTRACELLULAR VESICLE‐BASED IMMUNOTHERAPEUTIC STRATEGIES FOR GASTROINTESTINAL CANCERS AND DISEASES

EVs have been employed in cancer therapy through four primary strategies including therapeutic drug delivery vehicles, direct modulation of tumor immunity, therapeutic targets and engineered EVs. ¹⁷ In the context of cancer immunotherapy, EVs have gained significant attention, especially in GI cancers, due to their multifunctionality and excellent delivery properties. ³¹ As natural biological carriers, EVs can interact with the immune system through their membrane structure and cargo, which in turn activates antitumor immune responses and improves the TME. A major immunotherapeutic application of EVs is as vaccine delivery platforms. EVs can serve as carriers for cancer vaccines by transporting tumor‐associated antigens to antigen‐presenting cells, thereby eliciting tumor‐specific immune responses. In parallel, EVs derived from immune cells represent another important modality for immune modulation. Immune cell‐derived EVs, by carrying specific immune factors, help modulate the immune environment to suppress tumor growth or improve disease conditions. Beyond immune cell‐derived EVs, tumor cell‐derived EVs have also been explored for immunotherapeutic purposes. Similarly, tumor cell‐derived EVs hold promise in delivering antigens, miRNAs, or signaling molecules that directly target tumor cells and activate immune responses. In addition to their role in antigen delivery, EVs also function as versatile carriers for immunomodulatory agents. Moreover, EVs can effectively transport small molecules or RNAs to enhance immune modulation within the TME. To further optimize these immunotherapeutic functions, substantial efforts have been devoted to the engineering of EVs. Genetic or structural modifications can enhance EV immune activation and targeting, improving immunotherapy efficacy. Importantly, EV‐based platforms also enable rational combination strategies. Combining EVs with adjuvants that block immune checkpoints opens new avenues for multimodal therapies. ³² Building on these established strategies, recent advances have expanded EV‐based immunotherapy to more sophisticated and cell‐free therapeutic modalities. Recent advancements have also highlighted the potential of EV‐based strategies such as CAR‐T‐cell therapy, delivery of oncolytic viruses, and CRISPR gene editing, all of which further demonstrate the versatility and potential of EVs in GI cancer immunotherapy (Figure 1 , Table 1 ).

Figure 1.

Comparison between naive and engineered EVs (graph was created in bioRender.com).

Table 1.

Applications of EV‐based immunotherapy in gastrointestinal tumors and diseases

Therapy strategies	Type of cancers	Sources of EVs	Therapeutic molecule or function	Year of study
Extracellular vesicles (EVs)‐Based Vaccines	Colorectal cancer	Tumor cell‐derived EVs	Carry fibroblast activation protein (FAP) to reprogram tumor‐associated fibroblasts and enhance antitumor immunity	2021 33
	Colorectal cancer	Tumor cell‐derived EVs	Deliver miR‐124‐3p to suppress PD‐L1 expression in dendritic cells and enhance cytotoxic T‐cell activity	2021 34
	Colorectal cancer	Heat shock‐treated tumor‐derived EVs	Enriched in HSP70 to activate dendritic cells and promote Th1 immune responses	2018 35
	Colorectal cancer	Dendritic cell‐derived EVs	Deliver Hsp70 to enhance antigen cross‐presentation and activate CD8+ T cells	2009 36
	Liver cancer, Colorectal cancer	Tumor cell‐derived EVs	Carry IRF‐1 to induce interferon signaling and enhance NK cell‐mediated tumor killing	2017 37
	Liver cancer	Tumor cell‐derived EVs	Regulate Rab27a to suppress EV‐mediated immunosuppressive signaling	2023 38
	Liver cancer	Dendritic cell‐derived EVs	Deliver P47‐P, AFP212‐A2, and N1ND‐N peptides to stimulate tumor‐specific T‐cell responses	2022 39
	Liver cancer	Tumor cell‐derived EVs	Deliver N1ND peptide as an immune adjuvant to activate dendritic cells	2020 40
	Liver cancer	Dendritic cell‐derived EVs	AFP‐enriched exosomes from DCs act as a cancer vaccine, triggering antigen‐specific immune responses, reshaping the tumor microenvironment, and inhibiting HCC progression through T‐cell activation	2017 41
	Pancreatic cancer	Tumor cell‐derived EVs	Loaded with MART‐1 antigen and CCL22 siRNA to inhibit regulatory T‐cell recruitment	2022 42
	Esophageal cancer	Dendritic cell‐derived EVs	Carry A‐P peptide for targeted delivery to tumor cells and T‐cell activation	2022 43
Immune cell‐derived EVs therapy	Colorectal cancer	M2 macrophage‐derived EVs	Carry miR‐155‐5p, which promotes immune escape in colon cancer by targeting ZC3H12B, upregulating IL‐6, and enhancing tumor progression	2021 44
	Gastric cancer	M2 macrophage‐derived EVs	Transfer miR‐155‐5p to inhibit ZC3H12B and promote immune evasion	2021 45
	Gastric cancer	M1 macrophage‐derived EVs	Contain miR‐16‐5p to inhibit PD‐L1 expression and promote T‐cell infiltration	2020 46
	Gastric cancer	Dendritic cell‐derived EVs	Loaded with tumor antigens to enhance T‐cell priming	2015 47
	Pancreatic cancer	M2 macrophage‐derived EVs	Deliver lncRNA SBF2‐AS1, which modulates the miR‐122‐5p/XIAP axis to promote pancreatic cancer progression by repressing miR‐122‐5p	2020 48
	Chronic pancreatitis	Bone marrow‐derived dendritic cell EVs	EVs modulate immune response by reducing pro‐inflammatory cytokines (TNF‐α, TGF‐β) and enhancing antioxidant enzymes, improving pancreatic function	2023 49
	Esophageal cancer	M2 macrophage‐derived EVs	Transfer lncRNA AFAP1‐AS1, which downregulates miR‐26a and upregulates ATF2, promoting invasion and metastasis of esophageal cancer cells	2020 50
	Inflammatory bowel disease	M1 macrophage‐derived EVs	miR‐21a‐5p in exosomes modulates immune regulation by promoting ILC2 activation and inhibiting E‐cadherin, contributing to UC immunopathogenesis	2021 51
	Liver transplant	CD4 + CD25+ regulatory T cells	Deliver TGF‐β and IL‐10 to suppress cytotoxic T‐cell activity in the tumor microenvironment	2019 52
Tumor cell‐ derived EVs therapy	Colorectal cancer	Colorectal cancer cell‐derived EVs	Carry lncRNA RPPH1 to promote macrophage M2 polarization and metastasis	2019 53
	Pancreatic cancer	Pancreatic cancer cell‐derived EVs	Deliver miR‐212‐3p to suppress MHC II expression in dendritic cells	2015 54
EVs as carriers for immune modulators	Colorectal cancer	Mesenchymal stem cell‐derived EVs	Deliver paclitaxel for tumor suppression and modulate the tumor microenvironment by activating CD4+/CD8+ T cells, polarizing TAMs to M1, and reducing Tregs	2023 55
	Colorectal cancer	CD47‐overexpressing CT26 cell‐derived EVs	Encapsulate thermosensitive liposomes loaded with chemotherapy drugs	2021 56
	Pancreatic cancer	Bone marrow mesenchymal stem cell‐derived EVs	Deliver galectin‐9 siRNA and oxaliplatin prodrug to trigger immunogenic cell death and reverse tumor immunosuppression	2020 57
	Liver cancer	Milk‐derived EVs	Carry β‐catenin siRNA to regulate Wnt signaling to reduce tumor growth and enhance the therapeutic response to anti‐PD‐1	2019 58
Engineered EVs for immunotherapy	Colorectal cancer	HEK293 cell‐derived EVs	Surface‐engineered with PTGFRN to deliver IL‐12 for localized immune activation	2021 59
	Liver cancer	Mesenchymal stem cell‐derived EVs	Modified exosomes with cationized pullulan targeting asialoglycoprotein receptors on hepatocytes for enhanced liver injury treatment	2017 60
Combination therapy	Colorectal cancer	Cancer cell‐derived EVs	Hybridized with liposomes and PD‐L1 antibodies for dual immune checkpoint inhibition	2023 61
	Liver cancer	Cell membrane nanovesicles	Combines immune checkpoint blockade and oncolytic virotherapy by delivering oncolytic adenovirus via PD‐1 nanovesicles, enhancing T‐cell activation and antitumor immunity	2021 62
EVs‐delivered CRISPR gene editing	Liver cancer	Engineered HEK293T‐derived EVs	Deliver Cas9 ribonucleoproteins to knock out oncogenes (e.g., MYC)	2022 63
	Acute hepatic damage, Liver fibrosis and Liver cancer	Hepatic stellate cell‐derived EVs	EVs modified with DNA nanostructures deliver CRISPR‐Cas9 RNP for targeted genome editing, downregulating WNT10B to inhibit tumor growth	2020 64
	HCC	Normal epithelial cell ‐derived EVs engineered with HN3 (HLC9‐EVs)	Engineered EVs encapsulated CRISPR/Cas9 and sorafenib reversed sorafenib resistance by reduction of CD133 + population that contribute to the stemness of liver cancer cells	2023 65
EVs‐delivered oncolytic viruses	Pancreatic cancer	Tumor cell‐derived EVs	Encapsulate oncolytic virus (e.g., adenovirus) carrying amiR‐4 to enhance viral replication in tumors	2022 66
	Colorectal cancer, Gastric cancer	Tumor cell‐derived EVs	Deliver oncolytic adenoviruses, protecting them from host immunity, bypassing receptor barriers, and enhancing tumor cell cytolysis	2016 67

Extracellular vesicles‐based vaccines for gastrointestinal tumors and diseases

The development of tumor cell‐based vaccines faces significant challenges, including poor immunogenicity, tumor heterogeneity, an immunosuppressive TME, and ineffective delivery strategies. These limitations have prompted increasing interest in alternative vaccine platforms capable of eliciting robust and durable antitumor immune responses. Recently, naturally released EVs from cells have emerged as ideal drug carriers and vaccine platforms. EVs exhibit high organ‐specific targeting capability, can induce broader and more effective immune responses, and demonstrate superior tissue delivery efficiency. As a result, EV‐based vaccines have been increasingly recognized as a critical component in the advancement of cancer immunotherapy. ⁶⁸ Compared with cell‐based vaccines, EV vaccines produced under good manufacturing practice (GMP) standards offer advantages such as higher safety, ease of storage and transportation, and diverse sourcing.

Immune cell‐derived extracellular vesicles for treating gastrointestinal tumors and diseases

EVs derived from various cell types, including immune cells, cancer cells, epithelial cells, and mesenchymal cells, play crucial roles in modulating the proliferation and activity of target cells within both the innate and adaptive immune systems. Among these, immune cell‐derived EVs are of particular interest due to their intrinsic immunoregulatory properties. Immune cell‐derived EVs can specifically modulate the immune response by delivering novel antigens, immune modulators, or therapeutic agents. The entry of EVs into target cells often occurs through mechanisms like receptor‐mediated endocytosis, clathrin‐coated pits, lipid rafts, phagocytosis, clathrin‐independent endocytosis, or macropinocytosis. Upon internalization, the content of EVs can trigger intracellular signaling pathways or fuse with the target cell's membrane, thereby delivering their cargo to the cytoplasm, where they exert their biological effects. Importantly, advances in genetic engineering have further expanded the therapeutic potential of immune cell‐derived EVs. Genetically engineered immune cells can be optimized to enhance the antitumor activity of their secreted EVs, offering new opportunities for precision immunotherapy. ⁶⁹ , ⁷⁰

Tumor cell‐derived extracellular vesicles for treating gastrointestinal tumors and diseases

In contrast to immune cell‐derived EVs, tumor cell‐derived EVs provide a complementary strategy that exploits tumor‐intrinsic properties for therapeutic delivery. Tumor cell‐derived EVs have emerged as highly promising therapeutic vehicles in the treatment of GI‐related diseases. ⁷¹ , ⁷² These vesicles can carry a range of tumor‐associated molecules, including antigens, immunosuppressive factors, and oncogenes, which enable precise and efficient delivery to tumor sites. Through interactions with tumor‐specific surface molecules, tumor‐derived EVs (TDEs) exhibit enhanced targeting specificity. As a result, they can serve as effective carriers for anticancer agents, siRNAs, and miRNAs, facilitating targeted regulation of tumor cell gene expression and modulation of the TME. Furthermore, engineering these EVs can enhance their targeting ability, stability, and drug‐loading capacity, optimizing their antitumor effects.

Utilizing extracellular vesicles as carriers for immune modulators

In addition to serving as antigen carriers or tumor‐targeting vehicles, EVs are increasingly utilized as delivery systems for immune‐modulatory agents. EVs are increasingly recognized for their potential in tumor therapy, primarily for precision drug delivery, immune modulation, and TME regulation strategies. ⁷³ Due to their inherent capability to target and accumulate at tumor sites, EVs can effectively deliver antitumor agents, nucleic acid‐based therapies, or immune modulators directly to tumors, thereby enhancing therapeutic efficacy while minimizing off‐target effects. Beyond exogenous cargo delivery, EVs also exert endogenous immunoregulatory functions. EVs naturally carry immune‐regulatory molecules that influence immune cell activity, suppress tumor growth, and inhibit metastatic progression. Furthermore, EVs contribute to tissue repair processes following radiotherapy and chemotherapy. Despite their potential, challenges remain in optimizing the biodistribution, half‐life, and cell‐targeting capabilities of EVs in vivo. Moreover, the heterogeneity of EV composition and the lack of standardized protocols for detection and evaluation may impact the consistency and effectiveness of EV‐based therapies. Nonetheless, EVs continue to hold considerable promise as drug delivery vehicles and immune modulators, with substantial potential for advancing precision medicine and personalized cancer treatments in the future.

Developing engineered extracellular vesicles for immunotherapy

To overcome the intrinsic limitations of natural EVs, extensive efforts have focused on the development of engineered EVs. Engineered EVs have shown significant potential in tumor immunotherapy, primarily through various strategies to enhance their immune‐modulating effects. ⁷⁴ , ⁷⁵ First, they can boost immune cell function, such as overexpressing CD47 to promote macrophage phagocytosis or carrying immune adjuvants to stimulate dendritic cell (DC) maturation, increasing CD8+ and CD4+ T‐cell infiltration. Furthermore, EVs can also deliver siRNA to modulate tumor–immune interactions, suppressing TAM polarization and enhancing immunotherapy. Additionally, they serve as tumor vaccine carriers and efficient drug delivery platforms, improving tumor targeting. However, challenges remain in optimizing targeting, large‐scale production, purification, and quality control for clinical application.

Extracellular vesicle‐CAR‐T potential and challenges

Building on engineered EV platforms, EV‐based chimeric antigen receptor (CAR‐T) strategies have emerged as a cell‐free extension of adoptive cell therapy. While CAR‐T‐cell therapy has achieved remarkable success in hematologic malignancies, its efficacy in GI cancers remains limited by multiple barriers, including an immunosuppressive TME, insufficient tumor infiltration, and severe treatment‐related toxicities. ⁷⁶ To overcome these obstacles, emerging strategies such as engineered bacteria‐assisted CAR T‐cell therapy have been proposed, leveraging the tumor‐homing capacity and immunomodulatory properties of bacteria to reshape the TME and enhance CAR T‐cell antitumor activity. ⁷⁷ EV‐based CAR‐T therapy offers several advantages over traditional CAR‐T‐cell therapies, particularly due to its lower immunogenicity and toxicity. ⁷⁸ , ⁷⁹ , ⁸⁰ This strategy involves delivering antitumor molecules directly to tumor cells using EVs, which are naturally able to home to tumor sites. Compared with conventional CAR‐T cells, EV‐based CAR‐T therapy reduces the risk of cytokine release syndrome (CRS) and can penetrate tumor barriers more effectively. The nanoscale size of EVs enhances their ability to target and deliver molecules to solid GI tumors while evading immune system clearance, thereby improving antitumor efficacy. Furthermore, the short lifespan and non‐proliferative nature of EVs make them a ready‐to‐use, low‐toxicity option, reducing the risk of in vivo degradation and systemic toxicity typically associated with traditional CAR‐T therapies. Since EVs are derived from endogenous cells, they exhibit excellent biocompatibility and low toxicity, providing a safer treatment option. Additionally, EVs can be produced and stored on a large scale, independent of living cells. However, challenges such as optimizing biodistribution, targeting ability, stability, and overcoming immune escape mechanisms must be addressed to ensure successful clinical translation.

Extracellular vesicle‐delivered oncolytic viruses: Prospects

Another emerging EV‐based strategy involves the delivery of oncolytic viruses (OVs), which represent a distinct class of immunotherapeutic agents. Accumulating preclinical and clinical evidence demonstrates their potential across multiple malignancies, positioning oncolytic virotherapy as an important component of next‐generation cancer immunotherapy. However, the clinical efficacy of OVs is often limited by immune clearance and inefficient tumor delivery, prompting the exploration of novel delivery strategies. ⁸¹ The therapeutic strategy utilizing EVs to deliver OVs leverages EVs as carriers to provide more effective antitumor treatments. EV‐encapsulated OVs can evade immune recognition, reducing immune clearance reactions and enhancing virus infection rates and spread within tumor cells. ⁸² Furthermore, EVs provide an alternative entry route for viruses, ensuring efficient gene transfer to targeted cancer cells. Studies show that EVs secreted by oncolytic virus‐infected cancer cells (IEVs) carry viral genes and can initiate infection in other cancer cells, offering new directions for viral and gene therapies. However, further research is required to improve EV tumor targeting, ensure efficient viral load delivery, and avoid adverse effects on surrounding healthy cells. Additionally, optimizing EV release and enhancing its infectious effect in different types of tumors requires further exploration. Nevertheless, EV‐delivered OVs hold promising potential in tumor immunotherapy, particularly in enhancing oncolytic virus efficacy, reducing immune rejection, and improving targeting.

Extracellular vesicle‐delivered CRISPR gene editing

Beyond immunomodulation and viral delivery, EVs have also been explored as carriers for genome‐editing technologies. EV‐based CRISPR gene‐editing systems offer significant advantages, including high biocompatibility, stability, and low immune responses. EVs efficiently encapsulate CRISPR/Cas9 components within their lipid bilayer, ensuring sustained circulation, reduced clearance, and enhanced targeting delivery efficiency. Key strategies include loading Cas9 proteins and sgRNAs into EVs through cell transfection, employing fusion proteins, reversible protein–protein interactions, and specific signal peptides to improve loading efficiency. Additionally, light activation or chemical conjugation methods can regulate the loading and release of Cas9. ⁸³ However, despite these strategies improving the loading efficiency of CRISPR components, challenges remain, especially in targeting delivery of EVs, overcoming endogenous and exogenous barriers, and achieving precise release mechanisms for CRISPR components. Moreover, the design of EV delivery systems still faces technical obstacles, including subtype identification, standardized loading efficiency, and efficiency in nuclear delivery. Overall, although EV‐based delivery of CRISPR gene‐editing systems shows great potential, further optimization of the delivery systems and improvement of efficiency are necessary to address‐related technical challenges for clinical applications.

Combination therapy: The application of multistrategy treatment

Given the multifaceted functions of EVs, combination therapies represent a logical extension of EV‐based therapeutic strategies. EV‐based combination therapy mainly includes the co‐delivery of chemotherapy drugs, therapeutic nucleic acids, and immunotherapies. Additionally, through engineered modifications of EVs, dual or multiple drugs can be co‐delivered, improving pharmacokinetic properties, enhancing tumor accumulation, and improving the efficiency of targeted delivery. However, some challenges remain, particularly in the selection of combination therapy regimens and optimization of delivery routes. Not all combination strategies effectively enhance antitumor effects, and certain combinations may increase the risk of adverse reactions. Despite these challenges, EV‐based combination therapies have shown good efficacy in various cancer animal models, highlighting their immense potential for future clinical applications.

APPLICATION OF EXTRACELLULAR VESICLE‐BASED IMMUNOTHERAPY IN GASTROINTESTINAL TUMORS AND DISEASES

EV‐based immunotherapy represents a significant advance in the treatment of GI tumors, offering a versatile and precise approach that integrates various therapeutic strategies, including tumor vaccines, modulation of the immune microenvironment, and engineered delivery systems. ³¹ , ⁸⁴ These nanoscale vesicles, comprising exosomes and microvesicles, are crucial mediators of intercellular communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids, which can either promote or inhibit tumor progression depending on their origin and cargo. ⁸⁵ As an integral component of precision medicine, EVs offer promising therapeutic potential. The integration of multistrategy approaches will further enhance immunotherapy effectiveness, providing improved treatment options and prognoses for GI tumor patients (Figure 2 ).

Figure 2.

EV‐powered immunotherapies in gastrointestinal cancers and diseases (graph was created in bioRender.com).

Importantly, the biological effects of EVs are not uniformly antitumor. Depending on their cellular origin and molecular cargo, certain EV populations may actively promote tumor progression and metastatic dissemination. For example, platelet‐derived EVs have emerged as a potentially important protumorigenic component of the TME. In colorectal cancer (CRC), platelet‐derived exosomal LINC00183 has been reported to be markedly upregulated and transferable to tumor cells, where it interacts with ENO1 to enhance glycolysis, lactate accumulation, and invasive growth. ⁸⁶ Supporting evidence outside the GI setting further indicates that platelet‐derived exosomes can activate epithelial–mesenchymal transition‐associated programs and angiogenesis, reinforcing their prometastatic potential. ⁸⁷ Beyond platelet‐derived vesicles, EVs from other cellular sources may similarly facilitate metastatic progression in GI malignancies. Representative examples include GP73‐dependent hepatocyte‐derived exosomes enriched in NAV2, which promote CRC liver metastasis, ⁸⁸ myofibroblast‐like cancer‐associated fibroblast‐derived exosomal PWAR6, which facilitates CRC liver metastasis by reshaping glutamine competition within the metastatic niche, ⁸⁹ M2 macrophage‐derived exosomes that promote gastric cancer progression through MALAT1‐dependent metabolic reprogramming, ⁹⁰ and hepatocellular carcinoma‐derived exosomal SLC16A1‐AS1, which induces macrophage M2 polarization and supports malignant progression. ⁹¹ Collectively, these observations highlight the context‐dependent and dual role of EVs in GI tumor biology.

Colorectal cancer

EV‐based strategies have emerged as a multifaceted immunotherapeutic paradigm for colorectal cancer (CRC), encompassing tumor vaccine development, immune microenvironment reprogramming, and engineered combinatorial delivery systems. Early vaccine‐oriented studies demonstrated that TDEs could be rationally engineered to convert the immunosuppressive tumor stroma into an immune‐supportive niche. For instance, Hu et al. showed that exosome‐like nanovesicles modified with fibroblast activation protein (FAP) selectively targeted tumor‐associated fibroblasts, thereby reshaping the stromal compartment, enhancing antitumor immune infiltration, and inducing significant tumor regression in CRC mouse models. ³³ Building on the concept that stress‐associated danger signals enhance EV immunogenicity, heat shock‐treated TDEs enriched in HSP70 were shown to activate DCs and skew CD4⁺ T cells toward a Th1 phenotype, effectively suppressing tumor growth. ³⁵ Consistently, Cho et al. reported that Hsp70‐enriched exosomes induced potent antitumor immune responses independent of MHC restriction, underscoring their capacity to facilitate antigen cross‐presentation and robust CD8⁺ T‐cell activation. ³⁶ These findings collectively established the foundation for EV‐based cancer vaccines in CRC, highlighting the importance of EV cargo composition and immunostimulatory signals in shaping antitumor immunity.

Beyond vaccine applications, EVs critically regulate immune escape and immune suppression within the CRC TME. Tumor‐derived EVs enriched in miR‐124‐3p were shown to downregulate PD‐L1 expression in DCs, thereby restoring cytotoxic T lymphocyte activity and enhancing antitumor immune surveillance in CT26 tumor‐bearing mice. ³⁴ In contrast, immunosuppressive EV signaling also contributes to disease progression: M2 macrophage‐derived EVs carrying miR‐155‐5p promote immune evasion by targeting ZC3H12B, leading to elevated IL‐6 signaling and accelerated tumor growth. ⁴⁴ Similarly, CRC‐derived EVs transporting lncRNA RPPH1 induce macrophage M2 polarization and facilitate metastatic dissemination, emphasizing the bidirectional crosstalk between tumor cells and myeloid populations mediated by EVs. ⁵³

To overcome these immunosuppressive barriers, engineered and hybrid EV platforms have been developed to integrate immune modulation with chemotherapy or cytokine delivery. Mesenchymal stem cell‐derived EVs deliver paclitaxel not only enhancing drug delivery efficiency but also reprogramming the TME by activating CD4⁺ and CD8⁺ T cells, promoting TAM polarization toward the M1 phenotype, and reducing regulatory T‐cell infiltration. ⁵⁵ Furthermore, surface‐engineered EVs have enabled precise cytokine delivery; for example, IL‐12–displaying EVs derived from engineered HEK293 cells achieved potent immune activation with reduced systemic toxicity. ²⁴ In parallel, CD47‐overexpressing CT26‐derived EVs encapsulating thermosensitive liposomes facilitated immune evasion blockade while improving intratumoral drug retention, leading to enhanced tumor clearance and immune remodeling. ⁵⁶

Moreover, advanced hybrid systems combining EVs with immune checkpoint blockade have further amplified therapeutic efficacy. EV–liposome nanovesicles co‐delivering plasmids and PD‐L1 antibodies achieved dual immune checkpoint inhibition, resulting in robust immune activation and marked tumor growth suppression. ⁶¹ Finally, Additionally, TDEs have been exploited as protective carriers for oncolytic adenoviruses, shielding viral particles from neutralizing immunity and enhancing tumor‐selective cytolysis, thereby extending EV applications into combinatorial viro‐immunotherapy for CRC. ⁶⁷ Collectively, these studies demonstrate that EV‐based therapies in CRC have evolved from proof‐of‐concept tumor vaccines toward sophisticated, multifunctional immunotherapeutic platforms.

Pancreatic cancer

EV‐based therapeutic strategies have attracted increasing attention in pancreatic cancer, a malignancy characterized by aggressive biological behavior, profound immunosuppression, and a high proportion of late‐stage diagnosis. Early studies have demonstrated that TDEs can be engineered to actively modulate immune cell recruitment and function within the pancreatic TME. Tumor‐derived EVs loaded with the melanoma‐associated antigen MART‐1 and CCL22 siRNA were shown to suppress regulatory T‐cell (Treg) recruitment by silencing CCL22 expression, thereby enhancing antitumor immune responses and restoring immune surveillance in pancreatic tumor models. ⁴² In contrast to these immune‐stimulatory approaches, accumulating evidence indicates that pancreatic cancer‐derived EVs also play a critical role in immune evasion. Pancreatic cancer cell‐derived EVs carrying miR‐212‐3p downregulate MHC class II expression in DCs, impairing antigen presentation capacity and consequently blunting CD4⁺ T‐cell‐mediated immune activation. ⁵⁴ This EV‐mediated suppression of antigen presentation highlights a key mechanism by which pancreatic tumors evade immune recognition. Beyond tumor cell‐derived EVs, EVs originating from immune cells further exacerbate immunosuppressive signaling within the pancreatic TME. M2 macrophage‐derived EVs enriched in lncRNA SBF2‐AS1 promote pancreatic cancer progression by modulating the miR‐122‐5p/XIAP axis, in which SBF2‐AS1 represses miR‐122‐5p, leading to increased XIAP expression and enhanced tumor cell survival, thereby reinforcing immune suppression and tumor aggressiveness. ⁴⁸

To overcome these immune barriers, EVs have been increasingly explored as delivery vehicles for immune modulators and oncolytic therapeutics. Bone marrow mesenchymal stem cell‐derived EVs co‐delivering galectin‐9 siRNA and an oxaliplatin prodrug were shown to induce immunogenic cell death, reverse tumor‐associated immunosuppression, and promote systemic antitumor immune responses. ⁵⁷ In parallel, TDEs have been leveraged as protective carriers for OVs. EVs encapsulating oncolytic adenoviruses, such as adenovirus expressing amiR‐4, enhance viral stability and replication within pancreatic tumors, thereby improving oncolytic efficacy and extending EV applications into combinatorial viro‐immunotherapy for pancreatic cancer. ⁶⁶ Collectively, these studies highlight the versatility of EVs in modulating the immune environment, suppressing immune evasion, and delivering therapeutic agents, offering new avenues for pancreatic cancer treatment.

Liver cancer

EV‐based therapeutic strategies are rapidly emerging as versatile platforms for liver cancer treatment, particularly hepatocellular carcinoma (HCC), integrating cancer vaccination, immune modulation, targeted delivery, and genome editing. Early studies have demonstrated that TDEs can function as potent immune stimulators by delivering immunoregulatory molecules that activate innate and adaptive antitumor immunity. Tumor‐derived EVs carrying interferon regulatory factor‐1 (IRF‐1) were shown to activate interferon signaling and enhance natural killer (NK) cell‐mediated tumor cytotoxicity, resulting in suppressed tumor growth in HCC models. ³⁷ In parallel, TDEs enriched with N1ND peptides acted as immune adjuvants to promote DC activation and antigen presentation, thereby amplifying antitumor immune responses. ⁴⁰ Consistently, DC‐derived EVs have been exploited as cell‐free cancer vaccines in liver cancer. DC‐derived EVs delivering alpha‐fetoprotein (AFP) or tumor‐specific peptides effectively stimulated tumor‐reactive T‐cell responses, reshaped the immunosuppressive TME, and inhibited HCC progression in preclinical models. ³⁹ , ⁴¹ Beyond EV cargo loading, modulation of EV biogenesis has emerged as an additional immunoregulatory strategy. Regulating Rab27a expression in tumor cells was shown to reduce EV secretion and attenuate EV‐mediated immunosuppressive signaling, thereby restoring antitumor immune activity and enhancing immune surveillance in HCC. ³⁸ EVs released from CD4⁺CD25⁺ regulatory T cells deliver immunosuppressive cytokines, including TGF‐β and IL‐10, to inhibit cytotoxic T‐cell activity, highlighting a potential EV‐mediated mechanism contributing to immune tolerance, particularly in the context of liver transplantation. ⁵²

Beyond immune activation, EVs serve as delivery vehicles for immune modulators, OVs, and gene‐editing tools. Milk‐derived EVs encapsulating β‐catenin siRNA were shown to suppress Wnt/β‐catenin signaling and sensitize HCC tumors to anti‐PD‐1 immunotherapy, thereby enhancing therapeutic responsiveness. ⁵⁸ In parallel, mesenchymal stem cell‐derived EVs engineered with cationized pullulan exhibited enhanced liver targeting and delivery efficiency, improving the therapeutic precision of EV‐based interventions in HCC. ⁶⁰ Combination strategies have further leveraged PD‐1‐coated nanovesicles for the delivery of oncolytic adenoviruses, resulting in enhanced T‐cell activation and synergistic antitumor immune responses. ⁶² More recently, EV‐based platforms have been extended into genome‐editing applications. Hepatic stellate cell‐derived EVs modified with DNA nanostructures were used to deliver CRISPR‐Cas9 ribonucleoprotein complexes, enabling precise genome editing and downregulation of WNT10B, which effectively inhibited tumor growth in liver cancer models. ⁶⁴ Collectively, these studies demonstrate that EV‐based therapies in liver cancer have evolved from immune activation and vaccination strategies toward multifunctional platforms integrating immunotherapy, targeted delivery, and genome editing.

Gastric cancer

EV‐based therapeutic strategies are increasingly being explored in gastric cancer, particularly for immune modulation and oncolytic virotherapy. Accumulating evidence indicates that immune cell‐derived EVs play dual and context‐dependent roles in shaping the gastric TME. M2 macrophage‐derived EVs were shown to transfer miR‐155‐5p, which suppresses ZC3H12B expression, thereby promoting immune evasion and facilitating tumor progression. ⁴⁵ In contrast, EVs released from M1 macrophages contain miR‐16‐5p, which downregulates PD‐L1 expression in tumor cells, enhances cytotoxic T‐cell infiltration, and restores antitumor immune surveillance. ⁴⁶ Consistently, DC‐derived EVs loaded with tumor‐associated antigens have been demonstrated to improve T‐cell priming and activation, further reinforcing antitumor immune responses in gastric cancer models. ⁴⁷ Beyond immune modulation, tumor cell‐derived EVs have also been exploited as delivery vehicles for oncolytic virotherapy. Tumor‐derived EVs encapsulating oncolytic adenoviruses protect viral particles from host immune neutralization, bypass receptor‐dependent entry barriers, and enhance tumor‐selective cytolysis, thereby improving the therapeutic efficacy of oncolytic virotherapy in gastric cancer. ⁶⁷ These findings underscore the potential of EV‐based strategies in gastric cancer, integrating immune modulation and virotherapy for improved therapeutic outcomes.

Esophageal cancer

EV‐based therapies have emerged as promising strategies for esophageal cancer, particularly in cancer vaccine development and immune modulation. We previously highlighted DC‐derived EVs carrying the A‐P peptide as a novel esophageal cancer vaccine, enabling targeted tumor delivery and T‐cell activation. ⁴³ Notably, this study represented the first application of an EV‐based vaccine strategy in mutation‐independent tumors, highlighting the unique advantage of EVs in overcoming neoantigen heterogeneity (Figure 3 ). In parallel with therapeutic applications, EVs also contribute to tumor progression through immune cell‐mediated signaling. M2 macrophage‐derived EVs were shown to transfer the long noncoding RNA AFAP1‐AS1, which promotes tumor invasion by downregulating miR‐26a and upregulating ATF2, thereby facilitating malignant progression in esophageal cancer. ⁵⁰ Collectively, these studies demonstrate the dual roles of EVs in esophageal cancer as both therapeutic vehicles and mediators of tumor progression, underscoring the necessity for precise EV engineering to maximize therapeutic efficacy while minimizing protumorigenic effects.

Figure 3.

EV‐based vaccines for mutation‐independent cancer treatment (graph was created in bioRender.com).

Other GI diseases

Pancreatitis

EV‐based approaches are emerging as promising immunomodulatory strategies for pancreatitis, particularly in the management of chronic inflammatory injury and immune dysregulation. In chronic pancreatitis, bone marrow‐derived DC‐derived EVs were shown to alleviate disease progression by suppressing excessive inflammatory responses. Recent studies emphasize the critical role of exosomes in acute pancreatitis, highlighting their involvement in intercellular communication, regulation of inflammatory responses, and modulation of cell death pathways and macrophage polarization. ⁹² Specifically, these EVs downregulated pro‐inflammatory cytokines, including tumor necrosis factor‐α (TNF‐α) and transforming growth factor‐β (TGF‐β), while simultaneously enhancing antioxidant enzyme activity, thereby improving pancreatic tissue integrity and function in experimental models. ⁴⁹ This study highlights the therapeutic potential of DC‐derived EVs as cell‐free immune modulators capable of restoring inflammatory balance and attenuating chronic pancreatic injury.

Gastritis and gastric ulcers

EVs play a critical role in host immune responses during Helicobacter pylori‐associated gastritis and gastric ulceration, acting as mediators of immune activation, immune regulation, and antimicrobial defense. EVs derived from peripheral blood mononuclear cells (PBMCs) or macrophages of H. pylori‐infected patients exhibited significantly elevated levels of miR‐155 both in vitro and in vivo. ⁹³ These miR‐155‐enriched EVs were shown to initiate and sustain H. pylori‐specific immune responses while exerting potent bactericidal activity against H. pylori. Mechanistically, macrophage‐derived EVs carrying miR‐155 regulate cytokine secretion and intracellular signaling pathways, promote appropriate immune cell polarization, and contribute to immune tolerance within the gastric mucosa. Through reshaping the local immune microenvironment, these EVs effectively alleviate gastric inflammation and facilitate bacterial clearance, highlighting their potential as EV‐based immunotherapeutic agents for gastritis and gastric ulcer treatment.

Hepatitis

EVs have emerged as important modulators of hepatic inflammation and immune homeostasis in hepatitis, exerting protective effects through coordinated regulation of immune signaling, inflammatory cascades, and tissue repair processes. EVs derived from HepAD38 cells have been shown to actively participate in immune regulation by transporting bioactive cargos, including cytokines, lipid mediators, and small regulatory RNAs. These EVs reshape the hepatic immune microenvironment by delivering anti‐inflammatory factors such as interleukin‐10 (IL‐10) and transforming growth factor‐β (TGF‐β), thereby modulating T‐cell activation, regulating DC antigen presentation, and fine‐tuning immune responses. ⁹⁴ Through these mechanisms, HepAD38‐derived EVs effectively attenuate hepatic inflammation, delay fibrosis progression, and promote hepatocyte repair and regeneration, ultimately improving liver injury outcomes in hepatitis models. In parallel, mesenchymal stem cell‐derived EVs have demonstrated robust anti‐inflammatory properties in hepatitis. Bone marrow‐derived mesenchymal stem cell EVs (BMSC‐Exo) deliver miR‐223 to hepatocytes and immune cells, leading to suppression of the NLRP3 inflammasome pathway. By inhibiting NLRP3–caspase‐1 signaling and reducing the release of pro‐inflammatory cytokines, BMSC‐Exo markedly decrease serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, alleviate hepatic tissue damage, and preserve liver structural integrity, highlighting their therapeutic potential in inflammatory liver disease. ⁹⁵

Liver cirrhosis

Building on their immunomodulatory effects in hepatitis, EV‐based therapies have been increasingly explored for the treatment of liver cirrhosis and advanced fibrotic liver diseases. Recent studies show that EVs from human umbilical mesenchymal stem cells (hUC‐MSCs) offer potential for liver cirrhosis treatment. These EVs significantly reduce circulating fibrosis markers, including hyaluronic acid (HA) and TGF‐β1, suppress collagen deposition, and attenuate fibrosis progression by inhibiting the TGF‐β1/Smad signaling pathway and preventing epithelial–mesenchymal transition (EMT) in hepatocytes. ⁹⁶ Consistently, in carbon tetrachloride (CCl₄)‐induced liver fibrosis models, hUC‐MSC‐derived EVs alleviate hepatic inflammation, reduce collagen accumulation, and promote hepatocyte repair through paracrine mechanisms, thereby improving overall liver architecture and function. Beyond fibrosis control, these EVs also exhibit metabolic regulatory effects; by modulating miR‐627‐5p expression and immune signaling pathways, they improve lipid metabolism, ameliorate insulin resistance in non‐alcoholic fatty liver disease (NAFLD), reduce oxidative stress, and attenuate chronic inflammation. ⁹⁷

In addition to hUC‐MSC‐derived EVs, EVs released from mesenchymal stem cells broadly contribute to liver repair by orchestrating immune cell reprogramming. MSC‐derived EVs suppress hepatic inflammation and fibrosis, promote the expansion of regulatory T cells (Tregs), enhance anti‐inflammatory macrophage phenotypes, and modulate the activity of macrophages, T cells, and B cells. Through these coordinated immunoregulatory effects, MSC‐derived EVs facilitate tissue repair and regeneration, underscoring their therapeutic potential in liver cirrhosis, non‐alcoholic steatohepatitis (NASH), and related chronic liver diseases. ⁹⁸

Inflammatory bowel disease

EVs have emerged as critical mediators of immune dysregulation in inflammatory bowel disease (IBD), particularly through their ability to modulate innate lymphoid cell‐driven mucosal immune responses. EVs derived from M1 macrophages have been shown to transport miR‐21a‐5p, which directly downregulates E‐cadherin mRNA expression in intestinal epithelial cells. The loss of E‐cadherin disrupts its interaction with the inhibitory receptor KLRG1, thereby relieving suppression of type 2 innate lymphoid cells (ILC2s) and promoting their activation. ⁵¹ Therefore, aberrant ILC2 activation induces an excessive Th2‐skewed immune response, leading to pathological cytokine production, breakdown of epithelial junction integrity, and impairment of the intestinal mucosal barrier. This EV‐mediated M1 macrophage–ILC2 axis provides mechanistic insight into ulcerative colitis (UC) pathogenesis and highlights EV‐associated miR‐21a‐5p as a potential therapeutic target for restoring immune homeostasis in IBD.

Liver transplantation

Beyond inflammatory diseases, EV‐based strategies have also shown promise in promoting immune tolerance in the context of liver transplantation. EVs released from CD4⁺CD25⁺ regulatory T cells (Tregs) carry immunosuppressive cytokines, including transforming growth factor‐β (TGF‐β) and interleukin‐10 (IL‐10). These EVs suppress cytotoxic T‐cell activation, dampen alloimmune responses, and reduce graft rejection, thereby contributing to the establishment of transplant immune tolerance. ⁵² Importantly, EV‐mediated immunoregulation may offer a cell‐free alternative to conventional immunosuppressive regimens, potentially improving graft survival while minimizing systemic immunosuppression‐associated toxicity.

Organ regeneration in the GI tract

In addition to immune modulation, EVs play a pivotal role in GI organ regeneration, particularly in liver injury repair. Mesenchymal stem cell‐derived EVs (MSC‐EVs) exhibit pronounced regenerative capacity in liver ischemia–reperfusion injury (IRI). These EVs significantly reduce serum transaminase levels, attenuate hepatic necrosis, increase the proportion of Ki67‐positive proliferating hepatocytes, and suppress inflammation‐associated gene expression, collectively promoting hepatocyte survival and tissue repair. ⁹⁹ Comparative analyses further reveal that although fibroblast‐derived EVs contribute to liver regeneration during later stages of IRI, MSC‐EVs exert more robust and sustained reparative effects. This superiority positions MSC‐derived EVs as a particularly promising therapeutic strategy for enhancing liver regeneration and functional recovery following acute hepatic injury.

Gut microbiome

EVs derived from diverse biological sources have emerged as key modulators of gut microbiota composition, intestinal barrier integrity, and mucosal immune homeostasis, thereby exerting therapeutic effects in GI and systemic diseases. Plant‐derived EVs have demonstrated notable capacity to regulate gut microbial ecology and intestinal barrier function. Cranberry‐derived EVs enhance gut barrier integrity and microbial diversity, which is associated with protection against early‐onset ovarian failure and preservation of ovarian function. ¹⁰⁰ Similarly, grape‐derived EVs optimize colitis treatment by activating the aryl hydrocarbon receptor (AHR) signaling pathway, restoring gut metabolite profiles, and rebalancing microbiota composition. ¹⁰¹ EVs isolated from Houttuynia cordata further promote intestinal barrier repair and microbial homeostasis, thereby alleviating colitis‐associated inflammation. ¹⁰² Animal‐derived EVs, particularly those obtained from dairy products, offer advantages in biosafety, oral bioavailability, and translational feasibility. Goat milk‐derived EVs alleviate colitis by strengthening intestinal barrier function and reshaping gut microbiota structure, ¹⁰³ while cow milk‐derived EVs similarly reduce intestinal inflammation through modulation of microbial composition and immune responses in colitis models. ¹⁰⁴

Beyond plant‐ and animal‐derived systems, bacterial extracellular vesicles (bEVs) have recently emerged as a promising modality for microbiota‐targeted therapy in GI diseases. A recent study demonstrated that bEVs derived from Bifidobacterium longum NSP001 significantly ameliorated UC in mice by modulating T‐cell‐mediated immune responses through both microbiota‐dependent and microbiota‐independent mechanisms. ¹⁰⁵ Owing to their intrinsic compatibility with the gut environment, bEVs can efficiently deliver microbial–host signaling molecules, reshape mucosal immunity, and remodel microbial communities. Furthermore, a recent comprehensive review highlighted that engineered bEVs, leveraging advances in vesicle biogenesis, cargo loading, and targeting, could serve as scalable, low‐immunogenicity therapeutics for GI disorders, offering complementary advantages to plant‐ or animal‐derived EV systems. ¹⁰⁶ Nevertheless, challenges remain in standardizing production, ensuring batch reproducibility, and validating safety and efficacy in clinical settings.

Stem cell‐ and perinatal tissue‐derived EVs also exhibit robust potential in microbiome modulation and intestinal immune regulation. Mesenchymal stem cell (MSC)‐derived EVs restore gut microbiota balance, regulate macrophage polarization, and attenuate inflammation associated with extrahepatic injuries. ¹⁰⁷ MSC‐derived EVs enriched with Exendin‐4 further modulate gut metabolites, contributing to the alleviation of diabetic nephropathy. ¹⁰⁸ In addition, EVs derived from perinatal tissues, such as human umbilical cords, reshape the gut microbiome by reducing pro‐inflammatory bacterial populations while promoting beneficial taxa, thereby improving colitis outcomes. ¹⁰⁹

Irritable bowel syndrome and stem cell therapy

EVs have emerged as critical mediators in gut–brain interactions and show therapeutic potential in irritable bowel syndrome (IBS). Tea‐derived EVs have shown potential therapeutic effects in alleviating IBS. Research demonstrates that oral administration of tea‐derived ELNs can effectively improve IBS symptoms by modulating the corticotropin‐releasing hormone (CRH) pathway, which is associated with gut function. ¹¹⁰ Building on this concept, mesenchymal stem cell‐derived apoptotic vesicles (apoVs) have been shown to regulate the 5‐HT brain–gut axis in IBS mice, reducing serotonergic signaling in the brain and gut, restoring intestinal barrier integrity, improving neuronal activation, and alleviating visceral hypersensitivity. Furthermore, gut microbiota‐derived EVs are implicated in post‐infectious IBS (PI‐IBS) pathogenesis. ¹¹¹ Chlorogenic acid (CGA) treatment increases Bacteroides acidifaciens abundance and glycine levels, which reshape B. acidifaciens EVs to reduce intestinal inflammation and hypersensitivity, maintain mucosal barrier function, and modulate brain tissue signaling. ¹¹² Collectively, these studies underscore the role of EVs, from dietary, stem cell, and microbial sources, in modulating brain–gut homeostasis, highlighting novel non‐pharmacological and biologically targeted strategies for IBS management.

CLINICAL TRIAL PROGRESS OF EXTRACELLULAR VESICLES IN GASTROINTESTINAL CANCERS AND DISEASES

EVs have been extensively explored as therapeutic platforms for GI cancers and diseases in preclinical studies, generating essential mechanistic and translational evidence that supports their progression toward clinical evaluation (Figure 4 ). In vitro models, including conventional two‐dimensional cultures and three‐dimensional organoid systems, have been used to investigate EV‐mediated intercellular communication and therapeutic responses. ¹¹³ In vivo, animal models provide valuable insights, including syngeneic and orthotopic tumor models, genetically engineered mouse models, physically or virus‐induced mouse models, chicken chorioallantoic membrane model and patient‐derived xenograft models. ⁶³ , ¹¹⁴ , ¹¹⁵ , ¹¹⁶ Together, these preclinical frameworks provide a rational basis for the design and implementation of clinical trials, paving the way for the clinical translation of EV‐based therapies in GI diseases.

Figure 4.

Preclinical models and clinical trials of EV‐based therapies in gastrointestinal cancers and diseases (graph was created in bioRender.com).

In line with accumulating preclinical evidence, clinical trials are increasingly exploring EVs as novel therapeutic modalities for GI diseases (Table 2), with early studies primarily focusing on safety, feasibility, and proof‐of‐concept efficacy. In pancreatic cancer, EV‐based strategies have been developed to overcome therapeutic resistance, particularly through the delivery of functional nucleic acids. EV‐packaged microRNAs have been reported to enhance chemosensitivity, and notably, a phase I clinical trial (NCT03608631) is currently evaluating mesenchymal stem cell‐derived EVs loaded with KrasG12D siRNA in patients harboring KrasG12D mutations, representing a landmark attempt to translate EV‐mediated gene silencing into clinical oncology. ¹¹⁷ Similarly, in CRC, EVs are being investigated as biocompatible drug delivery vehicles and immune modulators. A phase I trial (NCT01294072) is assessing the safety and bioavailability of plant‐derived EVs for curcumin delivery, while ascites‐derived EVs combined with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) have demonstrated enhanced antitumor immune responses, highlighting the immunostimulatory potential of endogenous EVs. ¹¹⁸ Beyond oncology, EV‐based interventions are also advancing into clinical evaluation for chronic inflammatory liver and intestinal diseases. In cirrhosis, a phase II trial (NCT05871463) is recruiting patients to assess the anti‐inflammatory and tissue‐reparative effects of umbilical cord‐derived mesenchymal stem cell EVs, underscoring their promise as low‐immunogenic, cell‐free therapeutics. In inflammatory bowel disease (IBD), a completed clinical trial (NCT04879810) investigated ginger‐derived EVs loaded with curcumin in a cohort of 90 patients, providing clinical evidence for the anti‐inflammatory capacity and translational feasibility of orally administered EV‐based therapies. Collectively, these early‐phase clinical studies illustrate the multifunctionality of EVs, as drug carriers, gene regulators, and immune modulators, and underscore their growing translational relevance in the treatment of GI diseases.

Table 2.

Clinical trial studies of EVs in the treatment of gastrointestinal tumors and diseases

Type of diseases	Sources of EVs	Year of study	Number of clinical trials	Phases	Sample size
Pancreatic cancer	Cancer‐Associated Fibroblast‐derived EVs therapy	2023	ChiCTR2200061320	NA	59
	Mesenchymal stem cell (MSC)‐derived EVs as drug carriers	2021	NCT03608631	Phase I	15
Colorectal cancer	Plant‐derived EVs as curcumin delivery	2023	NCT01294072	Phase I	NA
	Ascites‐derived EVs combined with GM‐CSF therapy	2008	NA	Phase I	40 118
Advanced hepatocellular carcinoma, gastric cancer, and colorectal cancer	Macrophage‐derived EVs therapy	2022	NCT05375604	Phase I	9
Liver cirrhosis	MSC‐derived EVs	2023	NCT05871463	Phase II	15
Inflammatory bowel disease	Ginger‐derived EVs combined with curcumin	2022	NCT04879810	Completed	90

NA, not available; Completed, study status completed.

Overall, these preclinical and clinical advancements highlight the potential of EV‐based therapies to address various GI diseases, paving the way for more targeted, personalized, and effective treatments (Figure 5 ). As research progresses, the versatility and therapeutic promise of EVs in GI diseases are becoming increasingly apparent, offering new avenues for patient care and disease management.

Figure 5.

Key milestones in the EV‐based therapeutics for gastrointestinal cancers and diseases (graph was created in bioRender.com).

CURRENT CHALLENGES AND OPPORTUNITIES IN THE CLINICAL TRANSLATION OF EV‐BASED IMMUNOTHERAPEUTICS

Despite their considerable therapeutic promise in GI cancers and diseases, the clinical translation of EV‐based immunotherapeutics remains limited by several unresolved barriers (Figure 6 ). EVs possess a set of intrinsic properties that support their development as therapeutic platforms, including favorable biocompatibility, low immunogenicity, endogenous intercellular communication capacity, and the ability to traverse biological barriers. These features make EVs attractive for cargo delivery and immune modulation. However, such advantages are counterbalanced by persistent challenges in standardization and quality control, scalable production, targeting precision, and the biological complexity imposed by tumor heterogeneity. Increasing evidence indicates that clinically relevant EV development will require rigorous control of production, molecular characterization, stability, and batch‐to‐batch consistency, together with well‐defined reference markers and optimized storage conditions. ¹¹⁹ In parallel, donor‐cell variability, limited expansion capacity, and the intrinsic heterogeneity of EV populations continue to compromise yield, reproducibility, and analytical comparability across studies. ¹²⁰ , ¹²¹ In addition, native EVs often display limited targeting efficiency and rapid systemic clearance, whereas spatial and functional heterogeneity within tumors may further influence EV composition and therapeutic responsiveness. ¹²²

Figure 6.

Opportunities and challenges of EV‐based therapies in gastrointestinal cancers and diseases (graph was created in bioRender.com).

Several emerging strategies are beginning to address these limitations. On the manufacturing side, scalable production and purification approaches, including tangential flow filtration coupled with size‐exclusion chromatography and bioreactor‐based systems, are being developed to improve yield, purity, and process consistency. ¹²³ At the same time, engineering strategies such as producer‐cell modification, surface ligand display, and cargo loading are being actively explored to enhance tissue selectivity and therapeutic efficacy. ¹²⁴ Advances in single‐vesicle profiling, microfluidic platforms, super‐resolution imaging, and AI‐assisted analysis may further improve EV subclass resolution, quality assessment, and patient stratification, which will be particularly important for addressing heterogeneity at both the vesicle and tumor levels. ¹²¹ , ¹²⁵ , ¹²⁶ Taken together, the next stage of progress in EV‐based immunotherapeutics will depend not only on harnessing their intrinsic biological advantages but also on establishing reproducible production, more precise delivery, and a deeper understanding of disease‐specific EV behavior.

CONCLUSIONS

Overall, EV‐based therapeutics have emerged as a promising avenue for GI cancers and GI‐related diseases, owing to their integrated roles in intercellular communication, immune modulation, and cargo delivery. As highlighted throughout this review, EVs are not merely natural carriers, but biologically active mediators that influence the TME and shape therapeutic responses, thereby enabling diverse applications ranging from cancer vaccines and engineered immunotherapy to genome editing and combination strategies. Notably, their relevance extends beyond malignancy to inflammatory and regenerative GI disorders, underscoring the broad translational significance of EV biology. Although substantial work remains before these approaches can be fully implemented in clinical practice, current preclinical and early clinical evidence supports the view that EV‐based strategies may contribute to more precise, mechanism‐informed, and therapeutically adaptable interventions in GI diseases.

FUNDING

The work was supported in part by grants from the National Natural Science Foundation of China (82273183 and 82572981 to Hao Zhang; 82573209 to Songwang Cai); Major Project of the Open Joint Fund of the National Clinical Key Specialty Construction of Oncology, The First Affiliated Hospital of Henan University of Science and Technology (ZLKFJJ20230104 to Hao Zhang).

CONFLICT OF INTEREST

The authors declared no competing interests for this work.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.