Nanoporous membrane filtration was created by an integrated double-filtration microfluidic system for the separation and enrichment of exosomes from urine, and then quantitatively detected exosomes using a chip Enzyme-Linked Immunosorbent Assay (ELISA). Therefore, urine exosomes can be isolated, enriched, and quantified by the device to aid in the screening process for bladder cancer (32). Liu et al. designed and successfully developed an isolation tool called Exosome Total Isolation Chip (ExoTIC), which is simple, fast, economical, scalable, and high-yield for isolating exosomes from various body fluids of cancer patients (33). ExoTIC provides a lot of exosomes for downstream analysis and creates a standard microenvironment for subsequent research without being separated into separate modules.

DLD embeds the gradient column array in a microfluidic device to continuously separate suspended particles by diameter (34). The DLD clutch-column array platform has been applied to isolate circulating tumor cells (CTCs), and a method for sensitive detection of non-fluorescent tags with micron-scale protein and polymer vesicle properties using DLD clutch-column arrays has been established (35, 36). Moreover, the Exodisc (a microfluidic tangential flow filtration device) could effectively separate exosomes from body fluid, resulting in a higher yield of exosomes than traditional technologies (37).

The DEP device induces polarized particles to move in the non-uniform electric field and has proven to be an ideal choice for the transport, aggregation, separation and characterization of particles at the nanoscale in microfluidic systems (38). To address the difficulty in separating exosomes in plasma, Ibsen and his team demonstrated that the alternating current electromotor (ACE) microarray device enables rapid isolation and recovery of glioblastoma exosomes from undiluted plasma (39). On this basis, Ayala-Mar and his colleagues further designed a miniature device called the DC-iDEP, which consists of a channel and two electrically insulated columns in order to generate different dielectric electrophoresis forces on the suspended objects (38). Notably, Tayebi’s group integrated acoustic radiation force (ARF) with dielectrophoresis and positioned a high-frequency inter-digital transducer (>10MHz) into the flow pathway, generating a force field based on both acoustic radiation and dielectrophoresis. Lateral translation of particles in the medium was observed as a result of the interplay of fluid drag force, ARF, and dielectrophoresis force fields, leading to the isolation of exosomes (40).

Modification of probe molecules on the inner surface of microfluidic channels is one of the most common methods for separation and capture of exosomes by immunoaffinity (41). Thus, exosomes in the body fluid are captured and accumulated in the device channel, while the rest of the body fluid is released. The formation of AuNC-exosome-AuR complexes was utilized by Yang and his group to develop a microfluidic device for the isolation of exosomes (42). This device was successful in extracting 5×10 (9) particles from a 5mL urine sample within 30 minutes. However, the separation of captured exosomes still faces a significant challenge due to the tough bond between the antigen and antibody. Kang’s team combined the OncoBean microfluidic device with antibodies to capture and release circulating exosomes, creating the ExoBean system. This system consists of both different immune mode layers that can be mechanically rotated to improve the binding probability between antibodies and exosomes, thus achieving specific isolation of exosomes (43).

However, it is also important to recognize that exosomes purification technologies are constantly developing, and conventional biomarkers may only distinguish the exosomes with specific cargoes. Therefore, with the adoption of new technologies, it will be necessary to refine some findings in the future.

As we know, exosomes fixed in microfluidic chips could be specifically stained with fluorescent dyes (like PKH67, PKH26 and DiO) (44). Kanwar uses an “ExoChip”, staining exosomes with DiO and quantifying exosomes using a standard microplate meter (45). The combination of fluorescein and microfluidic technique also shows great potential for the detection of exosomes content. The Reversible Interaction Nanoscale Sorting Enrichment (RInSE) system utilizes the principle of inertial focusing in order to continuously isolate and analyze beads using flow cytometry. Exosomes were isolated using functional beads, and then introduced into the microchannel devised specifically to induce inertial focusing and facilitate buffer exchange. This method allowed for precise positioning of the beads, thus enabling continuous fluorescence analysis through flow cytometry (46).

In recent years, electrochemical detection has gradually developed into an effective way to detect exosome separation or accumulation, because electrochemical sensing devices can achieve high sensitivity and high efficiency detection through signal amplification (47, 48). Zhou’s group designed an electrochemical biosensor based on aptamers used for the quantification of exosomes with more objective and direct results. An aptamer specific to CD63 is fixed on the gold electrode surface, and the pre-labeled probe chain binds to the aptamer molecule fixed on the electrode surface. If exosomes are present, these beacons release the detection chain, resulting in a decrease in the electrochemical signal (49).

SPR is the resonance oscillation generated by the conduction electron at the interface of the particle’s negative conductivity and positive conductivity material excited by incident light (50, 51). SPR technology based on microfluidics has gradually come into view and become one of the main technical means in the field of exosome detection (52). Notably, Lee et al. described a method for Nanoplasma exosome (nPLEX) analysis as a new technique for quantification of exosomes. It works through the transmitted SPR of periodic nanopore arrays, in which each array is functionalized with antibodies for the analysis of various exosomal proteins (53).

Intrinsic heterogeneity is one of the main factors hindering exosome analysis in body fluids. Single exosome detection may provide more accurate information of tumor progression. Because conventional flow cytometry is not capable of sensitively identifying nanosized exosomes, researchers have utilized aldehyde/sulfate latex beads to bind to the vesicles. These vesicles are then stained with fluorescent antibodies and analyzed for their protein markers. The percentage of positive beads, in which both the anti-GPC-1 antibody and Alexa-488-tagged secondary antibody were introduced, is defined as the percentage of GPC-1 positive exosomes (54). Recent study demonstrated a TIRF-based single-vesicle imaging assay was developed. This assay delivered molecular beacon probes into exosomes, resulting in an amplified fluorescence of the target microRNA. Through the direct visualization of individual vesicles and in-situ quantitative analysis of miR-21 in human serum samples, researchers discovered that this assay outperformed conventional polymerase chain reaction (PCR) assays in the diagnosis of cancer patients from healthy donor (55).

MB is a probe that resembles a hairpin-shaped oligonucleotide. It is labeled with a fluorescent dye and a quencher at both ends. When MB hybridizes with the targeted sequence, it disrupts the hairpin structure and causes fluorescence to appear (56). MB-based biosensors were used to detect miR-375 and miR-574-3p in exosomes from human urine (57). As well as the expression level of miR-21, miR-375, and miR-27a were detected in exosomes from human serum (58). Furthermore, Lee et al. discovered that the hybridization of molecular beacons and miRNA-21 in exosomes from cancer cells and human serum produced strong fluorescent signals (59).

The occurrence of LSPR takes place when the frequency of the incident photon matches the overall vibration frequency of precious metal nanoparticles or metal conducting electrons. Joshi et al. devised a biosensor that utilizes LSPR for the label-free and nondestructive evaluation of exosomal miR-10b (60). In the subattomolar concentration range, this system displayed standard sensitivity to discriminate between miR-10b and miR-10a, despite having only a one-nucleotide difference. To accurately diagnose NSCLC, Wu et al. created an SPRi-based biosensor capable of detecting multiple exosomal miRNAs. They employed an Auon-Ag heterostructure and a DNA tetrahedral framework to amplify the SPR signal, enabling the identification of each exosomal miRNA with high sensitivity through different SPR signals (61).

Exosomal proteins, whether on the surface or within the membrane, provide abundant, stable, sensitive, and distinct information (142). Multitude evidence revealed that the proteins may be able to monitor progression of GI cancer. Wang et al. established a risk model with TNFRSF10B and ILF3 for ESCC, and the risk scores were positively correlated with the IC50 of multiple chemotherapeutic drugs by spearman analysis (143). As an oncofetal protein, exosomal glypican 3 (GPC3) is a predictive biomarker for primary gastro-esophageal adenocarcinoma (GEA), as patients with GEA had lower overall survival (OS) when the GPC3 levels were higher (144). In stage III and stage IV GC, patients with exosomal surface marker CD63 expression had significantly lower OS rates than those with CD63-negative expression (P < 0.0001 and P = 0.0063, respectively) (145). In addition, it has been proven that highly glycosylated CD133 in ascites-derived exosomes may serve as a prognostic biomarker for advanced PC (146). Members of Rab GTPases Rab27a and Rab27b control exosome release (147). Nambara et al. demonstrated that Rab27b knockdown could suppress peritoneal metastasis in the xenograft mouse model of GC, and they proved the correlation between elevated Rab27b and poor OS rates in 178 GC patients from the local hospital and 406 from the TCGA database (148). Liang et al. established a predictive model for CRC chemoresistance with EXOC2, EXOC3 and STX4 in exosomes, and the area under curve (AUC) of 5-year progression-free survival (PFS) was 0.804 (149). Another research group also found significant differences in the level of dipeptidyl peptidase IV (DPP4) in exosomes from 5-FU resistant and sensitive cells (151). After analysis of the small sample of clinical data, researchers pointed out the higher expression of DPP4, and the lower differentiation degree of colon cancer tissues, which predicted a poor prognosis.

Differential expression of miRNA in exosomes can serve as biomarkers to detect treatment responses for GI cancer. Yagi et al. compared miR-125b level in plasma exosomes between CRC clients with progressive and stable disease receiving FOLFOX-based first-line chemotherapy and discovered miR-125b was upregulated in patients with progressive disease (152). In addition, COX multivariate analysis revealed miR-125b as an independent predictor for PFS (P = 0.041). Hence, exosomal miR-125b might be a useful minimally invasive indicator for CRC chemoresistance. Xu et al. discovered that patients with HCC who were in the low or high miR-451a expression groups had significantly different survival times (P<0.05), suggesting that patients with lower expression levels of miR-451a had a worse prognosis (102). Furthermore, miR-196b-5p was highly enriched in serum exosomes of CRC patients, which promoted stemness and chemoresistance of CRC cells (153). Notably, OS was considerably lower in individuals with high than in low miR-196b-5p levels (P = 0.006). MiR-155 was identified as a clinical resistance predictor (154). High expression of miR-155 was found in the exosome generated from GEM-resistant cell lines, and PDAC patients with high expression of miR-155 had shorter disease-free survival time (P = 0.021) and OS time (P = 0.008) than PDAC patients with low expression. The studies have indicated that miR-454-3p, miR-7974, miR-3615, miR-130b-3p and miR-4326 can collectively predict the efficacy of oxaliplatin in treating CRC (AUC = 1). It is anticipated that these will become biomarkers for predicting treatment resistance (155). Additionally, ROC curve analysis indicated that the miR-500a-3p levels in plasma exosomes could differentiate between cisplatin resistant and sensitive group in stage III GC patients (AUC = 0.843), and miR-500a-3p overexpression suggested a dismal prognosis (157). In a clinical trial (NCT06388967), the researchers plan to test the previously identified combination of 13 exosomal miRNAs in a larger international cohort study to confirm the effectiveness of this method in accurately identifying PDAC at its early stages (158).

Several recent studies suggest that exosomal lncRNAs and circRNAs could be molecular markers for monitoring tumor progression. LncCACC was upregulated and enhanced CRC resistance to chemotherapy (159). Its expression in peripheral plasma exosomes of CRC patients could predict the chemotherapy effect. DACT3-AS1, transferred by exosomes, was decreased in GC tissues, which reduced oxaliplatin sensitivity and resulted in poor prognosis of GC patients (AUC = 0.6940) (83). Song et al. found that in GC patients receiving adjuvant chemotherapy, high levels of exosomal lncRNA-GC1 expression often indicated significantly shorter survival (P < 0.05) (161). In the clinical trial NCT05334849, the levels of lncRNA-GC1 in circulating exosomes were also measured to monitor and predict the response of GC patients to immunotherapy, and the expected results were shown (162). Transarterial chemoembolization (TACE) has been recommended by most clinical practice guidelines as standard treatment for patients with intermediate stage HCC due to its survival benefits (163). According to reverse transcription quantitative polymerase chain reaction (RT-qPCR) results, hsa-circRNA-G004213 was markedly increased after TACE (P < 0.01) (164). And researchers also demonstrated that exosomal hsa-circRNA-G004213 promoted the cisplatin sensitivity of HCC cells and was related to prognosis of liver cancer patients following TACE, which might be a biomarker for monitoring TACE effect. Recently, Shang et al. demonstrated that circHIPK3 was expressed more in the serum exosomes of GC patients who were resistant to cisplatin, indicating that exosomal circHIPK3 may be a biomarker for the clinical effectiveness of cisplatin chemotherapy (165). The aforementioned studies have partially validated the involvement of exosomes-based liquid biopsy in tumor resistance; nevertheless, additional samples and trials are required to validate their clinical value in tumor diagnosis and treatment.

As an endogenous bio-vehicle, exosomes have many advantages compared with conventional drug delivery systems such as synthetic polymers, liposomes, micelles, super magnetic particles, proteins, and recombinant viral vectors (167). It has been mentioned previously that exosomes are present in the majority of bodily fluids. Numerous edible plants have been shown to secrete exosomes, which create conditions for their abundant production (168). Further, exosomes are structurally stable and their membranes can protect the contents from degradation in harsh conditions. Notably, exosomes are also relatively non-immunogenic and non-toxic because of their similar composition to the body’s cells (169). Another benefit of exosomes is that they can deliver small molecular drugs or siRNAs across the blood-brain barrier, which is impossible with most nanocarriers (170). In addition, it is easier to modify drug carrying ability and tissue targeting specificity of exosomes (171). Therefore, exosomes deserve to be considered as potential vehicles for the delivery of small molecule drugs to treat cancer.

The delivery of therapeutic RNA, specifically siRNA, by exosomes to tumor cells is currently a popular area of cancer research. As the basis for cancer metastasis, c-Met is essential for tumor proliferation, invasion, and metastasis. Although the exact molecular mechanism is still unclear, Zhang et al. constructed si-c-Met loaded by exosomes and delivered it into GC cells, reversing the resistance to cisplatin in GC (172). Huang et al. loaded siRNAs targeting coiled-coil domain-containing protein 80 (CCDC80) into exosomes and delivered them to the CRC liver metastasis mouse model, which prolonged the overall survival of the mice (173). P21-activated kinase 4 (PAK4) was found to be oncogenic, and the encapsulating si-PAK4 in PANC-1 cell-derived exosomes successfully suppressed pancreatic tumors in vitro and in vivo (174). Yu et al. developed engineered exosomes carrying si-BRIX1, which could induce nucleolar stress and thereby enhance the efficacy of 5-FU against CRC (175). Studies have also shown that hsa_circ_0004085 could be packaged into exosomes, which effectively inhibited ER stress in the recipient cells and enhanced their resistance to oxaliplatin and 5-FU (176).

Liang et al. found an example of exosomes delivering anticancer drugs to overcome chemoresistance in CRC (177). They generated target-specific exosomes by engineered 293T cells and packaged 5-FU and miR-21 inhibitor oligonucleotide (miR-21i) into the exosomes. The engineered exosome delivery system markedly enhanced cytotoxicity and improved the effect of cancer treatment. CDK-004, consisting of exosomes carrying a STAT6 anti-sense oligonucleotide (ASO), was first introduced in a multi-center clinical trial in advanced HCC, and liver metastases from GC or CRC in 2023 (NCT05375604) (178). It was administered intravenously as a single agent and inhibited tumor development by repolarizing M2 macrophages. Although the clinical trial had to be terminated due to the company bankruptcy, it provided ideas for early clinical entry of exosome-based drugs in oncology. Research (NCT01294072) into the delivery of curcumin to colon tumor cells via plant-secreted exosomes to enhance its anti-tumor activity has also entered phase I clinical trials (179). The exosome-encapsulated 5-FU has also been proven to enhance CRC cell nuclear fragmentation and apoptosis in comparison to the exact same dose of free 5-FU (180). Wang et al. synthesized a novel modified exosome loaded with oxaliplatin, which could enhance the chemosensitivity of CRC by regulating mitochondrial function. The validity of these findings has been substantiated through in vitro experimentation and in orthotopic cancer models (181). Furthermore, M1 macrophage-derived exosomes loaded with GEM and Deferasirox could reduce cell viability and adhesion ability of GEM-resistant PC cells (182).

As previously mentioned, exosomes mediate intercellular communication, and tumor-derived exosomes (TDEs) play an essential part in tumorigenesis and development. Thus, it is feasible for targeted inhibition of exosomes, especially TDEs, to improve the efficacy of cancer therapy. A clinical trial (NCT02393703) has already been conducted to investigate whether exosome activity is associated with disease recurrence and outcomes in PC patients (183). Theoretically, disrupting any step in the process from their biogenesis in tumor cells to internalization by recipient cells can successfully result in TDE dysregulation, such as inhibiting TDE biogenesis and packaging, modulating TDE trafficking, and blocking TDE internalization (184). Up to now, genetic manipulation and pharmacological inhibition are the most researched methods for targeting the inhibition of TDEs (185). DDX3, a DEAD-box RNA helicase, was reported to suppress the release of HCC exosomes, which contributes to its anti-tumor property (186). He et al. proposed that targeting vesicle trafficking-related protein myoferlin could be a promising opportunity to treat CRC clinically, as myoferlin suppressed exosome secretion and internalization and thereby reduced the invasive capacity of tumor cells (187). Li and his team found Rab27B was overexpressed and reduced drug concentration in 5-FU resistant HCC cells by encouraging exosome release, while genetic knockdown of Rab27B restored sensitivity to chemotherapy drugs and improved their therapeutic effects (188). Recent studies have suggested that Rab31 also played the same roles in GC development and could be a possible target for therapeutic interventions (189). Besides, regenerating islet-derived protein 3 beta (REG3β) was proved to block the capture of TDEs by target cells, thereby reversing the malignant phenotype of PDAC cells (190).

Pharmacological inhibition is another form of targeting TDEs for tumor therapy. For instance, as a blocker of nSMase2, GW4869 has been applied as an exosome secretion inhibitor to regulate tumor progression in several experiments. Wang et al. injected GW4869 directly into the CRC tumor, which suppressed TDEs secretion and tumor growth (191). In addition, Richards discovered that GW4869 negatively affected exosome secretion from CAFs, resulting in the re-sensitization of PDAC cells to gemcitabine (192). M2 macrophage-derived exosomes were demonstrated to promote migration of GC cells, and GW4869 treatment significantly reduced the exosome-induced lung colonization in mice models of GC (193). In rat models, pantoprazole reduced the quantity of exosomes in the liver and serum, indicating the potential to reduce liver tumorigenesis (194). Additionally, there are many other compounds, like manumycin A, cannabidiol, tipifarnib, ketoconazole, neticonazole, climbazole, triadimenol, and simvastatin, were demonstrated to affect the exosome release (195, 196). Several direct exosome scavenging techniques have also been investigated, such as therapeutic plasma exchange, dialysis, hemofiltration, and hemoperfusion. However, the above treatments carry an increased risk of trauma and infection, which is an additional burden for cancer patients.

It has been reported that all clinically apparent tumors have evolved mechanisms of immune evasion, which are achieved by altering tumor cell self-modification and the tumor immune microenvironment (TIME) (197). Given these mechanisms, researchers have proposed several immunotherapies applied in cancer treatment, such as oncolytic virus therapies, cytokine therapies, adoptive cell transfer, immune checkpoint inhibitors, and cancer vaccines (198). Despite their clear efficacy, these therapies still have their own limitations and side effects. Exosome-based immunotherapy is considered to be a potential treatment option for GI cancer that overcomes the toxicity and instability of traditional tumor vaccines (199). Dendritic cells (DCs), professional antigen-presenting cells, can stimulate tumor antigen-specific T cells to trigger anti-tumor immune response (200). On the basis of maintaining the immunostimulatory function of DCs, DC-derived exosomes (DEXs) also show their stability, controllability, and low immunosuppression as anticancer vaccines (201). Wang et al. suggested that DEXs treated with hyperthermic CO2 reduced the expression of HSP70 and enhanced its own immune effect (202). Although definitive in vivo studies have not yet been performed, DEXs have been observed to trigger more potent major histocompatibility complex class I (MHC I)-restricted cytotoxic T lymphocytes (CTLs) response and maximally activated specific immune responses against HCC (203). In another study, Zhong et al. proved that microwave ablation (MWA) combined with DEXs increased the quantity of CD8+ T cells and decreased the level of FOXP3+ Treg cells in tumor sites compared to MWA alone, thereby reshaping TIME (204). The vivo studies also showed the combination therapy markedly suppressed HCC progression. In a recent study reported by Rezaei et al, DEXs loaded with miR-124-3p mimics reduced the CD4⁺/CD8⁺ cell ratio and FOXP3⁺ Treg/CD8⁺ cells ratio in tumor tissue, eliciting stronger tumor immunosuppression (205). Xiao et al. loaded TEXs into DC cells and inoculated them into PC mouse models in combination with drugs acting on MDSCs (206). The results showed that vaccination with DEXs could markedly prolong mice’s survival time, and co-inoculation triggered more activated T cells and more effective antitumor immune responses. Moreover, exosomes secreted by other cells may also participate in the treatment of GI cancer by regulating immune function. For example, Exosomes from heat-treated malignant ascites of GC patients induced CTL responses via increase of heat shock proteins and maturation of DC cells (207). In brief, exosomes offer a cell-free immunotherapy vaccination option that may be beneficial for treating drug-resistant GI cancer.