2.1 Types of EVs: exosomes, microvesicles, and apoptotic bodies

In the 1980s, Pan and Johnstone demonstrated that peptide-containing vesicles are discharged into the extracellular space from sheep reticulocytes. Since then, the term “exosome” has been applied to EVs.¹⁷ Depending on their size and biogenesis, the taxonomy of EVs typically includes three types of vesicles: exosomes (<150 nm in diameter), microvesicles (MVs) (100–1000 nm in diameter), and apoptotic bodies (ApoBDs) (0.8–5.0 µm in diameter).^18-20 Within the TME, EVs are crucial for intercellular communication, as well as for long-distance circulation. Although Wolf and colleagues initially regarded EVs as nothing more than cellular waste, emerging data in this area have demonstrated their importance as signaling molecules in physiological and pathological processes, such as cancer development (Figure 1).

Exosome biogenesis is a byproduct of the endosomal pathway. Early endosomes are formed when the cell membrane protrudes inward and their lumens fill with accumulating intraluminal vesicles (ILVs). The development of multivesicular bodies (MVBs) is then initiated by the maturation of early endosomes into late endosomes.²¹ Following their production, MVBs are subsequently transported by cytoskeleton filaments and proteins to the plasma membrane, where SNARE proteins assist in the release of the ILVs as exosomes.^{22, 23} The endosomal sorting complex required for transport (ESCRT) has been identified to be involved in MVB biogenesis, vesicle budding, and protein cargo sequestering and sorting.²⁴ There is also an ESCRT-independent pathway that leads to the creation of ILVs and MVBs. Exosomes are commonly referred to as “cargo,” as they encapsulate numerous substances, such as proteins (membrane proteins, cytosolic and nuclear proteins, extracellular matrix proteins), nucleic acids (mRNAs, noncoding RNAs [ncRNAs] and DNA), and metabolites.²¹ There are significant similarities in the types of lipids in EV membranes and the cells from which they originated, such as sphingomyelin, gangliosides, and disaturated lipids.²⁵ However, unlike their cell of origin, exosomes have lower concentrations of phosphatidylcholine and diacylglycerol.²⁶ In contrast, the proteins in exosomes are more diverse than the lipids, including biogenesis-related proteins and parental cell-specific proteins. In the case of tumor-derived exosomes, they store protumoral proteins and metabolites that promote tumor growth. Similar to protein composition, the nucleic acid composition of exosomes from tumor cells, such as mRNAs, microRNAs (miRNAs), and other small ncRNAs (sncRNA), reflects the characteristics of the parental cell.

MVs are generated through the direct process of budding from the plasma membrane of cells, as opposed to exosomes.²⁷ It is now believed that flippases, floppases, scramblases, and calpain change the composition of the phospholipid bilayer.²⁸ The cooperation of these enzymes results in the rearrangement of the components of the phospholipid bilayer and the reorganization of the actin cytoskeleton, enabling physical membrane bending as well as more effective MV formation.²⁹ The cellular absorption of MVs is hindered by their large size, posing a major obstacle. A recent study found that macropinocytosis, which involves the production of lamellipodia and plasma membrane ruffling, allows cells to ingest huge amounts of fluid outside the cells, hence enhancing the intake of cellular MVs.³⁰

Dying cells produce membrane-bound vesicles known as ApoBDs, formerly considered trash bags but later shown to transfer useful elements. Up to now, researchers have reported phosphatidylserine (PS) as the only marker for identifying ApoBDs.³¹ During the formation of ApoBDs, one of the initial and easily recognizable morphological alterations is the deformation of cells, which manifests as the formation of membrane blebs.³² Actomyosin-mediated contraction and increased hydrostatic pressure within the cell cause the formation of blebs.³³ Following repeated blebbing and retraction, ApoBDs are released and filled with cellular components and functional molecules, such as DNA, RNA, and proteins.^{34, 35} When apoptosis is initiated by therapy, different types of molecules from tumor cell-derived ApoBDs affect various signaling pathways, which in turn result in various biological effects and promote tumor progression.^{36, 37}

EVs are membrane-encapsulated vesicles that contain bioactive molecules ejected by donor cells. Recipient cells absorb EVs through receptor-ligand interactions, membrane fusion and phagocytosis.^{38, 39} The mechanism by which EVs are taken up by the recipient cell is nonrandom in conjunction with transmembrane proteins. Recent research has established that the tetraspanin Tspan8–CD49d complex plays a substantial role in facilitating the attachment of exosomes to recipient cells.⁴⁰ Additionally, intercellular adhesion molecule (ICAM)-1 is expressed as a receptor molecule on the membrane surface in a proinflammatory milieu, which improves exosome adherence to recipient cells.⁴¹ Further research is necessary to clarify the mechanisms by which EVs manipulate cancer cell targets and the factors that regulate their fusion.

2.2 Molecular cargo of EVs: proteins, nucleic acids (miRNAs, mRNAs, and DNA), lipids, and metabolites

A variety of substances, including proteins, nucleic acids (DNA, mRNA, and ncRNAs), lipids, and metabolites, can be autocrine and/or paracrine, serving as messengers between tumor cells and stromal cells. This section will address the contents of EVs in order to ascertain how EV payloads affect the biological processes in the TME.⁴²

3.1 EV-mediated transfer of oncogenic signals between cancer cells

Cancer cells display high heterogeneity in terms of molecular signatures within the same tumor site. Therefore, EVs released from cancer cells can function as a novel kind of messenger by transferring essential oncogenic signals to other cancer cells, which in turn promotes tumor growth (Figure 2). EGFR is an important oncogenic component of cell signaling pathways that regulate cell division and survival.⁹² Mutations in the EGFR gene result in the higher expression of EGFR proteins in certain types of cancer cells and transportation of EGFR via EVs induces an accelerated progression of cancer cells. For instance, glioma cells transfer oncogenic EGFR variant III (EGFRvIII) via EVs to other glioma cells lacking this receptor, promoting their morphological transformation and cell proliferation by activating the MAPK and AKT signaling pathway.⁹³ Another in vitro study also reported that EGFRvIII derived from EVs can stimulate the proliferation of human U87 glioma cells.⁹⁴ GC can spread to the liver more easily since exosomes carrying EGFR have an impact on the liver microenvironment.⁹⁵ In addition to promoting the proliferation of recipient cells, EVs also play pivotal roles in cancer chemoresistance and radiotherapy resistance by horizontal transfer of resistance-related molecules to nonresistant cancer cells. In a non-small cell lung cancer (NSCLC) model, Wu et al.⁹⁶ suggest that EV-mediated transfer of wild-type EGFR protein promotes osimertinib resistance to EGFR-mutated sensitive cancer cells by activating PI3K/AKT and MAPK signaling pathways.

EVs also contain other molecules that are critical for the oncogenic signaling pathway. A highly malignant pancreatic cell line PC-1.0 could enhance the proliferation of another moderately malignant cell line PC-1 through transferring oncogenic ZIP4 via EVs.⁹⁷ It has also been reported that EVs from KRAS mutant colon cancer cells transfer mutant KRAS to wild-type cells resulting in their enhanced three-dimensional growth.⁹⁸ In another lung cancer model, highly metastatic lung cancer cells 95D strongly accelerated the proliferation and migration of poorly metastatic lung cancer cells 95C, which was mediated by the EV-transferred hepatocyte growth factor (HGF).⁹⁹ Taken together, these studies demonstrated that tumor-derived EVs (TDEVs) can contribute to the horizontal propagation of oncogenic signals among subsets of cancer cells, which induces a more aggressive phenotype of the recipient cancer cells and results in tumor progression.

3.2 EV-mediated communication between cancer cells and the TME

Cancer cells also interact with noncancer cells in the TME by EV-mediated signaling, particularly with CAFs, a prevalent kind of stromal cells. The dynamic interaction between cancer cells and CAFs plays a crucial role in tumor formation and partially relies on the transmission of signals through EVs. On one hand, TDEVs are capable of transforming or activating CAFs. Webber et al.¹⁰⁰ reported that some cancer cells secrete EVs containing TGF-β which could transform normal stromal fibroblasts into CAFs. Multiple other studies also demonstrated that functional cargoes such as TGF-β, miR-1247-3p, and miR-125b transferred by EVs from liver, bladder, and breast cancer cells, respectively, induced the activation of CAFs.^101-103 As a result, the activated CAFs promote cancer cell proliferation and invasiveness by remodeling the extracellular matrix within the TME or releasing soluble factors.^{104, 105} On the other hand, EVs secreted by CAFs can also mediate tumor aggressiveness. EVs derived from CAFs isolated from human oral squamous cell carcinoma (OSCC) significantly induce migration and invasion of OSCC cells in vitro.¹⁰⁶ Similarly, breast-cancer-associated fibroblasts release EVs that activate Wnt-planar cell polarity signaling, enhancing the protrusive activity and motility of breast cancer cells.¹⁰⁷ To explore the role of EVs from CAFs involved in chemotherapy resistance, Bai et al.¹⁰⁸ demonstrated that EVs enriched with miR-522 are transmitted from CAFs to chemo-sensitive cancer cells in GC. Upon assimilation, miR-522 reduces the expression of arachidonate lipoxygenase 15 (ALOX15), thus decreasing the lipid peroxides accumulation and resulting in chemo-resistance of recipient cancer cells.¹⁰⁸ In addition to transferring functional biomolecules such as proteins and miRNAs, CAF-derived EVs can directly supply nutrients for starved cancer cells. It has been shown that EVs secreted by patient-derived CAFs contain intact metabolites including amino acids, lipids and TCA-cycle intermediates. These substances can be used by cancer cells to support their central carbon metabolism when they are lacking nutrients, ultimately promoting tumor growth.¹⁰⁹ In the TME, MSCs are highly regarded as a promising type of stem cells in the field of tissue engineering due to their convenient accessibility and their capacity to differentiate into many cell types such as adipocytes, osteoblasts, cardiomyocytes, and neurons.¹¹⁰ MSC-derived exosomes in the TME bestow colorectal stem cell characteristics by activating ERK1/2 and stimulating the Wnt signaling pathway, which raise the proportion of CSCs and promote the formation of tumor spheroid in vitro and tumorigenicity in vivo.¹¹¹ BM-MSCs released exosomes that contained miR-214 inhibited oxidative stress injury in CSCs, which ultimately assisted in the formation of tumors by means of CaMKII silencing.¹¹² Exosomes derived from tumor-associated MSCs exhibited elevated levels of miR-155. This, upon absorption by tumor cells, could lead to the inhibition of tumor suppressor genes SMARCA4 and augment the potential of tumor migration.¹¹¹ Furthermore, it has been discovered that glioma-associated human MSCs, which are a possible novel target in glioblastoma, can enhance the invasiveness of glioma stem cells through exosome-derived miR-1587.¹¹³ Nevertheless, it is intriguing that it presents antiangiogenic miRNAs in MSC exosomes, such as miR-16 and miR-100, which block angiogenesis in breast cancer cells by targeting VEGF.^{113, 114}

CSCs are a subset of heterogeneous cells residing in tumor sites possessing an unlimited capacity for self-renewal and diversification.¹¹⁵ EVs have a role in transferring information to facilitate the conversion between non-CSCs and CSCs.¹¹⁶ They could participate in maintaining the balance of CSCs such as lncRNA FMR1-AS1 in exosomes through TLR7-NFκB signaling activation.¹¹⁷ A number of processes, such as improved DNA repair efficiency and antiapoptotic ability, slowed cell cycle progression, drug efflux, and production of detoxifying enzymes, contribute to CSC therapeutic resistance, where EVs play a significant role in this process.¹¹⁸ As drug efflux pumps contribute to multidrug resistance (MDR) of cancer cells, the transfer of those transporters from drug-resistant cancer cells to low-expressing drug-sensitive cancer cells via EVs serves as an important scheme of resistance propagation. For instance, one of the drug efflux pumps P-glycoprotein plays a key role in maintaining intracellular drug concentration. Several studies found that EVs isolated from chemoresistant osteosarcoma, breast, ovarian, and PC cells transport P-glycoprotein to drug-sensitive cells and mediate the extrusion of drugs to enhance their chemoresistance.^119-122 Stromal cells are responsible for orchestrating a complex interaction with breast cancer cells in order to regulate the growth of therapy-resistant tumor-initiating cells. This is accomplished through the transfer of exosomes, which increases the expression of the IRDS genes (Interferon-Related DNA Damage Resistance Signature).¹²³ In summary, all these studies support the role of EVs in the crosstalk between cancer cells and stromal cells within the TME. Furthermore, the function of EVs between cancer cells and immune cells within the TME is discussed further below.

3.3 EV-mediated metabolic reprogramming in tumors

3.4 Role of EVs in promoting angiogenesis

Angiogenesis is a complex and dynamic process by which tumors develop new blood vessels to supply oxygen and nutrients, thus playing a critical role in tumor growth and progression. The VEGF which can induce vascular permeability and tube formation is the most typical regulator that initiates angiogenesis.¹⁵⁷ TDEVs can induce angiogenesis by horizontal transfer of VEGF directly or molecules regulating the VEGF pathway. For example, VEGF-A exhibits a higher capacity of permeability and angiogenic potential of human brain endothelial cells carried by glioblastoma stem-like cell-derived EVs.¹⁵⁸ A VEGF isoform localized on the surface of EVs can also promote tumor angiogenesis by stimulating endothelial cell migration and tube formation.¹⁵⁹ Another glioma cell U87-MG-derived EVs transfer linc-CCAT2 to endothelial cells, promoting human umbilical vein endothelial cells (HUVECs) migration, proliferation and tubular-like structure formation by upregulating VEGFA expression.¹⁶⁰ Similarly, EVs containing miR-25-3p can be transferred from CRC cells to endothelial cells, promoting vascular leakiness and enhancing CRC metastasis by regulating the expression of VEGFR2 which is the main signaling receptor for VEGF.¹⁶¹ There are also other factors transferred by EVs that can promote angiogenesis without stimulating the VEGF signaling pathway. For example, treated with anti-miR-9 or JAK inhibitor, EV-induced angiogenesis was suppressed by increasing the SOCS5 levels and deactivating the JAK–STAT signaling pathway.¹⁶² CCA-derived EVs transfer circ-CCAC1 from cancer cells to endothelial monolayer cells, disrupting endothelial barrier junction and promoting angiogenesis by increasing GRB2-like protein 2 expression.¹⁶³ Angiogenesis is also greatly impacted by TGF-β, especially TGF-β-enriched exosomes.¹⁶⁴ TGF-β+ EVs from HNSCC facilitate the transformation of nonactivated macrophages into the proangiogenic M2 phenotype.¹⁶⁵ Blocking TGF-β interactions could make these TGF-β-enriched exosomes attractive targets for antiangiogenic treatment. Similar to bevacizumab, a popular angiogenesis inhibitor that targets VEGF, RER is a recently created TGF-β inhibitor that binds to TGF-β and greatly reduces angiogenesis.¹⁶⁶ Apart from cancer cell-derived EVs, other cell types within the TME can also shed EVs to mediate angiogenesis. For example, miR-10a-5p from CAF-derived EVs promotes angiogenesis in vivo and in vitro by activating the Hedgehog signaling in cervical squamous cell carcinoma.¹⁶⁷ The delivery of miR-21 to multiple myeloma (MM) endothelial cells by CAF-derived EVs was demonstrated in another study, which subsequently facilitated angiogenesis.¹⁶⁸

There are substantial variations in the protein content of TDEVs in different types of cancer, including different proangiogenic factors. According to molecular characterization, EVs generated from glioblastoma have all the necessary components for stimulating angiogenesis including angiogenin, VEGF, TGF-β, IL-6, and IL-8.^{169, 170} Additionally, exosomes are exceptionally abundant in CD44 variant isoform 5, ICAM-1, and MMP-13 in nasopharyngeal carcinoma. Conversely, these exosomes exhibit downregulation of the angiosuppressive protein thrombospondin-1 (TSP-1).^{171, 172} By serving as a coreceptor for tissue plasminogen activator, exosomal annexin II stimulates angiogenesis in breast cancer while exosomes from bladder cancer exhibit overexpression of EGF-like repeats and discoidin I-like domain-3, which are critical for angiogenesis.^{173, 174} Moreover, the following proteins are found in exosomes produced by MM as proangiogenic factors: VEGF, basic fibroblast growth factor, MMP-9, HGF, and serpin E1.¹⁷⁵ Proangiogenic proteins such as endothelin-1, IL-8, and VEGF could be induced in lung cancer by exosome-derived sortilin.¹⁷⁶

Hypoxia is a crucial factor that affects the formation, release, and composition of EVs, as well as cancer angiogenesis. Synergistically, HIF-1 enhances the expression of proangiogenic factors, including VEGF, and angiopoietin 1/2, placental growth factor.¹⁷⁷ HIF-dependent VEGF stimulation and subsequently angiogenesis result from the loss of tumor suppressor genes such as p53, p21, pRb, or PTEN.¹⁷⁸ Under hypoxic conditions, certain important proteins such as HIF-1α, lysyl oxidases, plasminogen activator inhibitor 1, platelet-derived growth factors, TSP-1, caveolin-1, annexin II, and signal transducer and activator of transcription 3 are highly transported to exosomes.^179-184 These proteins play a crucial role in modifying the TME, facilitating the progression of the tumor, evading the immune system, promoting angiogenesis and developing resistance to therapy.

Collectively, all these studies suggest that EVs from distinct cells including tumor cells and stromal cells participate in the angiogenesis process by transferring functional cargoes which stimulate VEGF-dependent or independent signaling pathways, finally leading to tumor progression.

3.5 The functions of EVs in mediating immune evasion

EVs that originate from malignant cells are vital targets within the complex web of tumor immunity.¹⁸⁵ Supporting tumor cells against regulation by immune cells, enhancing tumor cell immune tolerance, and allowing for the evasion of immune surveillance are all attributes of these EVs, which can also inhibit immune function and promote the differentiation of regulatory T cells (Tregs), MDSCs, and tumor-associated macrophages (TAMs).^{186, 187} The purpose of this section is to shed light on the most important aspects of the current landscape of EVs that redefine the immune microenvironment (Figure 3).

4.1 EV-associated biomarkers for early cancer detection

Distinct cargo such as proteins, mRNAs, or metabolic profiles are shown in EVs comparing early-stage cancer patients to healthy controls. Given that the level of several proteins was higher in EVs isolated from the plasma or serum of tumor patients, they have been suggested as a potential biomarker for tumor diagnosis (Table 1).^248-250 In addition to plasma/serum, higher levels of associated proteins were observed in EVs in cerebrospinal fluid from brain tumors,²⁵¹ urine from balder cancer²⁵² and renal cell carcinoma,²⁵³ ascites from ovarian cancer,²⁵⁴ and tissue from the primary tumor site.^{251, 254} Even though protein-based biomarkers have gained a lot of attraction among EV biomarkers, developing them becomes more difficult when dealing with complicated samples such as serum or plasma. These samples contain a lot of nonvesicular proteins, which makes it difficult to isolate low-abundance protein complexes. Additionally, heterogeneous posttranslational modification adds another layer of complexity to these samples.²⁵⁵

Table 1 provides a summary of the EV-associated nucleic acids (mRNA, miRNA, DNA) that have been detected in body fluids for early tumor detection.³⁰³ The yield, purity, stability, and particularly the RNA content of EVs may be impacted by the RNA separation technique used. Thus, choosing the RNA separation technique based on the study's design and the body fluids’ accessibility is crucial.³⁰⁴ There is a great deal of interest in exploiting the DNA found in circulating EVs as liquid biopsies since it has been demonstrated that EVs contain transposable elements, including ssDNA, mtDNA, and genomic DNA (gDNA).³⁰⁵ When compared with DNA from cells without membranes, EVs have a higher concentration of tumor DNA since the defense against DNases keeps the DNA confined in the EV membranes relatively stable.³⁰⁶ Furthermore, EV DNA's short half-life allows for a precise depiction of the dynamic tumor hallmark, making it a valuable instrument for tracking the advancement of tumors over time and how they react to antitumor therapy.³⁰¹

Lipids and metabolites in EVs have demonstrated a growing amount of promise as biomarkers in tumor detection with the advancement of lipidomics and metabolomics. Several types of studies, including PC and pancreatic cancer studies, have involved exosomal metabolomic or lipidomic profiling, which might be used to obtain unique biomarker data.³⁰⁷ To identify PC biomarkers with enhanced sensitivity and specificity, Clos-Garcia et al.³⁰⁸ reported that a thorough examination of the composition of urine EVs may provide a window of opportunity. Consistent with the probable increase in androgen production, these authors detected elevated quantities of steroid hormones in urinary EVs, which supports the noninvasive use of urine EVs to provide information on metabolic changes in malignant tissue.³⁰⁸ A study reported that the amount of lipoprotein lipase (LPL) in EVs produced from ovarian cancer cells was shown to be considerably higher than in ovarian surface epithelial cells, indicating that LPL may be useful in the early detection of ovarian cancer.³⁰⁹ Researchers used liquid chromatography–mass spectrometry (LC–MS) to examine the lipidomic profile of exosomes formed from CRC cell lines and patients. According to the findings, there were notable differences in the lipidomic signature between exosomes derived from nonmetastatic and metastatic cell lines and patient plasma, especially in the case of glycerophospholipids and sphingolipids.³¹⁰ These results offer important perspectives on the possible use of clusters of lipid biomarkers instead of single molecules for the diagnosis of CRC. There is much need for new biomarkers for HCC surveillance in cirrhotic patients. Several modifications in the lipid content of exosomes linked to HCC have been explored to indicate the changes in ferroptosis, retrograde endocannabinoid signaling, and glycerophospholipid metabolism.³¹¹ To sum up, this study found altered pathways in exosomes that might facilitate the growth and progression of tumors, as well as potential biomarkers for the early detection of HCC. Ketone body metabolism and FAO show distinct signatures in patient serum-derived EVs, according to a comparison of healthy controls and patients receiving radiation therapy for head and neck cancer.^{88, 312} These illustrations demonstrate the significance of lipids and metabolites found in EVs for the potential of detecting early tumors and guiding therapy approaches.

Artificial intelligence (AI) has been a popular choice for various cancers' early detection. Within AI, machine learning (ML) is a subfield that learns from large amounts of data and uses algorithms to evaluate in order to create models that support decision-making and prediction. Through the analysis of exosome surface-enhanced Raman spectroscopic profiles, Shin et al.³¹³ employed AI to detect six types of solid tumors much earlier, while another study used a panel of exosome-associated proteins calculated by ML to distinguish between different kinds of tumors.³¹⁴ To quantitatively analyze the pleiotropic impact of EVs, Nagrath et al.³¹⁵ performed a novel computational methodology of the contribution of metabolite cargo to the metabolism of cancer cells delivered from CAFs, known as exosome-mediated metabolic flux analysis (Exo-MFA), based on an examination of the flux of 13C metabolic products. Exo-MFA is capable of predicting the rate of exosome internalization and determining the contribution of exosomal cargo to metabolites inside PDAC cells, and the results of this analysis strongly suggested that exosome-supplied metabolites can assist in PDAC metabolism during the initial phases of nutrient restriction.³¹⁵ Additionally, predictive panels were generated using an ML algorithm, which was also capable of distinguishing tumors from healthy controls.³¹⁶ In an effort to identify mutated proteins in circulating exosomal cargo, Kim et al.³¹⁷ implemented a deep learning algorithm and nanoplasmonic spectra. Different mutant variants of EGFR were identified in the blood of lung cancer patients using this model. Actually, confounding factors in cancer samples and data acquired from various centers should be taken into account by ML models in order to enhance cancer prediction.

4.2 EV cargo profiling for predicting treatment response and disease progression

In addition to their diagnostic potential, EVs also provide insights into the whole spectrum of cancer development, from the disease's earliest beginnings to metastases and treatment responses. An investigation revealed that the presence of Tim-3 and Galectin-9 proteins in EVs was linked to age, distant metastasis, and TNM staging in individuals with NSCLC.³¹⁸ EV cargos like NY-ESO-1, EGFR, PLAP, EpCam, and Alix had a substantial effect on overall survival (OS), which depended on their concentration in NSCLC.²⁶⁵ Hoshino et al.⁵⁶ have identified a set of tumor-type-specific EV-associated proteins in both tumor tissues and plasma, which can be used to accurately diagnose cancers of unknown origin. Furthermore, EV-mRNA can serve as a biomarker for prognosticating the impact of ICIs. Del et al.³¹⁹ conducted a study to find the relationship between the effectiveness of anti-PD-L1 immunotherapy and the quantity of EV-mRNA PD-L1 expressed in plasma. Amounts of studies suggested that certain immunotherapy patients—many of whom exhibit no response at all or continue to progress their disease—had rising exosomal PD-L1 levels over time. Finally, increased exosomal PD-L1 levels may result in poor efficacy both during and after treatment.^{153, 320, 321} Currently, numerous studies are endeavoring to address the issue of immunotherapy resistance by targeting exosomal PD-L1 and related modulators.^{235, 322}

Tumor-specific biomarkers have the potential to forecast the effectiveness of treatment and tumor progression. Compared with traditional biomarkers like CA199 and CEA, HOTTIP in serum EVs demonstrated superior accuracy in GC.³²³ Urinary EVs lncRNA PCAT-1 and MALAT1 overexpression in bladder cancer were linked to a poor recurrence-free survival rate and facilitated recurrence prediction.³²⁴ Higher circulating levels of EV lncRNA-ATB in HCC were linked to decreased OS and progression-free survival (PFS),³²⁵ while uroepithelial carcinoma patients may be at risk of metastasis if they have increased levels of circRNA PRMT5 in the serum and urine.³²⁶ Although the initial analysis of EV cargo focuses more on mRNA, miRNA and lncRNA, subsequent research has uncovered a plethora of other forms of ncRNAs, such as tRNA and tRNA fragments, Y RNA, piRNA, and rRNA, which may function as biomarkers for therapy response.³²⁷ Using LC–MS-based untargeted lipidomics, Tao et al.³²⁸ sought to identify potential metabolic biomarkers linked to tumor stage, CA199, CA242, and tumor diameter in pancreatic cancer. A plasma exosome-based metabolome marker pattern from a random forest model that can predict the recurrence of esophageal squamous cell carcinoma revealed a significant increase in palmitoleic acid in recrudescent individuals.³²⁹ The levels of metabolites carried in serum-derived exosomes for glycolysis, gluconeogenesis, the tricarboxylic acid cycle, pyruvate metabolism, and the mitochondrial electron transport chain were shown to be significantly different in head and neck cancer patients before and after radiotherapy, which mirrored putative radiotherapy-induced alterations in a variety of metabolic pathways.⁸⁸ In the end, the capacity to use EVs as dynamic markers of cancer development holds the potential to completely redefine cancer treatment, resulting in more accurate and effective therapeutic strategies.

4.3 Diagnostic potential of EVs in liquid biopsies and circulating tumor cells

EVs may prove to be a valuable noninvasive diagnostic and screening instrument in the early stages of the disease. Taking circulating EV proteins for example, pancreatic cancer patients can be distinguished from healthy donors using GPC1-positive circulating exosomes according to the study of Melo et al.²⁶² A study indicates that the decrease in gastrokine 1 protein in serum EVs could serve as a valuable diagnostic for identifying individuals with GC.³³⁰ Additionally, it has been claimed that novel diagnostic biomarkers for breast cancer have been discovered in the form of phosphorylated proteins from the plasma EVs of patients.³³¹ Concurrently, it has been discovered that miRNAs from EVs may serve as potential diagnostic indicators for many malignancies, such as ovarian,³³² hepatocellular,²⁷⁹ and breast cancers.³³³ A cluster of miRNAs identified using EV small RNA sequencing in both the ascites and plasma was found to exhibit a high level of diagnostic accuracy.³³⁴ Jin et al.²⁸⁶ conducted a study where they used miR-23a-3p, miR-486-5p, let-7b-5p, and let-7e-5p in exosomes to diagnose NSCLC at an early stage. The study achieved an AUC value of 0.899, a sensitivity of 80.25%, and a specificity of 92.31%.²⁸⁶ And the diagnostic efficacy of early-stage tumors can be enhanced by the combination of exosomes and serum markers. Based on plasma exosomes miR-15a-5p, the AUC for the diagnosis of stage I endometrial cancer was only 0.813. However, the AUC was increased to 0.899 when CEA and CA125 were included in the analysis.³³⁵ Another study discovered that the combination of 8exo-miRNA and 5cf-miRNA, along with CA199, effectively distinguished patients with tumors that had a CA199 level below 37 U/mL. This identification had an AUC value of 0.99.³³⁶ The comprehensive range of diagnostic capabilities of EVs in liquid biopsies and circulating tumor cells (CTCs) has been documented in Table 1. While EVs show promise for early tumor diagnosis, there are still constraints that need to be addressed in order to enhance the accuracy and dependability of EVs.

5.1 Strategies to inhibit EV biogenesis and secretion

The majority of present treatment approaches aim to reduce EV production, secretion, and uptake, impede EV-mediated intercellular communication, and eliminate a particular active molecule found in EVs (as shown in Table 2).

In terms of exosomes, mechanisms that produce or modify cell membrane asymmetry seem to be crucial for the formation of MVs. In actuality, cholesterol appears to play an important role in the budding of the cell membrane, as lipid rafts are essential for this process and their reduction decreases the release of MVs.^{364, 365} Pantethine may be employed to impede the discharge of MVs, which has the ability to impede the translocation of PS on the outer membrane leaflet, a crucial mechanism involved in the generation of EVs.³⁶⁶ Doxorubicin (Dox)-resistant cells generated more MVs upon stimulation with the agonist A23187, but Dox-sensitive MCF-7 cells did not. After receiving pantethine as a pretreatment, this effect was lessened, resulting in a 24% total MV reduction.³³⁷ Therefore, in order to gain a comprehensive understanding of pantethine and its potential as a selective inhibitor of MVs, it is necessary to expand upon the previous work. The tricyclic antidepressant imipramine has gained notice for its ability to inhibit the enzyme acid sphingomyelinase (aSMase). Since it improves membrane fluidity, exosome release, and EV synthesis, aSMase enzymes catalyze the hydrolysis of sphingomyelin to ceramide, a process engaged in the formation of both exosomes and MVs.³³⁷ It should be noted that while imipramine was shown to disrupt exosomes and MVs in PC-3, these vesicle types were not examined individually including EVs of both <150 and >150 nm-sized vesicles.³³⁸ It would be beneficial to conduct more comprehensive investigations of imipramine at subtoxic concentrations and to conduct a thorough characterization of the EVs.

GW4869, a neutral sphingomyelinase (nSMase) inhibitor, is the most extensively used pharmaceutical treatment for preventing exosome production. GW4869 has proven to be essential for immunological modulation due to its ability to facilitate the suppression of exosomes containing PD-L1, which rejuvenated T cells and boosted ferroptosis.³⁴⁰ Besides, the oxygen consumption rate in breast cancer cells could be partially restored by GW4869, demonstrating that exosomes released by CAFs were responsible for the decrease in mitochondrial respiratory function in tumor cells and could be targeted to deter tumor metabolism.¹²⁷ Deterred antitumor immunity could be alleviated by reducing the suppressive effects of exosomes. A set of semiconductor polymers encapsulating an exosome inhibitor GW4869 and ferroptosis inducer was utilized to fabricate phototheranostic metal-phenolic networks (PFG MPNs), which were used to alleviate exosomal silencing during DC maturation by augmenting immunogenic cell death.³⁴⁰ The fibroblast-derived conditioned medium enhanced the percentage and clonogenicity of CSCs. Treatment with GW4869, a compound that inhibits exosome production, resulted in a reduction of CSCs and increased susceptibility to 5-fluorouracil (5-Fu) or OXA.³³⁹ In NOD/SCID mice carrying ASPC1 and CAFs, the tumor's growth rate was significantly slower 10 days after cotreatment of gemcitabine and GW4869 than it was in the gemcitabine-only group. Although GW4869 has been investigated very thoroughly and shown to suppress exosomes, some studies have not described the exosomes’ characterization or their separation process. By targeting nSMase, more drugs like spiroepoxide and DPTIP have been explored to limit exosome secretion by preventing ceramide-modulated inward budding of MVBs and the consequent release from MVBs.^{343, 344} Apart from that, antidiabetic drugs (Glibenclamide)³⁴⁵ and anti-inflammatory drugs (Indomethacin)³⁴⁶ are implicated in lipid transport. These compounds have the potential to serve as inhibitors of exosome release, as lipids are fundamental to the formation of MV and exosomes.

MVBs can, as previously indicated, fuse with lysosomes or release exosomes through fusion with the cell membrane. Many proteins interact with actin and microtubules of the cytoskeleton to govern their translocation toward the cell membrane. It appears that calpains can encourage MVs shedding because of their involvement in cytoskeleton remodeling. Therefore, it has been observed that blocking them with calpain inhibitors lowers the number of MVs released from cells.³⁶⁷ Calpeptin is the most extensively researched inhibitor of calpain, which has been widely utilized as an MV inhibitor. Calpeptin has been reported to ameliorate MV-mediated anticancer drug resistance. Jorfi et al.³⁴⁷ showed that the accumulation of methotrexate and docetaxel within the cells was made possible by calpeptin's inhibition of MVs release. This accumulation led to a significant reduction in cell proliferation and an increase in cell death compared with the conditions without calpeptin treatment in vitro and in vivo of PC.³⁴⁷ Nevertheless, given the positive results of the calpeptin experiments that have been published thus far, more research on the substance's capacity to prevent EV release is necessary. ROCK1 and ROCK2 play a part in the formation of EVs, which could be inhibited by Y-27632, a selective, reversible, competitive kinase-activity inhibitor.^{348, 368} Mechanistically, ROCK1 and ROCK2 govern actin cytoskeletal remodeling and actomyosin contraction by activating adducin, which keeps the actin network together.¹³ Cerione et al.³⁴⁸ explored that ras homolog family member A (RhoA) knockdown prevented the release of EVs from cancer cells. It is intriguing that the presence of EVs along their membrane was eliminated by treatment with Y-27632, which confirmed the role of Rho-GTPases in the formation of EVs.³⁴⁸ Furthermore, the activation of ERK, which is required for microvesiculation, can be effectively prevented by protein kinase inhibitors such as MEK 1 and MEK 2 inhibitor U0126.³⁵⁰ Inhibition of Rac1, a member of the Rho family has been shown to exert exosomal targeting.³⁵¹ As a powerful and selective inhibitor of Ras farnesyltransferases, manumycin A, has been studied as an exosome secretion inhibitor due to Ras's role in exosome release. The biogenesis of exosomes that are dependent on ESCRT was specifically inhibited by manumycin A, as reported by Datta et al.³⁵² The production of exosomes was reduced by manumycin A in PC cell lines, with the effect being even more pronounced when manumycin A was used in conjunction with GW4869.³⁵² The drug Tipifarnib is another example that employs this comparable mechanism to prevent the discharge of EVs.³⁵³ A family of Ca²⁺-dependent enzymes known as the peptidylarginine deiminases (PADs) is responsible for protein deimination. When PAD enzyme activity was pharmacologically inhibited with chloramidine, significantly no deimination of cytoskeletal actin and then less MV release occurred.³⁵⁴ Another primary regulator of the mechanism that drives the release of EVs is the calcium-mediated activation of protein kinase C (PKC) and the externalization of PS.¹³ A study showed that treating PC-3 cell lines with bisindolylmaleimide-1 (BIM-1), a cell-permeable and reversible PKC inhibitor, reduced EV release by 75%.³⁵⁵ Reduced PS externalization has been suggested as the mechanism by which BIM-1 lowers EV secretion levels. Sulfisoxazole³⁵⁶ and macitentan,³²² two endothelin A receptor antagonists, have recently become available as novel treatments for preventing exosome secretion, inhibiting several Rab proteins as well as elements of the ESCRT-dependent pathway, such as ALIX and VPS4B. The syndecan-syntenin-ALIX pathway, which is associated with exosome biogenesis, is disrupted by SyntOFF through binding to the PDZ domain of syntenin. In breast cancer patients, this perturbation leads to a reduction in proliferation and metastasis, as well as a decrease in exosome secretion.³⁵⁷

Given the complexity of the EV release mechanism, different drugs acting on various targets involved in the same signal cascade can impede the same production pathway. Studies indicate that Ca²⁺ is an essential element in the process of Ca²⁺-dependent membrane fusion and exosome production.^{369, 370} Store-operated calcium channel blockers including ketotifen, an antihistamine reduced the release of exosomes from a variety of cancer cells by 70% through preventing the influx of calcium into cells.³⁵⁸ A derivative of amiloride, dimethyl amiloride is a drug used to treat high blood pressure and has been suggested as a potential exosome blocker because of its role of blocking the H⁺/Na⁺ and the Na⁺/Ca²⁺ channels.³⁵⁹

Exosome absorption in the receiving cell occurs in a nonrandom manner in conjugation with transmembrane proteins. Several mechanisms, including phagocytosis, macropinocytosis, caveolin-dependent endocytosis, and clathrin-mediated endocytosis (CME), can lead to the process.³⁷¹ All in all, the potential of these drugs (such as free heparan sulfate chain, dynasore, methyl-β-cyclodextrin, and gefitinib) to impede the uptake of EVs has been examined as shown in Table 2.^360-363 These results imply that focusing on different stages of exosome biosynthesis, secretion and uptake may be beneficial for the exploration of efficient drugs.

5.2 Utilizing EVs as drug delivery vehicles for targeted therapy

It is possible for EVs to avoid being taken up by macrophages, prolong their stay in the bloodstream, and pass through the extracellular matrix and vascular walls. Moreover, EVs possess low immunogenicity and good biocompatibility, enabling their stability and widespread distribution in biological liquids.²¹ Apart from that, they can also enrich the tumor, penetrate the tumor cells, and release carried drugs. The utilization of natural or gene-engineered EVs as drug-delivery vehicles offers special advantages because of their diverse biological features (Figure 4).³⁷²

Since many anticancer chemotherapeutic drugs target intracellular targets, their ability requires them to traverse through cell membranes. The drugs that are loaded in EVs are safeguarded by the lipid bilayer of EVs, which facilitates their entry into cells by interacting with the recipient cells through membrane proteins. Their minimal immunogenicity and toxicity are the most significant advantages of EVs, as they can significantly avoid immune system clearance.³⁷³ To enhance the selectivity of Dox toward tumor tissues rather than cardiomyocytes, Wei et al.³⁷⁴ coincubated Dox with BM-MSC-exosomes to create BM-MSC-derived exosome-loaded Dox (Exo–Dox) and showed noticeably higher cellular absorption efficiency and anticancer effects compared with free Dox. In addition, it also showed lower uptake efficiency and toxic effects in cardiomyocytes.³⁷⁴ They also indicated that Exo–Dox may accumulate in the tumor site in vivo, attracting osteosarcoma cells via the SDF1–CXCR4 axis. When comparing the Exo–Dox group to the free Dox group, there was a significant decrease in both Ki67-positive cells and cardiotoxicity.³⁷⁵ The existence of MDR decreases the tumor response rate to treatment using traditional chemotherapy drugs. Paclitaxel (PTX) loaded into sonication-treated exosomes demonstrated significant loading and prolonged drug release. PTX-loaded exosomes (exoPTX) exhibited notable accumulation within tumor cells and demonstrated an increase in cytotoxicity toward drug-resistant tumor cells. In vivo, exoPTX also displayed colocalization with the tumor and further inhibition in a pulmonary-metastasis mouse model of LLC.³⁷³ After genetic modification of human primary CD8+ T-cell-derived exosomes, they were impregnated with IL-2 and cetuximab (CTX), an anti-EGFR antibody, which has the potential to augment tumor cytotoxicity and refine cancer targeting.³⁷⁶ These suggest that EVs have significant potential for the delivery of therapeutic agents in the treatment of drug-resistant malignancies, as they facilitate in vivo targeting and enhance the antitumor effects.

Apoptosis is induced by TNF-related apoptosis-inducing ligand (TRAIL) in cancer cells. Unfortunately, because of their limited targeting and short half-life in vivo, TRAIL-based drugs, such as recombinant human soluble TRAIL, have not yet shown adequate therapeutic efficacy in clinical settings.³⁷⁷ Triptolide (TP) was loaded into exosomes released from TRAIL-overexpressing macrophages Raw264.7 to form TP-based TRAIL-engineered exosomes (TRAIL–Exo/TP). In vitro and vivo experiments revealed that TRAIL–Exo/TP significantly inhibited the development and promoted apoptosis of melanoma, in comparison with free TP and TP-loaded exosomes alone. On the other hand, TRAIL–Exo/TP was found to be biologically safe and did not induce systemic toxicity or myelosuppression.³⁷⁸ Exosomes containing TRAIL have been demonstrated in other recent preclinical investigations to cause apoptosis while preventing the growth of cancer in vitro as well.^{379, 380} To effectively deliver TP into the tumor, Gu et al.³⁸¹ found another method using arginine–glycine–aspartate (cRGD)-modified exosomes. This showed strong tumor targeting and a longer half-life of exosome-delivered drugs, which reduced systemic toxicity.

In contrast to small-molecule medicines, degradation occurs in biomacromolecules such as proteins, peptides, and nucleic acids in vivo. Additionally, they would contend with a number of biological obstacles to cell-to-cell communication, including cell membranes and endosomes, which restrict the use of biomolecules in anticancer therapy.³⁸² Exosomes carrying the biomolecules are highly advantageous for the delivery of biomolecular therapeutics such as exosomes loaded with Survivin-T34A.³⁸² It enhanced tumor cell apoptosis when treating pancreatic cancer cells in conjunction with gemcitabine through inhibiting survivin in tumor cells. Choi et al.³⁸³ explored a novel method for delivering target proteins within cells using optically reversible protein–protein interactions (EXPLORs). It has been demonstrated that treating recipient cells with protein-loaded EXPLORs greatly increases the intracellular levels of cargo proteins.³⁸³ Delivery of nucleic acid drugs in exosomes such as antisense oligonucleotides (ASOs) has been demonstrated to significantly decrease the target gene expression.³⁸⁴ Significant anticancer activity is produced by exosomes loaded with the ASO of C/EBPβ or STAT6 (exoASO), which transforms M2 into M1 macrophages.³⁸³ CRISPR/Cas9 has been widely used in genome editing based on common viral vectors or nonviral vectors. Both vectors have drawbacks like immunogenicity and insertional mutagenesis, organ toxicity and low biocompatibility respectively.³⁸⁵ Exosomes offer a promising substitute for CRISPR/Cas9 gene delivery methods. To overcome these disadvantages, McAndrews et al.³⁸⁶ demonstrated that exosomes carrying CRISPR/Cas9 plasmid DNA may specifically target pancreatic cancer cells with mutant KRAS G12D, leading to the deletion of target genes and the subsequent inhibition of tumor growth. Hao et al.³⁸⁷ reported that Erastin (iron ion inducers) and RB (photosensitizers) were successfully enclosed within exosomes through the process of sonication, which exhibited a potent induction of ferroptosis in tumors following exposure to radiation. To take advantage of the killing effect that the zinc oxide nanocrystal cargo conveys against lymphoma cells, EVs that are engineered from healthy cells and loaded with inorganic nanoparticles and monoclonal antibodies can be triggered on-demand by an external stimulus.³⁸⁸ The recently developed iRGD-tagged exosomes expressing EBV–miR-BART1-5p are a potentially useful tool for inhibiting tumor cell-mediated vasculogenic mimicry and endothelial sprouting angiogenesis in nasopharyngeal carcinoma.³⁸⁹ Photothermal and hyperthermia eradicate cancer cells and acquire exosomes containing significantly more immune-activating molecules in breast cancer. This indicates that thermal treatments, particularly those using photothermal-derived exosomes, enhance the vulnerability of malignancies to T cells, thereby inhibiting the growth of tumors.³⁹⁰ EV drug delivery has gained significant momentum during the past decades, developing into a potentially effective biological therapeutic approach. Modern methodologies allow scientists to precisely target therapy by inserting therapeutic chemicals onto the surface of EVs.

EVs that have been modified through the application of bioengineering techniques in order to improve their drug-loading efficiency, targeting capability, and resistance to clearance are referred to as engineered EVs.³⁹¹ Exogenous loading methods mean the process of loading pharmaceuticals directly into pre-separated exosomes by the utilization of membrane penetration methods while endogenous loading methods are based on the parent cell modification through direct transfection and coincubation.^{392, 393} However, both of them have disadvantages, including the small size of exosomes limiting their ability to load drugs and the high level of technical skill and equipment support needed for exogenous loading. Moreover, it also faces poor exosome-storage stability resulting from exosomes’ propensity to aggregate and degrade over extended periods of storage. As for endogenous loading methods, higher costs and the verification of drug-loading efficacy remain hurdles. It also has trouble choosing the best drugs and donor cells.³⁹⁴

5.3 Modulating EV-mediated intercellular communication to enhance treatment responses

Certain cancer patients benefit from extended PFS as a result of ICI-based treatments; however, a significant proportion of patients experience relapses due to acquired resistance. Consequently, it is critical to clarify the underlying mechanisms and devise approaches to overcome ICI resistance (Table 3).^{395, 396} An increasing amount of data currently points to links between poor prognosis, ICI resistance and substantial infiltration of immunosuppressive M2-like TAMs.^{397, 398} In terms of the underlying mechanisms, M2-derived exosomes can confer ICI resistance by delivering apolipoprotein E to cancer cells, downregulating MHC-I expression and reducing intrinsic tumor immunogenicity.³⁹⁹ To determine whether TAM-derived exosomes stimulated tumor immune evasion, Jiang et al.⁴⁰⁰ reported that LINC01232 enhanced the transcription of NBR1 by mechanistically binding to E2F2 and facilitating its entry into the nucleus of glioma cells. The expression of MHC-I on the surface of tumor cells is induced, resulting in the evasion of CD8+ CTL immune attack by tumor cells.⁴⁰⁰ The distinct exosomes that contain BMI1 stimulate the growth and metastasis of CCA via autocrine and paracrine processes. Furthermore, one study showed that by encouraging repressive H2A ubiquitination in CCA cells, BMI1 suppresses chemokines that recruit CD8+ T cells, implicating a potential novel combination therapy of anti-PD-1 and BMI1 inhibitors for CCA.⁴⁰¹ Anti-PD-1 treatment sensitivity could also be compromised by exosomal circZHF451 through the induction of M2 macrophage polarization in lung adenocarcinoma (LUAD). These researches elucidated a novel mechanism by which anti-PD-1 resistance develops and identified a potential biomarker for predicting anti-PD-1 efficacy.²³⁰

BC cells acquired PTX resistance in response to gp96 exosomes derived from PTX-resistance-BC, whereas CD8+ T cells isolated from human peripheral blood mononuclear cells experienced pyroptotic cell death.⁴⁰² Cisplatin resistance was enhanced in NSCLC cells derived from PD-L1-containing exosomes.²⁰⁸ By means of exosomal miR-21 delivery, tumor cells can inhibit the activity of inflammasomes in macrophages in response to chemotherapy in malignancies undergoing snail-induced EMT.⁴⁰⁴ It has been reported that EVs containing miR-182 promote HCC resistance to cisplatin by regulating TP53INPI.⁴⁰⁵ A similar transformation of the chemoresistance phenotype has also been reported in cisplatin-resistant lung and breast cancers by transferring miR-100-5p and miR-423-5p, respectively.^{406, 407} The actin-associated protein plasma gelsolin can be secreted and transported via EVs from chemoresistant ovarian cancer cells to their chemosensitive counterparts to confer cisplatin resistance.⁴¹¹ In addition to PTX and cisplatin, several frequently used chemotherapeutic medicines have been found to exhibit resistance through EV transportation such as gemcitabine,^412-414 carboplatin,⁴¹⁵ OXA,⁷³ 5-Fu,¹³⁸ Dox,⁴¹⁶ temozolomide,^417-419 melphalan, and bortezomib.¹³⁶ Taking gemcitabine for example, macrophage-derived EVs secrete CHI3L1 and FN1 as their most prevalent cargo proteins. Through the ERK signaling pathway, they could play a crucial role in transferring gemcitabine resistance characteristics to cancer cells through intercellular communication in pancreatic cancer.⁴¹²

Molecularly targeted therapies employ various mechanisms to combat cancer, including impeding the growth and division of cancer cells by disrupting signaling pathways, inhibiting the formation of blood vessels that support tumor growth, transferring cytotoxic substances directly to cancer cells, and depriving cancer cells of essential hormones necessary for their growth.⁴²⁵ Identifying the mechanism of resistance to targeted therapy and developing strategies to overcome it will be a highly effective approach to tumor treatment. In NSCLC, osimertinib, a third-generation EGFR-TKI, can inhibit both sensitive EGFR mutations and acquired EGFR T790M mutations.⁴²⁶ While osimertinib has shown considerable effectiveness and acceptable safety, the problem of resistance is still unavoidable. Exosomes originating from osimertinib-resistant cells were shown to be internalized by macrophages, resulting in macrophage polarization toward the M2 phenotype.⁴²⁰ However, there was a significant correlation between the prolongation of PFS following gefitinib treatment and plasma exosomal Docking Protein 3 (DOK3), a molecule implicated in B-cell receptor signaling in lung cancer. These results imply that patients are more likely to benefit from gefitinib therapy if they have active T- and B-cell immunity.⁴²¹

Radiation therapy is used prevalently as a therapeutic modality for the management of cancer. The intrinsic metabolism of tumors is important for their resistance to ionizing radiation, making radiotherapy less effective. The exosomal proteins ALDOA and ALDH3A1 were critical signaling components involved in the mechanism by which irradiated lung cancer cells promoted the migration of recipient cells by accelerating glycolysis.⁴²⁷ Valya et al.⁴²⁸ reported that irradiation induced the export of miR-603 via EVs, which promoted the CSC state and acquired radioresistance in glioblastomas. Similarly, mi-208a can be transported by EVs and affect the proliferation and radiosensitivity of lung cancer cells by targeting p21.⁴²⁹

Studies have demonstrated that cargos of EVs or drugs targeting EVs have a synergistic effect with traditional antitumor therapy through enhancing immune cell activation and inducing cancer cell death.⁴³⁰ Botulinum neurotoxin type A, which has been reported to have antitumor effects, augments the efficacy of PD-1 inhibitors on MC38 tumors through reducing serum exosome levels and improving the population of tumor-infiltrating CD4+ and CD8+ T lymphocytes.⁴³¹ The United States Food and Drug Administration-approved antagonist macitentan, a dual endothelin receptor A/B (ETA/ETB) antagonist used to treat pulmonary arterial hypertension, significantly increased antitumor efficacy by inhibiting exosome secretion and increasing CD8+ T-cell quantity and activity while lowering Treg numbers in tumors and draining lymph nodes when combined with an anti-PD-L1 drug.³²² One study reported that sulfisoxazole-mediated inhibition of tumor-derived exosomal PD-L1 has the capacity to impede immune evasion. Combining sulfisoxazole with anti-PD-1 therapy in animal models led to a significant decrease in exosomal PD-L1 levels in the blood and the activation of CD8+ cytotoxic T cells.⁴³² PS, which is expressed on the surface of exosomes derived from human TMEs, has been causally associated with T-cell immunosuppression. Richard et al.⁴³³ synthesized a novel compound consisting of (ZnDPA)6-DP-15K designated as ExoBlock, which is a multivalent PS binder that substantially inhibits the immunosuppressive activity of exosomes, leading to an increase in both the quantity and functionality of CD4+ and CD8+ T cells in ovarian tumors and melanoma. The nonsteroidal anti-inflammatory drug sulindac has been demonstrated to impede the development and progression of CRC with the mechanism of a reduction in exosomal PD-L1. As a result, sulindac can potentially enhance the overall efficacy of anti-PD-L1 therapy.⁴³⁴ However, to confirm the efficacy of these pharmaceuticals in combination with PD-1 inhibitors, additional clinical trials are needed.

6.1 EV-based liquid biopsies for personalized cancer treatment

A major advancement in contemporary medicine is personalized cancer therapy, which entails creating treatment plans based on the features that make each patient's cancer distinct.⁴³⁵ Imatinib is a medication that may be the embodiment of customized treatments.⁴³⁶ The treatment for chronic myelogenous leukemia (CML), imatinib, selectively deactivates a mutant tyrosine kinase called BCR–ABL, which is encoded by the fusion of two genes.⁴³⁷ This genetic construct causes uncontrollable growth and malignant transformation in cells as a result of an aberrant chromosomal translocation. Therefore, a medication that targets this gene's product ought to destroy leukemia cells only while sparing healthy cells. That's exactly what it accomplishes, demonstrating remarkable efficacy and minimal harm. Another excellent example of personalized cancer treatment is using immunotherapies, including PD-1 blocking antibodies, in a customized manner, giving pembrolizumab to patients whose tumors express PD-L1 at measurable levels.^{438, 439}

EVs have been the subject of a significant amount of effort to convert them into cancer biomarkers. Nevertheless, the recent focus is on the potential of these biomarkers to transform the field of personalized treatment. There are two primary applications for EVs in adaptive medicine: adjusting the initial treatment plan as needed during therapy using routine liquid samples to track a patient's reaction; making the best clinical decisions for each patient.⁴⁴⁰ Several studies demonstrated that specific drugs have the potential to cause cancer cells to alter their EV emission characteristics following CTX therapy, which could indicate drug-related therapeutic stress.⁴⁴¹ For instance, EGFR, p-EGFR, and gDNA-containing exosome-like EVs were released in large quantities when treated with the EGFR inhibitor CTX.⁴⁴² As a result, it is possible to characterize and use these EV emission profiles to assess the effectiveness of specific treatments in individuals.

6.2 EV-based vaccines and immunotherapeutic strategies

Therapeutic cancer vaccines have been investigated as a potential advanced weapon to provide clinical advantages for cancer patients.⁴⁴³ Dex is thought to be a viable approach for DC-based immunotherapy because it maintains the essential immunostimulatory characteristics of DCs.⁴⁴⁴ TDEVs produce tumor-specific antigens that may also be recognized and processed by DCs, leading to the activation of DCs and the stimulation of antigen-specific CD8+ T lymphocytes, which in turn exhibit antitumor effects. Following Dex treatment, the number of PD-1+ CD8+ T cells significantly increased, suggesting that PD-1 inhibitors combined with Dex may have combined antitumor effects.⁴⁴⁵ As a potential antitumor vaccine, the human endogenous MUC1 protein has been utilized to conjugate with Dex. The antibodies induced by this vaccine exhibited robust affinity toward tumor cells expressing MUC1 and elicited antigen-specific CTLs and robust antibody response, leading to tumor eradication.⁴⁴⁶ In contrast, immature DCs promote inflammation and tumor progression because of their inability to stimulate cytotoxic T-cell responses. In Ewing sarcoma, EVs reduced the expression of costimulatory molecules associated with DC maturation (CD74, CD86, and HLA-DR) while also inducing cytokine release, including IL-6, IL-8, and TNF of CD33+ myeloid cells and CD14+ monocytes. EVs inhibited the proliferation of CD4+ and CD8+ T cells and IFN-γ release. As a result, antigen-presenting cell differentiation and function may be compromised by EVs, thereby diminishing adaptive immunity.⁴⁴⁷ The advancement of Dex therapy is confronted with a formidable obstacle. To facilitate the extensive application of Dex, it is imperative to devise an advanced technique for its synthesis that incorporates a greater quantity of pure exosomes. Dex has numerous complex mechanisms and effects on the immune system that have not been fully elucidated. Because Dex vaccine research is still in its infancy, additional investigations are necessary to improve the understanding and application of Dex.⁴⁴⁸

Mature Dex (mDex) has been shown to ameliorate the immunosuppressive TME (Table 4). Mechanistically, mDex has been found to have a greater capacity to stimulate naïve DCs and T cells than exosomes isolated from immature DCs, as evidenced by their increased expression of MHC and costimulatory molecules. mDex in combination with PLX-3397, a small molecule inhibitor of CSF-1/CSF-1R, enhanced CD8+ T-cell infiltration and depleted TAMs and MDSCs in the TME of mouse melanoma model, resulting in prolonged survival and delayed tumor growth.⁴⁴⁹ It was suggested that immunotherapy based on mDex provides a therapeutic approach for the management of solid malignancies.

In addition to Dex, alternative mechanisms by which EVs induce an antitumor immune response have been demonstrated. Tumor-derived circulating exosomal MHC-I is crucial for immune system activation. Combining immune checkpoint inhibition with MHC-I overexpression in exosomes allowed glioma cells to present antigens again and stimulated CD8+ T cells to mount a powerful antitumor immunological response.⁴⁵⁵ Shin et al.⁴⁵⁶ reported that EVs derived from CD4+ T cells enhance the proliferation and activity of CD8+ T cells, thereby augmenting their antitumor response. Emerging evidence has indicated that CD8+ T cells may also perform exosome delivery to recipient tumor cells, thereby inducing the release of specific cargoes, including mRNAs, miRNAs, proteins, and lipids to kill tumors.⁴⁵⁷ VδT cells are MHC-unrestricted lytic innate-like T cells. They have tremendous immunotherapeutic potential against malignancies. Vδ2 T cells can be found in the peripheral circulation and lymphoid tissues. A T-cell-mediated antitumor response was produced by Vδ2 T-cell-derived exosomes, which led to more effective suppression of EBV-associated malignancies.⁴⁵⁸ CAR-T cells are potentially effective new treatments for cancers, partially because of the EVs released by CAR-T cells. Exosomes harboring CARs express a high concentration of cytotoxic biological molecules and prevent the growth of tumors.⁴⁵⁹ In addition, the M1 macrophage-derived exosomes can reinforce the CTL response by acting as immune adjuvants.⁴⁶⁰ It has been demonstrated that inducing the active release of EVs by NK cells could be an effective therapeutic strategy. NK cells and NK-derived EVs were significantly more abundant in NSCLC patients than in healthy donors. Additionally, the number of NK cells and the quantity of CTCs exhibited a negative correlation.⁴⁶¹

6.3 Clinical trials utilizing EVs as therapeutic agents or biomarkers

Since EVs are rich in parent cell-derived proteins, they provide a unique window into cancer processes. The capacity of EVs to deliver accurate and trustworthy information is what supports the increasing acknowledgment of their potential in therapeutic applications.⁴⁶² Proteins enclosed within protective lipid bilayers make them more accurate diagnostic tools with a lower chance of false positives, which is especially helpful in situations involving heterogeneous cell populations such as tumors. The usefulness of proteins in EVs as biomarkers has already been shown in a few clinical studies, such as thyroglobulin in the urine of follicular thyroid cancer patients (NCT05463107) or integrins in CRC patient blood (NCT04394572) as shown in Table 4. Actually, among the numerous proteins, the identification of the most specific protein remains a critical issue that must be resolved in the future.

As we showed in the above in vitro and in vivo study, nucleic acids (mRNA, miRNA, lncRNA, circRNA) in EVs have the potential to function as biomarkers. Clinical trials for EVs as diagnosis and therapeutic agents are currently underway, and we have updated them in Table 4 to reflect their potential medicinal benefits. After being internalized by recipient cells, mRNA produced from EVs can be translated into proteins, which can control the biological activities of receptor cells in addition to serving as a signal carrier. New forms of RNA are constantly appearing, joining the already established mRNAs and ncRNAs.⁴⁶³ These include chimeric RNAs, tRNAs, and piRNAs, which are also explored in recent clinical trials (ChiCTR2000031507).

A phase I trial investigates the optimal dosage and side effects of exosomes from mesenchymal stromal cells carrying KrasG12D siRNA (iExosomes) for the treatment of patients with metastasic pancreatic cancer (NCT03608631). The purpose of another clinical trial is to determine whether plant exosomes can more efficiently transport curcumin, which has a potent antitumor growth inhibitory effect, to both normal colon tissue and colon cancers (NCT01294072). In phase II clinical research, Chaput et al.⁴⁵² determined the clinical benefit of IFN-γ-Dex loaded with cancer antigens in patients with NSCLC that is incurable and does not exhibit tumor progression as maintenance immunotherapy following induction chemotherapy. Since the sources of EVs are diverse, they can be used as a variety of medication carriers. Examining the safety and effectiveness of EVs as medication delivery vehicles and investigating their possible therapeutic applications in a range of diseases are the goals of further clinical trials.

7.1 Standardization of EV isolation and characterization protocols

Numerous biofluids, including blood, urine, sweat, and milk, are frequently reported to contain EVs.⁴⁶⁴ However, protein complexes and biomacromolecules can make it difficult to separate and collect from these complex biofluids.⁴⁶⁵ Numerous methods have been developed according to density, including two widely used techniques: density gradient centrifugation and differential ultracentrifugation (Figure 4).⁴⁶⁶ Notably, there are certain restrictions associated with these methods. The structural integrity of exosomes may be compromised by the intense centrifugal forces during ultracentrifugation, which requires expensive apparatus and huge sample volumes. Compared with centrifugation, size-based isolation such as sequential filtration and size-dependent microfluidics has many benefits.⁴⁶⁷ Reducing the size of the device results in lower sample volume needs, higher throughput, and enhanced sensitivity. Because of its adaptability and label-free ability to separate exosomes by size, the microfluidic method has great promise for clinical applications.⁴⁶⁸ Many researchers prefer polymers like polyethylene glycol (PEG)-based methods for exosome separation recently.⁴⁶⁹ Sedimentation of EV products after incubation in a PEG solution is observed near the tube base after low-speed centrifugation. Although this kind of coprecipitation technique provides a simple isolation process, its wider utility may be limited by its relatively poor throughput and the possibility of chemical contamination. Another technique known as immunoaffinity-based capture (IAC) takes advantage of the binding affinity between the receptors and ligands interactions. As a result, exosomes can be specifically isolated from bodily fluids, thereby facilitating the use of IAC in isolating tumor-specific exosomes for liquid biopsies and disease diagnostics.⁴⁷⁰ Studies have demonstrated that melanoma-specific exosomes may be successfully separated and isolated with an efficiency of about 95% using the IAC-based technique by recognizing melanoma-associated antigens.⁴⁷¹ Likewise, exosomes unique to AML were successfully extracted from cell culture supernatants using an anti-CD34 antibody-based IAC technique. Just 10 µL aliquots of the CD34 microbeads were able to capture all the exosomes present in 100–1000 µL of AML solution, demonstrating the remarkable effectiveness of microbeads used in this method.⁴⁷² Although there are numerous benefits to using IAC for exosome separation, such as its high efficiency and purity, the method incurs substantial expenses due to the utilization of antibodies, and magnetic beads. In general, it is imperative to take into account the specific needs of the application in order to determine the most appropriate method for isolating exosomes from complex biofluids. In this manner, it can be guaranteed that the selected method will satisfy the appropriate criteria such as purity, compatibility, efficacy, and yield for subsequent analyses.

Further validation of characterization is necessary for extracted and purified exosomes, and it offers a valuable means of confirming the efficacy of their extraction process and supplying a solid foundation for their potential uses. The prevailing techniques employed to quantify exosomes include measuring the total protein and particle amount. In addition, qualitative characterization necessitates the use of imaging techniques and biophysical features.⁴⁷³ The surface morphology information can be shown by electron microscopy. The particle concentration and size distribution can be found by nanoparticle tracking analysis and dynamic light scattering, which measures the exosome size and potential distribution.⁴⁷⁴ Exosomal proteins can be analyzed using several techniques such as enzyme-linked immunosorbent assays, chromatography, flow cytometry, protein blotting, and MS.⁴⁷⁵ Recently, there have been advancements in protein detection methods, including electrochemical detection, colorimetric detection, surface-enhanced Raman scattering, fluorescence detection, surface plasmon resonance detection, and CRISPR/Cas detection.³⁰³ Moreover, MS methods are also used for the examination of exosomal lipids. In order to establish the nature of exosomes as enclosed vesicles with a lipid bilayer structure, it is necessary to analyze and identify at least one protein that binds to the membrane or lipids, as well as one protein existed in the cytoplasm. For the analysis of exosomal RNA and DNA, techniques such as digital PCR, real-time fluorescence quantitative PCR, and NGS sequencing can be utilized.⁴⁷⁶ Similarly, novel techniques for detecting nucleic acids, including single vesicle analysis, CRISPR/Cas-assisted detection and thermophoretic detection have recently been developed.³⁰³ As a result, advancements in exosome detection technologies have made them more accessible for diagnostic purposes. It is worth mentioning that exosomes can alter their physical or biological characteristics. To summarize, a thorough analysis of exosomes allows for the identification of their characteristics from several perspectives, hence offering substantial evidence for their future use.

7.2 Safety considerations and regulatory challenges for EV-based therapies

Numerous technical obstacles that impede the development of EV-based therapies have appeared. As for the isolation methods, newer techniques like size exclusion chromatography and tangential flow filtration have the potential to handle higher amounts of EVs.⁴⁷⁷ It is also imperative to figure out EVs populations with inherent heterogeneity which could exhibit maximum functional activity by enhancing isolation procedures and developing the potency assays capability. Further complexity arises when production is highly needed for therapeutic applications including bioreactors and media supplements (e.g., fetal bovine serum containing exosomes). To take safety into consideration, using suitable preclinical models and careful cargo selection will be necessary to determine the safest cell source for exosome isolation in order to reduce immunogenicity and undesired cargo.⁴⁷

A fuller comprehension of the delivery pathways is necessary for attempts to direct EVs to certain areas for therapeutic intervention. In recent breakthroughs, targeting specificity has been demonstrated in EV surfaces with the use of RNA aptamers or nanobody fragments.⁴⁷⁸ EVs have demonstrated potential for increasing tropism toward specific cells or organs through engineering; however, the production of EVs with therapeutic potential remains a challenge.

As widely known, EV components, encompassing glycans, proteins, nucleic acids, or lipids, exhibit great promise in cancer treatment. However, there is a long way to go to translate these findings into therapeutic applications. As a result, it is crucial to standardize clinically appropriate EV preparations, especially when it comes to figuring out how EVs work, how much is a good dose, and how they work.

7.3 Challenges for harnessing EVs for precision medicine and targeted drug delivery

EVs have shown great promise recently as drug carriers for targeted tumor therapy, but their practical applicability is still limited by a number of issues. The safety of EVs should be taken into account when they are employed as delivery vehicles due to their biological activity. TDEVs contain constituents that stimulate tumor proliferation, invasion, and metastasis. As such, there are potential dangers associated with hastening tumor progression.³⁹⁴ As a result, additional clinical research on EV treatment is required, with an emphasis on the toxicological features and safety of clinical trials. The content and properties of EVs are influenced by both upstream and downstream alterations of the source cells, leading to a range of therapeutic outcomes. Consequently, minimizing batch variation while preserving stable EV structure and characteristics may be achieved by managing the stability of the culture environment and creating efficient EV processing procedures.⁴⁷⁹ A solution to this issue might be the creation of more effective techniques for EV extraction and purification or the investigation of novel EV sources. The mechanisms behind the selective cellular uptake of EVs and the patterns of their intracellular distribution remain poorly understood, potentially resulting in off-target occurrence. When executing membrane-disrupting manipulations, such as cargo delivery to EVs, there is a danger of changing the orientation of membrane proteins. This could lead to identification by the immune system and subsequent negative reactions. The process of isolating, loading drugs, and modifying delivery methods is converted into the release of drugs at the location of the lesion, thereby eliminating the potential risk of altering the features and characteristics of EVs efficiently.⁴⁸⁰

EVs are difficult and expensive to mass-produce using current isolation methods as mentioned above. Moreover, it is yet unknown what the requirements are for production, loading efficiency, purification, storage, dosage, and clinical application.⁴⁸¹ Despite promising potential, only a small number of EV-based treatments have made it into clinical trials due to the disparity between laboratory and clinical applications.⁴⁸² Thus, more attention should be paid to safety and toxicological profiles, more effective isolation methods and production, specific drug delivery, optimizing modification techniques, standardizing from production to application, and comprehensive mechanisms of intracellular distribution and selective cellular uptake in EV-based drug delivery research.