Skip to main content
Download PDF

The detection, biological function, and liquid biopsy application of extracellular vesicle-associated DNA

Biomarker Research volume 12, Article number: 123 (2024) Cite this article

Abstract

Cell-derived extracellular vesicles (EVs), which carry diverse biomolecules such as nucleic acids, proteins, metabolites, and lipids reflecting their cell of origin, are released under both physiological and pathological conditions. EVs have been demonstrated to mediate cell-to-cell communication and serve as biomarkers. EV-associated DNA (EV-DNA) comprises genomic and mitochondrial DNA (i.e., gDNA and mtDNA) fragments. Some studies have revealed that EV-DNA can represent the full nuclear genome and mitochondrial genome of parental cells. Furthermore, DNA fragments loaded into EVs are stable and can be transferred to recipient cells to regulate their biological functions. In this review, we summarized and discussed EV-DNA research advances with an emphasis on EV-DNA detection at the population-EV and single-EV levels, gene transfer-associated biological functions, and clinical applications as biomarkers for disease liquid biopsy. We hope that this review will provide potential directions or guidance for future EV-DNA investigations.

Background

EVs are particles that are released from cells and delimited by a lipid bilayer membrane [1, 2]. EVs can either pinch off the surface of the plasma membrane via outward budding (e.g., microvesicles with a diameter of 100–1000 nm or apoptotic bodies with a diameter of 1000–5000 nm) or can be generated inside multivesicular endosomes or multivesicular bodies (MVBs) via double invagination of the plasma membrane and then are released to the extracellular space through the exocytosis pathway (i.e., exosomes with a diameter of 50–150 nm) (Fig. 1) [3, 4]. These EVs enclose many constituents of parent cells, including nucleic acids, proteins, and metabolites, and display a wide range of sizes. EVs are implicated in cell-to-cell communication, allowing cells to exchange components and influencing various pathophysiological processes in both parent and recipient cells [5,6,7,8,9,10].

Fig. 1
figure 1

Schematic illustration of heterogeneous EVs carrying diverse DNA fragments. EVs can be released by plasma budding or form in the multivesicular body (MVB) followed by release through the exocytosis pathway

Full size image

EVs are present in various tissues (e.g., the brain, melanoma, adipose, and liver) [11,12,13,14,15,16] and body fluids (e.g., plasma, urine, breast milk, ascites, saliva, cerebrospinal fluid, and bile) [17,18,19,20,21]. The lipid membrane of EVs can protect their cargoes against degradation, particularly for nucleic acids [22,23,24,25]. Compared with EV-associated RNA (EV-RNA), DNA molecules were later found within EVs and have been less investigated. However, EV-DNA has attracted increasing interest, and corresponding advances are ongoing. Single-strand DNA (ssDNA), double-strand DNA (dsDNA) or chromatin DNA, and mtDNA, have been detected inside and/or outside EVs enriched from in vitro cultured cells and biofluids such as plasma [26,27,28,29,30,31,32], serum [33, 34], urine [35], gastric juice [36], saliva [37], pleural effusion [38], and lymphatic drainage [39]. In this review, we summarize and discuss advances in EV-DNA-associated detection, biological function, and liquid biopsy applications.

EVs are a highly heterogeneous population manifesting in their size, content (cargo), source (cell of origin), and functional impact on recipient cells [3]. There is a lack of defined nomenclature for EV populations. In addition to biogenesis-related terms such as exosome, microvesicle, and ectosome, operational terms, such as small EVs (sEVs) and large EVs, are commonly used to denote EV subtypes in published papers [1]. sEVs are usually obtained with a diameter of generally < 200 nm after separation via methods such as differential ultracentrifugation (dUC) or filtration [1, 2]. Compared with large EVs, sEVs are widely prepared as starting materials and are studied more in terms of EV-DNA characterization, functions, and applications. As such, this review pays more attention to DNA fragments derived from a mixed population of sEVs without further demonstration of their intracellular origin. In addition, as there is no strict consensus on upper and lower size cut-offs [1], the term EVs is used in the manuscript except that sEVs need to be highlighted.

EV-DNA detection

Typically, EV-DNA detection requires three steps: EV isolation, DNA extraction, and DNA characterization (Fig. 2). On the basis of physical characteristics (such as size and density) and biochemical properties (e.g., surface protein markers), a variety of approaches have been developed to isolate EVs; these approaches have been summarized in many reviews [40,41,42,43,44,45,46] and are not introduced here. As current studies have performed DNA analyses on EV populations and single EVs, we reviewed EV-DNA detection at the population-vesicle and single-vesicle levels.

Fig. 2
figure 2

Schematic workflows of EV-DNA detection at the population-EV and single-EV levels. In general, EVs are first isolated from culture media or body fluids, followed by DNA enzyme digestion, EV lysis, DNA extraction, and DNA analysis. When DNA analysis is performed via nanoflow cytometry (nFCM), EV lysis and DNA extraction are not needed, but DNA staining is needed. When DNA cargoes are profiled in a single EV via hydrogel-based droplet digital multiple displacement amplification (ddMDA), EV lysis is still needed, while DNA extraction is not needed

Full size image

EV-DNA detection at the population-vesicle level

Cells can release a large number of EVs in culture media or biofluids (e.g., approximately 109/mL in peripheral blood) [47, 48]. However, EVs are relatively small in size, and the amount of fragmented gDNA or mtDNA is limited [49]. Thus, EV isolation and DNA extraction methods are concerned. Currently, dUC is widely used to isolate or concentrate EVs from culture media or body fluids. For EV-DNA isolation, various commercial kits that are applied to extract DNA from cells, blood or tissues have also been used to isolate DNA from EVs, such as the QIAamp Micro Kit (Qiagen), the QIAamp DNA Mini Kit (Qiagen), the DNeasy Blood and Tissue Kit (Qiagen), the MagAttract HMW DNA Kit (Qiagen), and the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma‒Aldrich) [26, 27, 30,31,32,33,34,35]. These DNA isolation kits use a silica membrane or magnetic beads to selectively bind DNA and do not require toxic phenol‒chloroform extraction or ethanol precipitation. In addition, few EV-DNA isolation kits that integrate EV and DNA isolation have also been developed on the market; for example, the EV-DNA isolation kits of Duolaimi Biotechnology Co., Ltd. (Wuhan, China) & GeMExo Biotech Corp. (http://www.dlmbiotech.com) were used. Unfortunately, few comparative studies have been performed on existing EV and DNA isolation methods to explore the optimal procedure for EV-DNA isolation from culture media, body fluids, or tissues.

For DNA analysis, DNA enzymes, including DNase I, S1 nuclease, and dsDNase, are used to determine which forms of DNA fragments (e.g., ssDNA or dsDNA) are loaded into EVs [50,51,52,53,54]. To determine the location of DNA fragments inside or outside EVs, isolated EVs are treated with or without DNA enzymes before EV lysis. For quantitation, the EV-DNA concentration can be determined via ultraviolet absorbance-based Thermo Scientific™ NanoDrop™ Spectrophotometer (with a detection range of 2‒12,000 ng/µL) or a fluorescence-based fluorometer [28]. Comparatively, fluorometric quantitation with a DNA-binding fluorescent dye is more sensitive and specific for the nucleic acid of interest. However, before testing, samples for fluorometric quantitation need to be processed with kits such as the Invitrogen™ Qubit™ dsDNA HS Assay Kit (with a detection range of 0.1‒120 ng), the Invitrogen™ Quant-iT™ PicoGreen™ dsDNA Assay Kit (with a detection range of 50 pg‒2 µg), and the Promega™ QuantiFluor™ dsDNA System (with a detection range of 0.01‒200 ng, https://www.promega.com.cn). In addition, the length of EV-DNA fragments can be determined via agarose gel electrophoresis with a DNA ladder/marker, Agilent Bioanalyzer 2100 instrument, or Agilent 4200 TapeStation instrument. Sequencing results revealed that EV-DNA fragments isolated from EV populations span all chromosomes and mitochondrial DNA [51, 55].

Although DNA fragments have been detected from EVs, there is no consensus on EV-DNA parameters including location, form, concentration, and size. It seems that large EVs or EV surfaces carry complex DNA fragments with greater weights [52, 56, 57]. In addition, factors that can cause DNA damage and cellular damage or stress increase the amount of DNA fragments loaded into EVs from culture media or body fluids [58,59,60,61,62,63,64,65]. Takahashi et al. reported that abnormal accumulation of harmful DNA in the cytosol could activate the DNA damage response and trigger an aberrant immune response via the cGAS (cylic GMP-AMP synthase)-STING (a stimulator of interferon genes) signaling pathway [66]. Thus, EV-mediated DNA excretion from cells is presumed to be helpful for maintaining cellular homeostasis. Similarly, EV-mediated mtDNA release may be necessary to maintain subcellular mitochondrial homeostasis [67]. However, these assumptions need to be proven in the future.

Meanwhile, some studies have explored how nuclear DNA and mtDNA fragments are loaded into EVs [68, 69]. In 2019, Yokoi et al. preliminarily explored the mechanism by which nuclear contents are loaded into exosomes [68]. They reported that MVBs and the tetraspanin CD63 biomarker directly interact with the micronuclei (MN), suggesting that gDNA-containing exosomes are likely produced following MN collapse, where their nuclear contents are shuttled into MVBs via tetraspanins [68]. Recently, Zhang et al. reported that the transcription factor FOXM1 interacts with LC3 in the nucleus and transfers specific chromatin DNA fragments, including the DUX4 gene and telomere DNA, to EVs through secretory autography during the lysosome inhibition process [69]. This finding revealed for the first time how chromatin DNA fragments are specified to EVs. With respect to how mtDNA fragments are loaded into EVs, mitochondrial-derived vesicles (MDVs) seem to be involved in this process [70]. MDVs have been proposed as another method for controlling mitochondrial quality in addition to the mitochondrial–lysosome axis [71, 72]. Like MN-mediated DNA fragments packaged into MVBs [68], mtDNA fragments are likely routed to exosomes after MDVs interact with MVBs; however, this hypothesis needs to be proven. In addition, EVs are enriched in lipids such as cholesterol, phospholipids, and sphingolipids [73, 74]. Cytoplasmic DNA is taken up in intraluminal vesicles possibly via its interaction with the lipid raft-like region of the MVB membrane [75].

However, Jeppesen et al. argued that exosomes do not carry DNA or DNA-binding histones [76]. To obtain exosomes, they first used the dUC method to isolate crude sEVs, followed by purification with high-resolution iodixanol density gradient fractionation. Then, immunoaffinity beads targeting exosomal tetraspainins are used to specifically isolate exosomes from other types of sEVs, which fail to collect marker-negative sEV subpopulations [76]. Through the use of the same method for isolating sEVs from fresh human plasma, Lichá et al. reported that 60–75% of the DNA remained on the surface of sEVs and that a portion of the DNA was localized inside the sEVs [52]. In addition, Zhang et al. used asymmetric flow-flow fractionation to identify two sEV subpopulations with diameters of 90–120 nm and 60–80 nm as well as nonmembranous nanoparticles termed ‘exomeres’ (~ 35 nm), and reported that these particles have unique DNA profiles [77]. Altogether, DNA fragments can be detected from bulk EVs; however, the high intrinsic heterogeneity of EV populations may lead to various and even contentious results in terms of EV-DNA features. It is necessary to develop optimal enrichment methods to obtain more homogeneous EV subpopulations for accurate characterization of EV-DNA fragments.

EV-DNA analysis at the single-vesicle level

Currently, technologies for single EV analysis, such as nanoflow cytometry (nFCM), atomic force microscopy, droplet digital polymerase chain reaction (ddPCR), digital ELISA, and immunofluorescence imaging, have been developed to facilitate in-depth comprehension of various EV subtypes with differential physical properties, molecular compositions, or biological roles [41, 49, 78,79,80,81,82]. However, few studies have been carried out to analyze EV-DNA at the single-EV level thoroughly. On the basis of a laboratory-built nFCM, Liu et al. detected single EVs bearing DNA fragments labeled with the membrane-permeable nucleic acid stain SYTO™ 16 (Fig. 2) [83]. This laboratory-built nFCM method can analyze single EVs as small as 40 nm in diameter and single DNA fragments of 200 bp. In addition, in combination with enzymatic treatment, the results revealed that (1) naked DNA or DNA associated with nonvesicular entities was abundantly present in EV samples prepared from cell culture media by dUC; (2) the quantity of EV-DNA in individual EVs exhibited high heterogeneity, and the population of DNA-positive EVs varied from 30 to 80% depending on the cell type; (3) external EV-DNA was mainly localized on relatively small EVs (e.g., < 100 nm for the HCT-15 cell line), and the secretion of external DNA-positive EVs could be significantly reduced by exosome secretion pathway inhibition; (4) internal EV-DNA was mainly packaged inside the lumen of relatively large EVs (e.g., 80–200 nm for the HCT-15 cell line); (5) dsDNA was the predominant form of both the external and internal EV-DNA; (6) histones (H3) were not found in EVs, and EV-DNA was not associated with histone proteins; and (7) genotoxic drugs induced an increased release of DNA-positive EVs and the number of both external DNA-positive EVs and internal DNA-positive EVs as well as the DNA content.

Additionally, Jiao et al. developed a hydrogel-based droplet digital multiple displacement amplification (ddMDA) approach for the comprehensive analysis of EV-DNA at the single-EV level [84]. EV samples were prepared via dUC, and then, single EVs were dispersed in thousands of cross-linked poly(ethylene glycol) hydrogel droplets and lysed for DNA amplification and identification (Fig. 2). The results revealed that (1) 5 − 40% of EVs were associated with DNA, and significant differences existed not only between normal and tumor cells but also between tumor cells treated with anticancer drugs and untreated cells; (2) compared with EVs with a mean diameter of 109.7 ± 59.1 nm, EVs with a mean diameter of 170.4 ± 95.6 nm presented a greater proportion of DNA-containing EVs and a more substantial presence of intraluminal DNA; (3) these DNA-containing EVs carry multiple DNA fragments on average; and (4) both dsDNA and ssDNA were detected at the single-EV level. These two studies used EVs isolated from in vitro culture media as the starting material for EV-DNA analysis at the single-EV level. The features of EV-DNA fragments derived from body fluids or tissues remain to be examined at the single-EV level.

Taken together, current methods for EV-DNA detection at either the population-EV or single-EV level require isolation of EVs from culture media or body fluids in advance (Fig. 2). Hence, the EV isolation method is critical and determines which type of EV is used for subsequent DNA analysis. It is unknown whether the characteristics of isolated EV-DNA fragments are consistent with those of the original culture media and body fluids. The optimal EV isolation method for accurate characterization of EV-DNA should be explored.

Biological functions of EV-DNA

EVs released from donor cells can be taken up and convey their molecular cargoes, including DNA, to recipient cells via receptor‒ligand interactions, endocytosis and/or phagocytosis or even membrane fusion, thereby exerting effects on recipient cells. Accumulating evidence has revealed that EV-DNA can be transferred to recipient cells through horizontal transmission and even vertical transmission from parents to offspring. After uptake, on the one hand, EV-DNA can offer additional gene materials to recipient cells, leading to changes in gene transcription, protein translation, and/or phenotype (Fig. 3). On the other hand, EV-DNA serves as a signal molecule to activate cytoplasmic DNA-sensing pathways (e.g., cGAS-STING and the AIM2 inflammasome) and drive the immune or inflammatory response in recipient cells (Fig. 3).

Fig. 3
figure 3

Schematic overview of DNA-containing EVs acting on recipient cells. Heterogeneous-source DNA-containing EVs can be transferred to recipient cells and transform or affect their biological responses: (1) EV-DNA may translocate to the recipient cell nucleus and/or mitochondria and be integrated into the host genome, resulting in changes in the gene expression and/or phenotype of recipient cells; (2) EV-DNA may activate cytosolic DNA sensors of recipient cells, triggering the innate immune or inflammatory response

Full size image

EV-DNA serves as an additional gene material contributing to changes in gene expression and/or the phenotype of recipient cells

In 2012, Waldenström et al. reported for the first time that DNA-stained (with acridine orange) EVs derived from the culture media of cardiomyocytes were transferred to target fibroblasts and could be seen in the fibroblast cytosol and even in the nuclei [85]. However, it is not clear whether EV-DNA transfer into recipient cells is functional. Since then, increasing evidence indicates that EVs contain functional genes or chromosomal DNA fragments and telomeres and transfer them to recipient cells, resulting in changes in gene expression and/or phenotypes [86,87,88,89,90,91,92,93,94]. Cai et al. reported that an endogenous promoter of the AT1 (angiotensin II type 1) receptor, NF-kB, could be recruited to the transferred DNAs in the nucleus and increase the transcription of the AT1 receptor in recipient HEK293 cells [86]. In addition, unique BCR/ABL hybrid gene-containing EVs derived from human chronic myelogenous leukemia K562 cells were found to be transportable to HEK293 cells or neutrophils, resulting in the expression of the BCR/ABL hybrid gene mRNA and protein in the recipient cells [86]. K562 cell-derived EVs were injected into the tail vein of Sprague‒Dawley rats and immunodeficient NOD/SCID mice, and some characteristics of chronic myeloid leukemia, such as fever, thinning, splenomegaly, and neutrophilia, but reduced neutrophil phagocytic activity were observed [87]. Lanna et al. also reported that EV-mediated telomere transfer from antigen-presenting cells (APCs) to T cells (primarily naïve and central memory cells) during initial synaptic contact with APCs could elongate the telomeres of T cells and make them stemlike and/or central long-lived memory cells, conferring long-lasting immune protection [88].

Additionally, EVs bearing tumor-associated oncogene DNA or mutated gene DNA were found to be able to drive a protumorigenic phenotype in normal recipient cells [89,90,91,92]. EVs derived from patients with dedifferentiated liposarcoma (DDLPS) or DDLPS cell lines carry MDM2 oncogene DNA. These EVs can be transferred to preadipocytes, leading to impaired p53 activity in preadipocytes and increased proliferation, migration, and production of matrix metalloproteinase 2 [89]. Domenis et al. showed that mutated dsDNA (TP53 c.818G > A and KRAS c.35G > T) in EVs derived from the human colon cancer cell line SW480 could be actively transcribed in normal CCD841-CoN colon epithelial and THLE-2 hepatic cells, thereby transforming normal cells and modifying their phenotypes, such as proliferation and migration [90, 91].

With respect to cancer therapy, studies have shown that EV-mediated mtDNA transfer helps cancer cells acquire resistance [55, 93]. Hormonal therapy can induce oxidative phosphorylation-deficient breast cancer cells. However, the impaired metabolism in cancer cells could be rescued via the transfer of mtDNA-laden EVs derived from cancer-associated fibroblasts, promoting escape from metabolic quiescence and hormonal therapy-resistant metastatic breast cancer [55]. Additionally, EVs from chemoresistant triple-negative breast cancer cells can transfer mtDNA to sensitive cancer cells by increasing mtDNA levels with mutations in the mtND4 gene (which is responsible for tumorigenesis), thus leading to acquired chemoresistance [93].

More recently, Bolumar et al. discovered that maternal endometrial EVs could mediate vertical DNA transmission to preimplantation embryos and demonstrated that the internalization of EV-derived nuclear-encoded (n)DNA/mtDNA by trophoblast cells of murine embryos was associated with a reduction in mitochondrial respiration and ATP production [94]. This finding suggested that EV-mediated vertical transmission of maternal DNA was associated with altered embryo bioenergetics during the periconception period. Taken together, these results indicate that EVs bearing DNA fragments from donor cell nuclear DNA or cytoplasmic mtDNA can be internalized and incorporated into the genome of recipient cells, resulting in corresponding functional or phenotypic changes.

EV-DNA serves as a signal molecule triggering innate immunity in immune cells

The innate immune response is the first line of defense against infection by bacterial and fungal pathogens. Abnormal DNA in the cytosol of cells can be sensed by DNA sensors such as toll-like receptor 9, cGAS, and STING and absent in melanoma 2 (AIM2), which mediates type I interferon (IFN) production and inflammasome activation [95,96,97,98,99]. With respect to tumor treatment, Kitai et al. reported that cancer cells treated with the antitumor drug topotecan (TPT) secreted DNA-loaded EVs, which could activate dendritic cells (DCs) via a STING-dependent pathway and produce inflammatory cytokines [100]. In vivo, TPT administration inhibited tumor growth in tumor-bearing mice, which was accompanied by the infiltration of activated DCs and CD8+ T cells [100]. For radiotherapy, mouse breast carcinoma cells treated with 8 Gy X 3 released dsDNA-containing EVs [101]. Likewise, EV-DNA was shown to stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN [101]. In vivo, irradiated tumor cell-derived EVs were found to elicit tumor-specific CD8+ T-cell responses and significantly better protect mice from tumor development than EVs from untreated tumor cells in a prophylactic vaccination experiment [101]. Furthermore, Lv et al. reported that nonionizing ultraviolet radiation and ionizing radiation (X-ray and Boron neutron capture therapy) had different effects on EV-DNA fragments derived from tumor cells [102]. Boron neutron capture therapy induced more DNA fragments in tumor cell-derived EVs. These DNA-loaded EVs were also shown to activate the DNA-sensing pathway in DCs and enhance their functions, including antigen presentation and migration capacity [102]. After these EV-educated DCs are vaccinated, the effector T cells significantly expand and infiltrate into tumors [102]. These results suggest that EV-DNA derived from treated cancer cells can activate immune cells and elicit protective antitumor immunity.

In addition, EV-DNA was found to be involved in intestinal immune and inflammatory responses [103,104,105]. Lian et al. reported that chemotherapeutic irinotecan (CPT-11) can induce the packaging of a large amount of gDNA and mtDNA into the EVs of intestinal cells, which can activate the AIM2 inflammasome in innate immune cells (e.g., macrophages and DCs), promote the secretion of the mature cytokines IL-1β and IL-18, and cause intestinal toxicity [103]. In addition to chemotherapy-induced intestinal immune-dyfunctional response, EV-DNA has also been shown to participate in the development of inflammatory bowel disease (IBD), such as Crohn’s disease [104, 105]. A high content of EV-DNA, including mtDNA and nuclear DNA fragments, was detected in the plasma or colon lavage of murine colitis and Crohn’s disease patients and was positively correlated with disease activity [104]. Zhao et al. discovered that EVs from the plasma of active human Crohn’s disease and LPS-damaged colon epithelial cells could trigger STING activation and increase inflammation in macrophages, whereas the effect disappeared after removal of EV-DNA via sonication and dsDNase to digest dsDNA in or out of the EVs [104]. In IBD patients, gut microbiota-derived EV-DNA was also shown to induce barrier function damage and inflammatory responses in epithelial cells via the cGAS/STING pathway [105].

Additionally, EV-mediated DNA transfer has been shown to play an important role in pathogen infection progression [106,107,108]. EVs from malaria parasite (Plasmodium falciparum)-infected red blood cells contain parasite gDNA, which can be internalized by monocyte cells and stimulate STING-TBK1-IRF3-dependent gene induction [108]. Additionally, Torralba et al. reported that the interaction of T cells with antigen-bearing DCs could initiate the antipathogenic programs of DCs [109]. T-cell-derived EVs contain gDNA and mtDNA, which can stimulate the cGAS/STING pathway and induce the expression of IRF3-dependent interferon-regulated genes in DCs [109]. T-cell EV-treated DCs were more resistant to subsequent viral infections. These findings indicate that the interaction of T cells with DCs has physiological consequences for DC functions. T cells prime DCs through EV-mediated DNA transfer, suggesting a specific role for antigen-dependent contacts in conferring protection to DCs against pathogen infection.

Taken together, these findings indicate that EV-DNA transfer from donor cells to recipient cells has physiological and pathological significance. However, the detailed mechanisms underlying EV-DNA uptake are poorly understood. Recipient cell responses to EVs have been shown to rely on the donor cell source and dose [110]. The EV dose had a more significant effect than the cell source; however, EV cell source-specific responses were observed at low doses [110]. In addition, as EVs convey complex molecular cargoes to neighboring or distant cells, some new phenotypic and molecular responses in recipient cells may be attributed to other regulatory molecules in EVs or synergistic effects of all cargoes in EVs. It is strongly necessary to deplete the DNA cargo within EVs to determine EV-DNA-induced function.

Liquid biopsy application of EV-DNA

Liquid biopsy is defined as the sampling and analysis of components (e.g., circulating tumor cells, circulating cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating tumor RNA, and exosomes present in body fluids such as blood, urine, and saliva [111,112,113]. Compared with tissue biopsy, liquid biopsy is minimally invasive or even noninvasive, which facilitates its routine clinical use in patients for disease diagnosis and dynamic monitoring of disease progression and treatment response. For EVs, the membrane bilayer structure has a protective effect on the encapsulated cargoes against degradation. DNA in serum EVs was reported to be stable for 1 week at 4 °C, 1 day at room temperature, and after repeated freeze‒thaw cycles (less than three times) [24]. Clearly, DNA fragments inside EVs are more stable than cfDNA. cfDNA was reported to have a short half-life of 2–2.5 h [114]. Furthermore, EVs can reflect their cell of origin and are associated with the physiological and pathological status of the body. Given these advantages, EV-DNA has been considered an alternative resource for gene detection and screening EV-DNA-based markers for disease diagnosis and monitoring (Fig. 4) [115,116,117,118,119,120,121,122]. Many studies have explored the potential of EV-DNA-based liquid biopsy for the treatment of cancers such as lung cancer, pancreatic cancer, urinary cancer, and nervous system tumors [123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152].

Fig. 4
figure 4

Schematic illustration of EV-DNA-based gene analyses for liquid biopsy. EV-DNA isolated from body fluids can be used for various gene analyses (including mutation, copy number variation, epigenetic modification, etc.) with the help of diverse PCR and sequencing techniques

Full size image

Lung cancer

Current studies have shown that samples collected from lung cancer patients, including blood plasma, pleural effusion, and bronchoalveolar lavage fluid (BALF), contain EV-DNA with detectable epidermal growth factor receptor (EGFR) mutations (e.g., exon 19 deletion, p.L858R, p.T790M) (Table 1) [38, 123, 124, 126, 127]. In both plasma and BALF samples from non-small cell lung cancer (NSCLC) patients, EV-DNA-based analysis showed greater agreement with conventional tissue biopsy than did cfDNA-based liquid biopsy [123]. In particular, the test results of the BALF samples were significantly greater than the results of the plasma samples for both ctDNA and EV-DNA, indicating that proximal biofluids better represented the tumor status [123]. Furthermore, the use of EV-DNA from BALF samples was more effective than tissue biopsy for detecting the p.T790M mutation in patients who developed resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) [123]. In addition to mutation analysis, Batochir et al. identified specific methylation patterns in the EV-DNA of lung cancer BALF and reported that combinations of seven epigenetic biomarkers (including HOXA9, HOXD3, PCDH1, NID2, NPTX2, RASSF1A, and SFRP2) were capable of discriminating between lung cancer and benign lung diseases suspected of lung malignancy [125].

Table 1 EV-NA- and cfDNA-based gene analyses in lung cancer
Full size table

With respect to the specimen of pleural effusion, the results revealed that (1) EV-DNA- and cfDNA-based EGFR genotyping for patients who were either EGFR-TKI naïve without EGFR-TKI treatment or who acquired resistance to EGFR-TKI also showed good agreement with tissue biopsy; (2) the detection rate of p.T790M using EV-DNA was more efficient than that using cell blocks or cfDNA [126]. In 18 patients who acquired resistance to EGFR-TKIs with the p.T790M mutation, EGFR genotyping via EV-DNA, cfDNA, and cell blocks detected this mutation in 13, 11, and 3 patients, respectively [126]. Targeted next-generation sequencing (NGS) results revealed that the genetic profiles of EV-DNA and cfDNA isolated from pleural effusions of stage IV lung adenocarcinoma patients were comparable, except for copy number variations (CNVs), which presented lower similarities between these two samples [38].

In addition to comparisons between EV-DNA and cfDNA, EV-associated nucleic acids (EV-NAs), including DNA and RNA, were extracted from the blood plasma samples of NSCLC patients, and their clinical utility was explored. ddPCR-based EGFR mutation test results showed that short-length EV-NAs (~ 200 bp) contained more detectable tumor-derived nucleic acids than EV-DNA (~ 200 bp in length or full length) or cfDNA. Short-length EV-NAs and cfDNA generally showed good concordance with the tissue EGFR results. The sensitivity of liquid biopsy using EV-NAs was greater than that of liquid biopsy using cfDNA [127]. Overall, EV-DNA, especially proximal biofluid-derived EV-DNA, is promising as an alternative source for lung cancer liquid biopsy.

Pancreatic cancer

Human blood samples are often collected from pancreatic cancer patients for the detection of cancer-related mutations in EV-DNA [128,129,130,131,132,133] (Table 2). The concentration of EV-DNA in serum was reported to be greater than that in plasma; however, the mutant allele fraction (MAF) of KRAS in EV-DNA in serum was lower [129]. In addition, the levels of tumor-derived mutant KRAS DNA were highest in association with large EVs and sEVs early and with sEVs and soluble proteins late in disease progression, indicating that sEVs were the most enriched in tumor-derived DNA throughout disease progression [130].

Table 2 EV-DNA- and cfDNA-based gene analyses in pancreatic cancer
Full size table

ddPCR-based gene testing revealed that mutant KRAS EV-DNA was present in 7.4%, 66.7%, 80%, and 85% of age-matched healthy controls and localized, locally advanced, and metastatic pancreatic ductal adenocarcinoma patients, respectively [133]. Similarly, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals [133]. A greater percentage of pancreatic ductal adenocarcinoma patients presented detectable KRAS mutations in EV-DNA than in cfDNA. In 48 clinically annotated serum samples from pancreatic ductal adenocarcinoma patients, dPCR analyses of EV-DNA revealed the KRASG12D mutation in 39.6% of patients and the TP53R273H mutation in 4.2% of patients [128]. KRASG12D and TP53R273H mutations were detected in EV-DNA from intraductal papillary mucinous neoplasm patients and chronic pancreatitis patients. Notably, KRAS mutations were identified in EV-DNA from healthy controls, indicating the need for careful consideration and application of liquid biopsy findings [128].

In addition to EV-DNA detection for the diagnosis of pancreas-associated pathologies, the clinical utility of EV-DNA and cfDNA KRAS MAFs in patients with localized and metastatic pancreatic ductal adenocarcinoma was determined, and the results were compared [132]. Compared with standard readouts, such as imaging and carbohydrate antigen 19 − 9, the dynamics of KRAS mutation detection in circulating nucleic acids, including EV-DNA and cfDNA, could be correlated with disease progression. In 34 patients with potentially resectable tumors, an increase in EV-DNA after neoadjuvant therapy was significantly associated with disease progression, whereas ctDNA did not correlate with outcomes [132]. The concordance rates of KRAS mutations present in surgically resected tissue and detected in liquid biopsy samples were greater than 95%. These findings suggest that EV-DNA-based mutation analyses have great potential for pancreatic cancer diagnosis and monitoring.

Urinary cancer

Among urinary cancers, body fluids from urothelial bladder carcinoma (UBC), prostate cancer, and renal cell carcinoma have been reported to contain EV-DNA [35, 56, 57, 134, 135] (Table 3). For prostate cancer, large EVs carry most of the tumor DNA in patient blood plasma, whereas negligible amounts of DNA are presented in the sEVs from the same patients [56, 57]. Whole-genome sequencing (WGS) revealed that plasma EV-DNA from patients with metastatic prostate cancer could represent tumor genomic features and reflect genetic aberrations in the cell of origin [134]. With respect to UBC, cfDNA and EV-DNA extracted from urine samples from patients undergoing surgical treatment with somatic mutations and CNV similar to those of tumor tissues were measured [35]. Nonetheless, these findings are preliminary, and more tests are needed to explore the clinical utility of EV-DNA as an alternative biomarker for urinary cancer. In addition, as urine is a specific sample for urinary cancers and can be easily collected in a truly noninvasive manner, urine-derived EV-DNA gene analysis may be more effective for detecting urinary cancers.

Table 3 EV-DNA- and cfDNA-based gene analyses in urinary cancer
Full size table

Nervous system tumors

For nervous system tumors, glioma- and neuroblastoma-derived EV-DNA have been analyzed to identify tumor-associated genes, gene mutations, and gene modifications such as methylation [136,137,138,139,140]. In glioblastoma multiforme, the NANOG and SOX2 genes were detected in EV-DNA isolated from conditioned culture media [136, 137]. Genome-wide methylation profiling of glioblastoma cell-derived EVs revealed that EV-DNA could reflect genome-wide methylation profiles [138]. Rosas‑Alonso et al. detected of O6-methylguanine-DNA methyltransferase methylation in the plasma EV-DNA of glioblastoma patients with a remarkable sensitivity of 85.7%, and suggested EV-DNA-based liquid biopsy for monitoring disease progression in IDH-wild type glioblastoma patients [139]. Regarding neuroblastoma, EVs from blood plasma samples from patients were determined to contain dsDNA [140]. Whole exome sequencing results revealed that such EV-DNA carried tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF), and represented the entire exome [140]. Furthermore, neuroblastoma-derived EV-DNA was useful for identifying variants responsible for acquired resistance, such as mutations in the ALK, TP53, and RAS/MAPK genes that occur in relapsed patients [140].

In addition to the abovementioned tumors, biofluids from other cancers, such as lymphoma [28], pediatric acute myeloid leukemia [30], colorectal cancer [141,142,143], colon cancer [144], breast cancer [145, 146], gastric cancer [36], oropharyngeal squamous cell carcinoma [147], epithelial ovarian cancer [148], pheochromocytoma [149], paraganglioma [149], melanoma [150], hepatocellular carcinoma [151], and osteosarcoma [152], are sporadically reported having EV-DNA molecules.

More recently, some studies have shown that EV-DNA exists in samples of noncancerous diseases [153, 154]. The copy number of mtDNA in the blood EVs of patients with cardiovascular disease was found to be greater than that in healthy subjects [153]. Song et al. also detected mutations in the plasma EV-DNA of patients with pulmonary nodules and developed a compact 21-gene panel for the differential diagnosis of malignant PN and benign PN [154]. At the NCBI website (http://clinicaltrials.gov), 9 clinical trials have been registered for exploring the role of EV-DNA in cancer screening (NCT06192875), cancer diagnosis (NCT03236675, NCT05854563, NCT04164134, and NCT04742608), occurrence and development of gastric cancer (NCT05956847) and acute respiratory distress syndrome caused by extrapulmonary sepsis (NCT05061212), and treatment monitoring (NCT03228277 and NCT03217266) (Table 4). Among them, only two trials, NCT04164134 and NCT03228277, were completed, but no results were posted. Much more effort is still needed to validate EV-DNA-based biomarkers in independent cohorts or prospective trials and establish EV-DNA-based liquid biopsies for cancer or noncancerous diseases in the future.

Table 4 EV-DNA associated clinical trials registered at the NCBI website
Full size table

Conclusions and outlooks

In summary, attractive advances have been made in the field of EV-DNA research. Nuclear gDNA and/or mtDNA fragments have been discovered in EVs isolated from culture media and various biofluids. Furthermore, EV-DNA has been shown to play diverse roles in multiple physiological and pathological processes and potentially serves as an alternative gene material for disease liquid biopsy. However, technologies that enable the isolation of homogeneous EV subpopulations from either culture media or biofluids are lacking. dUC has been extensively used to isolate and distinguish sEVs from large EVs, but the obtained EVs are still a mixed population that cannot better reflect their biogenesis, cell or tissue origin. It is essential to develop novel approaches capable of separating EV subpopulations from each other. In addition, as there are no standard protocols for EV and DNA isolation, EV-DNA obtained via different approaches may present controversial characteristics. The optimal procedures for EV-DNA isolation should also be investigated to define EV-DNA features well.

In addition to the discovery and validation of DNA within EVs, how EV-DNA is formed and released from donor cells followed by uptake, internalization, and function in recipient cells remains to be further explored to better understand the molecular mechanisms behind EV-DNA functions. For tissue-derived EVs, studies focused on DNA are lacking and should be carried out in the future. With respect to liquid biopsy, DNA inside EVs seems to be more stable than cfDNA without a lipid bilayer coating. More studies should be carried out to investigate the translation potential of EV-DNA for disease diagnosis and monitoring. Notably, disease-specific samples such as BALF for NSCLC, urine for bladder cancer, and bile for hilar cholangiocarcinoma are likely more efficient for liquid biopsy. In addition to mutations, other cancer-associated gene aberrations and epigenetic modifications remain to be comprehensively profiled in EV-DNA from different body fluids. Taken together, continuous research is still needed to comprehensively characterize EV-DNA features, deeply parse EV-DNA functions, and better apply EV-DNA for disease liquid biopsy.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

EVs:

Extracellular vesicles

sEVs:

Small EVs

MVBs:

Multivesicular bodies

MN:

Micronuclei

MDVs:

Mitochondria-derived vesicles

EV-DNA:

EV-associated DNA

EV-RNA:

EV-associated RNA

EV-NAs:

EV-associated nucleic acids

ssDNA:

Single-strand DNA

dsDNA:

Double-strand DNA

gDNA:

Genomic DNA

mtDNA:

Mitochondrial DNA

cfDNA:

Cell-free DNA

ctDNA:

Circulating tumor DNA

cGAS:

cylic GMP‒AMP synthase

STING:

Stimulator of interferon genes

AIM2:

Absent in melanoma 2

IFN:

Type I interferon

TPT:

Topotecan

DCs:

Dendritic cells APCs

APCs:

antigen-presenting cells

AT1:

Angiotensin II type 1

EGFR:

Epidermal growth factor receptor

EGFR-TKIs:

Epidermal growth factor receptor-tyrosine kinase inhibitors

dUC:

Differential ultracentifugation

nFCM:

Nanoflow cytometry

ddMDA:

Droplet digital multiple displacement amplification

PCR:

Polymerase chain reaction

qPCR:

Quantitative PCR

dPCR:

Digital PCR

ddPCR:

Droplet digital PCR

NGS:

Next-generation sequencing

WGS:

Whole-genome sequencing

sWGS:

Shallow WGS

CNVs:

Copy number variations

MAF:

Mutant allele fraction

DDLPS:

Dedifferentiated liposarcoma

IBD:

Inflammatory bowel disease

NSCLC:

Non-small cell lung cancer

BALF:

Bronchoalveolar lavage fluid

UBC:

Urothelial bladder carcinoma

PN:

Pulmonary nodules

References

  1. Welsh JA, Goberdhan DCI, O’Driscoll L, Buzas EI, Blenkiron C, Bussolati B et al. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J Extracell Vesicles. 2024;13:e12404.

  2. Thery C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7:1535750.

    Article  PubMed  PubMed Central  Google Scholar 

  3. van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19:213–28.

    Article  PubMed  Google Scholar 

  4. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367:eaau977.

  5. Marar C, Starich B, Wirtz D. Extracellular vesicles in immunomodulation and tumor progression. Nat Immunol. 2021;22:560–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Maacha S, Bhat AA, Jimenez L, Raza A, Haris M, Uddin S et al. Extracellular vesicles-mediated intercellular communication: roles in the tumor microenvironment and anti-cancer drug resistance. Mol Cancer. 2019;18:55.

  7. Yin Y, Chen H, Wang Y, Zhang L, Wang X. Roles of extracellular vesicles in the aging microenvironment and age-related diseases. J Extracell Vesicles. 2021;10:e12154.

  8. Grange C, Bussolati B. Extracellular vesicles in kidney disease. Nat Rev Nephrol. 2022;18:499–513.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Yu H, Feng H, Zeng H, Wu Y, Zhang Q, Yu J, et al. Exosomes: the emerging mechanisms and potential clinical applications in dermatology. Int J Biol Sci. 2024;20:1778–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yang G, Waheed S, Wang C, Shekh M, Li Z, Wu J. Exosomes and their bioengineering strategies in the Cutaneous Wound Healing and related complications: current knowledge and future perspectives. Int J Biol Sci. 2023;19:1430–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Crescitelli R, Lässer C, Lötvall J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat Protoc. 2021;16:1548–80.

    Article  CAS  PubMed  Google Scholar 

  12. Huang Y, Cheng L, Turchinovich A, Mahairaki V, Troncoso JC, Pletnikova O, et al. Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles. J Extracell Vesicles. 2020;9:1785746.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Crescitelli R, Lasser C, Jang SC, Cvjetkovic A, Malmhall C, Karimi N, et al. Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. J Extracell Vesicles. 2020;9:1722433.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Crewe C, Joffin N, Rutkowski JM, Kim M, Zhang F, Towler DA, et al. An endothelial-to-adipocyte extracellular vesicle axis governed by metabolic state. Cell. 2018;175:695–e70813.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ishiguro K, Yan IK, Patel T. Isolation of tissue extracellular vesicles from the liver. J Vis Exp. 2019;150.

  16. Li SR, Man QW, Gao X, Lin H, Wang J, Su FC, et al. Tissue-derived extracellular vesicles in cancers and non‐cancer diseases: Present and future. J Extracell Vesicles. 2021;10:e12175.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Guo M, Wang J, Zhao Y, Feng Y, Han S, Dong Q, et al. Microglial exosomes facilitate alpha-synuclein transmission in Parkinson’s disease. Brain. 2020;143:1476–97.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Leung LL, Riaz MK, Qu X, Chan J, Meehan K. Profiling of extracellular vesicles in oral cancer, from transcriptomics to proteomics. Semin Cancer Biol. 2021;74:3–23.

    Article  CAS  PubMed  Google Scholar 

  19. Erdbrugger U, Blijdorp CJ, Bijnsdorp IV, Borras FE, Burger D, Bussolati B, et al. Urinary extracellular vesicles: a position paper by the Urine Task Force of the International Society for Extracellular Vesicles. J Extracell Vesicles. 2021;10:e12093.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Bortot B, Apollonio M, Rampazzo E, Valle F, Brucale M, Ridolfi A, et al. Small extracellular vesicles from malignant ascites of patients with advanced ovarian cancer provide insights into the dynamics of the extracellular matrix. Mol Oncol. 2021;15:3596–614.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zong H, Yu W, Lai H, Chen B, Zhang H, Zhao J, et al. Extracellular vesicles long RNA profiling identifies abundant mRNA, circRNA and lncRNA in human bile as potential biomarkers for cancer diagnosis. Carcinogenesis. 2023;44:671–81.

    Article  CAS  PubMed  Google Scholar 

  22. Schulz-Siegmund M, Aigner A. Nucleic acid delivery with extracellular vesicles. Adv Drug Deliver Rev. 2021;173:89–111.

    Article  CAS  Google Scholar 

  23. Ma C, Ding R, Hao K, Du W, Xu L, Gao Q, et al. Storage Stability of blood samples for miRNAs in glycosylated extracellular vesicles. Molecules. 2024;29:103.

    Article  CAS  Google Scholar 

  24. Jin Y, Chen K, Wang Z, Wang Y, Liu J, Lin L et al. DNA in serum extracellular vesicles is stable under different storage conditions. BMC Cancer. 2016;16:753.

  25. Ding T, Diao Y, Zeng X, Zhou L, Wu G, Liu J, et al. Influential factors on urine EV DNA methylation detection and its diagnostic potential in prostate cancer. Front Genet. 2024;15:1338468.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Fernando MR, Jiang C, Krzyzanowski GD, Ryan WL. New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes. PLoS ONE. 2017;12:e0183915.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Soltész B, Urbancsek R, Pös O, Hajas O, Forgács IN, Szilágyi E, et al. Quantification of peripheral whole blood, cell-free plasma and exosome encapsulated mitochondrial DNA copy numbers in patients with atrial fibrillation. J Biotechnol. 2019;299:66–71.

    Article  PubMed  Google Scholar 

  28. Barıs IC, Hacıoglu S, Turk NS, Cetın GO, Zencır S, Bagcı G, et al. Expression and DNA methylation profiles of EZH2-target genes in plasma exosomes and matched primary tumor tissues of the patients with diffuse large B-cell lymphoma. Clin Transl Oncol. 2021;23:1152–66.

    Article  PubMed  Google Scholar 

  29. Montermini L, Meehan B, Garnier D, Lee WJ, Lee TH, Guha A, et al. Inhibition of oncogenic epidermal growth factor receptor kinase triggers release of exosome-like extracellular vesicles and impacts their phosphoprotein and DNA content. J Biol Chem. 2015;290:24534–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bernardi S, Zanaglio C, Farina M, Polverelli N, Malagola M, Russo D. dsDNA from extracellular vesicles (EVs) in adult AML. Ann Hematol. 2021;100:1355–6.

    Article  PubMed  Google Scholar 

  31. Lazo S, Noren Hooten N, Green J, Eitan E, Mode NA, Liu QR et al. Mitochondrial DNA in extracellular vesicles declines with age. Aging Cell. 2021;20:e13283.

  32. Byappanahalli AM, Omoniyi V, Noren HN, Smith JT, Mode NA, Ezike N, et al. Extracellular vesicle mitochondrial DNA levels are associated with race and mitochondrial DNA haplogroup. iScience. 2024;27:108724.

    Article  CAS  PubMed  Google Scholar 

  33. Kahlert C, Melo SA, Protopopov A, Tang JB, Seth S, Koch M, et al. Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J Biol Chem. 2014;289:3869–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Tsilioni I, Theoharides TC. Extracellular vesicles are increased in the serum of children with autism spectrum disorder, contain mitochondrial DNA, and stimulate human microglia to secrete IL-1β. J Neuroinflamm. 2018;15:239.

  35. Lee DH, Yoon H, Park S, Kim JS, Ahn Y-H, Kwon K, et al. Urinary exosomal and cell-free DNA detects somatic mutation and copy number alteration in urothelial carcinoma of bladder. Sci Rep. 2018;8:14707.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Yamamoto H, Watanabe Y, Oikawa R, Morita R, Yoshida Y, Maehata T, et al. BARHL2 methylation using gastric wash DNA or gastric juice exosomal DNA is a useful marker for early detection of gastric cancer in an H. pylori-independent manner. Clin Transl Gastroen. 2016;7:e184.

    Article  Google Scholar 

  37. Han P, Bartold PM, Salomon C, Ivanovski S. Salivary outer membrane vesicles and DNA methylation of small extracellular vesicles as biomarkers for periodontal status: a pilot study. Int J Mol Sci. 2021;22:2423.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Song Z, Cai Z, Yan J, Shao YW, Zhang Y. Liquid biopsies using pleural effusion-derived exosomal DNA in advanced lung adenocarcinoma. Transl Lung Cancer Res. 2019;8:392–400.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. García-Silva S, Benito-Martín A, Sánchez-Redondo S, Hernández-Barranco A, Ximénez-Embún P, Nogués L, et al. Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAFV600E mutation. J Exp Med. 2019;216:1061–70.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Yan H, Li Y, Cheng S, Zeng Y. Advances in analytical technologies for extracellular vesicles. Anal Chem. 2021;93:4739–74.

    Article  CAS  PubMed  Google Scholar 

  41. Zhang J, Wu J, Wang G, He L, Zheng Z, Wu M, et al. Extracellular vesicles: techniques and biomedical applications related to single vesicle analysis. ACS Nano. 2023;17:17668–98.

    Article  CAS  PubMed  Google Scholar 

  42. Li P, Kaslan M, Lee SH, Yao J, Gao Z. Progress in exosome isolation techniques. Theranostics. 2017;7:789–804.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Lin S, Yu Z, Chen D, Wang Z, Miao J, Li Q, et al. Progress in microfluidics-based exosome separation and detection technologies for diagnostic applications. Small. 2020;16:e1903916.

    Article  PubMed  Google Scholar 

  44. Shen J, Ma Z, Xu J, Xue T, Lv X, Zhu G, et al. Exosome isolation and detection: from microfluidic chips to nanoplasmonic biosensor. ACS Appl Mater Interfaces. 2024;16:22776–93.

    CAS  Google Scholar 

  45. Kumar K, Kim E, Alhammadi M, Reddicherla U, Aliya S, Tiwari JN, et al. Recent advances in microfluidic approaches for the isolation and detection of exosomes. TrAC Trend Anal Chem. 2023;159:116912.

    Article  CAS  Google Scholar 

  46. Zhou Y, Liu H, Chen H. Advancement in exosome isolation and label-free detection towards clinical diagnosis. TrAC Trend Anal Chem. 2024;179:117874.

    Article  CAS  Google Scholar 

  47. Johnsen KB, Gudbergsson JM, Andresen TL, Simonsen JB. What is the blood concentration of extracellular vesicles? Implications for the use of extracellular vesicles as blood-borne biomarkers of cancer. Biochim Biophys Acta Rev Cancer. 2019;1871:109–16.

    Article  CAS  PubMed  Google Scholar 

  48. Witwer KW, Buzas EI, Bemis LT, Bora A, Lasser C, Lotvall J et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013; 2.

  49. Morales R-TT, Ko J. Future of Digital assays to resolve clinical heterogeneity of single extracellular vesicles. ACS Nano. 2022;16:11619–45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Balaj L, Lessard R, Dai L, Cho Y-J, Pomeroy SL, Breakefield XO, et al. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat Commun. 2011;2:180.

    Article  PubMed  Google Scholar 

  51. Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 2014;24:766–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Lichá K, Pastorek M, Repiská G, Celec P, Konečná B. Investigation of the presence of DNA in human blood plasma small extracellular vesicles. J Mol Sci. 2023;24:5915.

    Article  Google Scholar 

  53. Kalluri R, LeBleu VS. Discovery of double-stranded genomic DNA in circulating exosomes. Cold Spring Harbor Symp Quant Biol. 2017;81:275–80.

    Article  Google Scholar 

  54. Chang X, Fang L, Bai J, Wang Z. Characteristics and changes of DNA in extracellular vesicles. DNA Cell Biol. 2020;39:1486–93.

    Article  CAS  PubMed  Google Scholar 

  55. Sansone P, Savini C, Kurelac I, Chang Q, Amato LB, Strillacci A, et al. Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer. Proc Natl Acad Sci U S A. 2017;114:E9066–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Vagner T, Spinelli C, Minciacchi VR, Balaj L, Zandian M, Conley A, et al. Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma. J Extracell Vesicles. 2018;7:1505403.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Lázaro Ibáñez E, Sanz Garcia A, Visakorpi T, Escobedo Lucea C, Siljander P, Ayuso Sacido Á, et al. Different gDNA content in the subpopulations of prostate cancer extracellular vesicles: apoptotic bodies, microvesicles, and exosomes. Prostate. 2014;74:1379–90.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Domenis R, Cifù A, Curcio F. The influence of a Stressful Microenvironment on Tumor exosomes: a focus on the DNA Cargo. J Mol Sci. 2020;21:8728.

    Article  CAS  Google Scholar 

  59. Szatmári T, Balázs K, Csordás IB, Sáfrány G, Lumniczky K. Effect of radiotherapy on the DNA cargo and cellular uptake mechanisms of extracellular vesicles. Strahlenther Onkol. 2023;199:1191–213.

    Article  PubMed  Google Scholar 

  60. Németh A, Orgovan N, Sódar BW, Osteikoetxea X, Pálóczi K, Szabó-Taylor KÉ, et al. Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA. Sci Rep. 2017;7:8202.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Sadikot RT, Bedi B, Li J, Yeligar SM. Alcohol-induced mitochondrial DNA damage promotes injurious crosstalk between alveolar epithelial cells and alveolar macrophages. Alcohol. 2019;80:65–72.

    Article  CAS  PubMed  Google Scholar 

  62. Sheller-Miller S, Urrabaz-Garza R, Saade G, Menon R. Damage-associated molecular pattern markers HMGB1 and cell-free fetal telomere fragments in oxidative-stressed amnion epithelial cell-derived exosomes. J Reprod Immunol. 2017;123:3–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Ariyoshi K, Miura T, Kasai K, Fujishima Y, Nakata A, Yoshida M. Radiation-induced bystander effect is mediated by mitochondrial DNA in exosome-like vesicles. Sci Rep. 2019;9:9103.

    Article  PubMed  PubMed Central  Google Scholar 

  64. Gardner JJ, Cushen SC, Oliveira Da Silva RDN, Bradshaw JL, Hula N, Gorham IK, et al. Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells. Am J Physiol-Cell Ph. 2024;326:C1776–88.

    Article  CAS  Google Scholar 

  65. Carpenter MA, Thyagarajan A, Owens M, Annamraju R, Borchers CB, Travers JB, et al. The acid sphingomyelinase inhibitor imipramine enhances the release of UV photoproduct-containingDNAin small extracellular vesicles in UVB ‐irradiated human skin. Photochem Photobiol. 2024. https://doi.org/10.1111/php.13932.

    Article  PubMed  Google Scholar 

  66. Takahashi A, Okada R, Nagao K, Kawamata Y, Hanyu A, Yoshimoto S, et al. Exosomes maintain cellular homeostasis by excreting harmful DNA from cells. Nat Commun. 2017;8:15287.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Suárez H, Andreu Z, Mazzeo C, Toribio V, Pérez Rivera AE, López Martín S, et al. CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells. J Extracell Vesicles. 2021;10:e12082.

    Article  PubMed  PubMed Central  Google Scholar 

  68. Yokoi A, Villar-Prados A, Oliphint PA, Zhang J, Song X, De Hoff P, et al. Mechanisms of nuclear content loading to exosomes. Sci Adv. 2019;5:eaax8849.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Zhang Y, Ding N, Li Y, Ouyang M, Fu P, Peng Y, et al. Transcription factor FOXM1 specifies chromatin DNA to extracellular vesicles. Autophagy. 2024;20:1054–71.

    Article  CAS  PubMed  Google Scholar 

  70. Liu D, Dong Z, Wang J, Tao Y, Sun X, Yao X. The existence and function of mitochondrial component in extracellular vesicles. Mitochondrion. 2020;54:122–7.

    Article  CAS  PubMed  Google Scholar 

  71. Picca A, Guerra F, Calvani R, Bucci C, Lo Monaco M, Bentivoglio A, et al. Mitochondrial dysfunction and aging: insights from the analysis of extracellular vesicles. J Mol Sci. 2019;20:805.

    Article  CAS  Google Scholar 

  72. Gagliardi S, Mitruccio M, Di Corato R, Romano R, Aloisi A, Rinaldi R, et al. Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells. Cell Commun Signal. 2024;22:165.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Donoso Quezada J, Ayala Mar S, González Valdez J. The role of lipids in exosome biology and intercellular communication: function, analytics and applications. Traffic. 2021;22:204–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Skotland T, Hessvik NP, Sandvig K, Llorente A. Exosomal lipid composition and the role of ether lipids and phosphoinositides in exosome biology. J Lipid Res. 2019;60:9–18.

    Article  CAS  PubMed  Google Scholar 

  75. Janas T, Janas MM, Sapon K, Janas T. Mechanisms of RNA loading into exosomes. FEBS Lett. 2015;589:1391–8.

    Article  CAS  PubMed  Google Scholar 

  76. Jeppesen DK, Fenix AM, Franklin JL, Higginbotham JN, Zhang Q, Zimmerman LJ et al. Reassessment of exosome composition. Cell. 2019; 177: 428 – 45.e18.

  77. Zhang H, Freitas D, Kim HS, Fabijanic K, Li Z, Chen H, et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat Cell Biol. 2018;20:332–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Ferguson S, Yang KS, Weissleder R. Single extracellular vesicle analysis for early cancer detection. Trend Mol Med. 2022;28:681–92.

    Article  CAS  Google Scholar 

  79. Welsh JA, Arkesteijn G, Bremer M, Cimorelli M, Dignat-George F, Giebel B, et al. A compendium of single extracellular vesicle flow cytometry. J Extracell Vesicles. 2023;12:e12299.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Bordanaba-Florit G, Royo F, Kruglik SG, Falcon-Perez JM. Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles. Nat Protoc. 2021;16:3163–85.

    Article  CAS  PubMed  Google Scholar 

  81. Banijamali M, Hojer P, Nagy A, Haag P, Gomero EP, Stiller C, et al. Characterizing single extracellular vesicles by droplet barcode sequencing for protein analysis. J Extracell Vesicles. 2022;11:e12277.

    Article  PubMed  Google Scholar 

  82. Saftics A, Abuelreich S, Romano E, Ghaeli I, Jiang N, Spanos M, et al. Single Extracell VEsicle Nanoscopy J Extracell Vesicles. 2023;12:e12346.

    Article  PubMed  Google Scholar 

  83. Liu H, Tian Y, Xue C, Niu Q, Chen C, Yan X. Analysis of extracellular vesicle DNA at the single-vesicle level by nano‐flow cytometry. J Extracell Vesicles. 2022;11:e12206.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Jiao Y, Gao L, Zhang T, He Z, Zheng S-Y, Liu W. Profiling DNA cargos in single extracellular vesicles via hydrogel-based droplet digital multiple displacement amplification. Anal Chem. 2024;96:1293–300.

    Article  CAS  PubMed  Google Scholar 

  85. Waldenstrom A, Genneback N, Hellman U, Ronquist G. Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells. PLoS ONE. 2012;7:e34653.

    Article  PubMed  PubMed Central  Google Scholar 

  86. Cai J, Han Y, Ren H, Chen C, He D, Zhou L, et al. Extracellular vesicle-mediated transfer of donor genomic DNA to recipient cells is a novel mechanism for genetic influence between cells. J Mol Cell Biol. 2013;5:227–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Cai J, Wu G, Tan X, Han Y, Chen C, Li C, et al. Transferred BCR/ABL DNA from K562 extracellular vesicles causes chronic myeloid leukemia in immunodeficient mice. PLoS ONE. 2014;9:e105200.

    Article  PubMed  PubMed Central  Google Scholar 

  88. Lanna A, Vaz B, D’Ambra C, Valvo S, Vuotto C, Chiurchiu V, et al. An intercellular transfer of telomeres rescues T cells from senescence and promotes long-term immunological memory. Nat Cell Biol. 2022;24:1461–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Casadei L, Calore F, Braggio DA, Zewdu A, Deshmukh AA, Fadda P, et al. MDM2 derived from dedifferentiated liposarcoma extracellular vesicles induces MMP2 production from preadipocytes. Cancer Res. 2019;79:4911–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Domenis R, Cifu A, Fabris M, Niazi K, Soon-Shiong P, Curcio F. Tumor exosomes mediate the horizontal transfer of DNA gene mutation. FASEB J. 2020; 34.

  91. Domenis R, Cifù A, Mio C, Fabris M, Curcio F. Pro-inflammatory microenvironment modulates the transfer of mutated TP53 mediated by tumor exosomes. Int J Mol Sci. 2021;22:6258.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Valcz G, Újvári B, Buzás EI, Krenács T, Spisák S, Kittel Á, et al. Small extracellular vesicle DNA-mediated horizontal gene transfer as a driving force for tumor evolution: facts and riddles. Front Oncol. 2022;12:945376.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Abad E, Lyakhovich A. Movement of mitochondria with mutant DNA through extracellular vesicles helps cancer cells acquire chemoresistance. ChemMedChem. 2022;17:e202100642.

    Article  CAS  PubMed  Google Scholar 

  94. Bolumar D, Moncayo-Arlandi J, Gonzalez-Fernandez J, Ochando A, Moreno I, Monteagudo-Sanchez A, et al. Vertical transmission of maternal DNA through extracellular vesicles associates with altered embryo bioenergetics during the periconception period. eLife. 2023;12:RP88008.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Briard B, Place DE, Kanneganti T-D. DNA sensing in the innate immune response. Physiology. 2020;35:112–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Motwani M, Pesiridis S, Fitzgerald KA. DNA sensing by the cGAS-STING pathway in health and disease. Nat Rev Genet. 2019;20:657–74.

    Article  CAS  PubMed  Google Scholar 

  97. De Gaetano A, Solodka K, Zanini G, Selleri V, Mattioli AV, Nasi M, et al. Molecular mechanisms of mtDNA-mediated inflammation. Cells. 2021;10:2898.

    Article  PubMed  PubMed Central  Google Scholar 

  98. Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G, Caffrey DR, et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 2009;458:514–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Fernandes-Alnemri T, Yu J-W, Datta P, Wu J, Alnemri ES. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature. 2009;458:509–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Kitai Y, Kawasaki T, Sueyoshi T, Kobiyama K, Ishii KJ, Zou J, et al. DNA-containing exosomes derived from cancer cells treated with topotecan activate a STING-dependent pathway and reinforce antitumor immunity. J Immunol. 2017;198:1649–59.

    Article  CAS  PubMed  Google Scholar 

  101. Diamond JM, Vanpouille-Box C, Spada S, Rudqvist N-P, Chapman JR, Ueberheide BM, et al. Exosomes shuttle TREX1-sensitive IFN-stimulatory dsDNA from irradiated cancer cells to DCs. Cancer Immunol Res. 2018;6:910–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Lv L, Zhang J, Wang Y, Liang H, Liu Q, Hu F et al. Boron neutron capture therapy-derived extracellular vesicles via DNA accumulation boost antitumor dendritic cell vaccine efficacy. Adv Sci. 2024:2405158.

  103. Lian Q, Xu J, Yan S, Huang M, Ding H, Sun X, et al. Chemotherapy-induced intestinal inflammatory responses are mediated by exosome secretion of double-strand DNA via AIM2 inflammasome activation. Cell Res. 2017;27:784–800.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Zhao F, Zheng T, Gong W, Wu J, Xie H, Li W, et al. Extracellular vesicles package dsDNA to aggravate Crohn’s disease by activating the STING pathway. Cell Death Dis. 2021;12:815.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Nie S, Zhang Z, Ji Y, Ding Q, Gong J, Xiao F, et al. CRIg + macrophages deficiency enhanced inflammation damage in IBD due to gut extracellular vesicles containing microbial DNA. Gut Microbes. 2024;16:2379633.

    Article  PubMed  PubMed Central  Google Scholar 

  106. Bazié WW, Boucher J, Goyer B, Kania D, Traoré IT, Somé DY, et al. HIV replication increases the mitochondrial DNA content of plasma extracellular vesicles. Int J Mol Sci. 2023;24:1924.

    Article  PubMed  PubMed Central  Google Scholar 

  107. De Carolis S, Storci G, Ceccarelli C, Savini C, Gallucci L, Sansone P, et al. HPV DNA associates with breast cancer malignancy and it is transferred to breast cancer stromal cells by extracellular vesicles. Fron Oncol. 2019;9:860.

    Article  Google Scholar 

  108. Sisquella X, Ofir-Birin Y, Pimentel MA, Cheng L, Abou Karam P, Sampaio NG, et al. Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors. Nat Commun. 2017;8:1985.

    Article  PubMed  PubMed Central  Google Scholar 

  109. Torralba D, Baixauli F, Villarroya-Beltri C, Fernández-Delgado I, Latorre-Pellicer A, Acín-Pérez R, et al. Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts. Nat Commun. 2018;9:2658.

    Article  PubMed  PubMed Central  Google Scholar 

  110. Hagey DW, Ojansivu M, Bostancioglu BR, Saher O, Bost JP, Gustafsson MO, et al. The cellular response to extracellular vesicles is dependent on their cell source and dose. Sci Adv. 2023;9:eadh1168.

    Article  CAS  PubMed  Google Scholar 

  111. Heitzer E, Haque IS, Roberts C, Speicher MR. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet. 2019;20:71–88.

    Article  CAS  PubMed  Google Scholar 

  112. Lone SN, Nisar S, Masoodi T, Singh M, Rizwan A, Hashem S, et al. Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments. Mol Cancer. 2022;21:79.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Ignatiadis M, Sledge GW, Jeffrey SS. Liquid biopsy enters the clinic - implementation issues and future challenges. Nat Rev Clin Oncol. 2021;18:297–312.

    Article  PubMed  Google Scholar 

  114. Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet. 1999;64:218–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Ghanam J, Chetty VK, Barthel L, Reinhardt D, Hoyer P-F, Thakur BK. DNA in extracellular vesicles: from evolution to its current application in health and disease. Cell Biosci. 2022;12:37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Malkin EZ, Bratman SV. Bioactive DNA from extracellular vesicles and particles. Cell Death Dis. 2020;11:584.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Kahlert C. Liquid biopsy: is there an advantage to analyzing circulating exosomal DNA compared to cfDNA or are they the same? Cancer Res. 2019;79:2462–5.

    Article  CAS  PubMed  Google Scholar 

  118. Hur JY, Lee KY. Characteristics and clinical application of extracellular vesicle-derived DNA. Cancers. 2021;13:3827.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. García-Silva S, Gallardo M, Peinado H. DNA-loaded extracellular vesicles in liquid biopsy: tiny players with big potential? Front Cell Dev Biol. 2021;8:622579.

    Article  PubMed  PubMed Central  Google Scholar 

  120. Amintas S, Vendrely V, Dupin C, Buscail L, Laurent C, Bournet B, et al. Next-generation cancer biomarkers: extracellular vesicle DNA as a circulating surrogate of tumor DNA. Front Cell Dev Biol. 2021;8:622048.

    Article  PubMed  PubMed Central  Google Scholar 

  121. Sharma A, Johnson A, Exosome DNA. Critical regulator of tumor immunity and a diagnostic biomarker. J Cell Physiol. 2020;235:1921–32.

    Article  CAS  PubMed  Google Scholar 

  122. Tsering T, Li M, Chen Y, Nadeau A, Laskaris A, Abdouh M, et al. EV-ADD, a database for EV‐associated DNA in human liquid biopsy samples. J Extracell Vesicles. 2022;11:e12270.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Hur JY, Kim HJ, Lee JS, Choi C-M, Lee JC, Jung MK, et al. Extracellular vesicle-derived DNA for performing EGFR genotyping of NSCLC patients. Mol Cancer. 2018;17:15.

    Article  PubMed  PubMed Central  Google Scholar 

  124. Moldovan N, Verkuijlen S, van der Pol Y, Bosch L, van Weering JRT, Bahce I, et al. Comparison of cell-free and small extracellular-vesicle-associated DNA by sequencing plasma of lung cancer patients. iScience. 2024;27:110742.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Batochir C, Kim IA, Jo EJ, Kim E-B, Kim HJ, Hur JY, et al. Discrimination of lung cancer and benign lung diseases using BALF exosome DNA methylation profile. Cancers. 2024;16:2765.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Lee JS, Hur JY, Kim IA, Kim HJ, Choi CM, Lee JC, et al. Liquid biopsy using the supernatant of a pleural effusion for EGFR genotyping in pulmonary adenocarcinoma patients: a comparison between cell-free DNA and extracellular vesicle-derived DNA. BMC Cancer. 2018;18:1236.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Kim Y, Shin S, Kim B, Lee K-A. Selecting short length nucleic acids localized in exosomes improves plasma EGFR mutation detection in NSCLC patients. Cancer Cell Int. 2019;19:251.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Yang S, Che SP, Kurywchak P, Tavormina JL, Gansmo LB, Correa DSP, et al. Detection of mutant KRAS and TP53 DNA in circulating exosomes from healthy individuals and patients with pancreatic cancer. Cancer Biol Ther. 2017;18:158–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Wang Z-Y, Wang R-X, Ding X-Q, Zhang X, Pan X-R, Tong J-H. A protocol for cancer-related mutation detection on exosomal DNA in clinical application. Front Oncol. 2020;10:558106.

    Article  PubMed  PubMed Central  Google Scholar 

  130. Hagey DW, Kordes M, Görgens A, Mowoe MO, Nordin JZ, Moro CF, et al. Extracellular vesicles are the primary source of blood-borne tumour‐derived mutantKRAS DNA early in pancreatic cancer. J Extracell Vesicles. 2021;10:e12142.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, et al. Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer. PLoS ONE. 2023;18:e0291623.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Bernard V, Kim DU, San Lucas FA, Castillo J, Allenson K, Mulu FC et al. New York, NY. Circulating nucleic acids are associated with outcomes of patients with pancreatic cancer. Gastroenterology (1943). 2019; 156: 108 – 18.e4.

  133. Allenson K, Castillo J, San Lucas FA, Scelo G, Kim DU, Bernard V, et al. High prevalence of mutantKRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients. Ann Oncol. 2017;28:741–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Casanova-Salas I, Aguilar D, Cordoba-Terreros S, Agundez L, Brandariz J, Herranz N, et al. Circulating tumor extracellular vesicles to monitor metastatic prostate cancer genomics and transcriptomic evolution. Cancer Cell. 2024;42:1301–e127.

    Article  CAS  PubMed  Google Scholar 

  135. Arance E, Ramírez V, Rubio-Roldan A, Ocaña-Peinado FM, Romero-Cachinero C, Jódar-Reyes AB, et al. Determination of Exosome mitochondrial DNA as a biomarker of Renal Cancer aggressiveness. Cancers. 2022;14:199.

    Article  CAS  Google Scholar 

  136. Vaidya M, Bacchus M, Sugaya K. Differential sequences of exosomal NANOG DNA as a potential diagnostic cancer marker. PLoS ONE. 2018;13:e0197782.

    Article  PubMed  PubMed Central  Google Scholar 

  137. Vaidya M, Sugaya K. Differential sequences and single nucleotide polymorphism of exosomal SOX2 DNA in cancer. PLoS ONE. 2020;15:e0229309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Maire CL, Fuh MM, Kaulich K, Fita KD, Stevic I, Heiland DH, et al. Genome-wide methylation profiling of glioblastoma cell-derived extracellular vesicle DNA allows tumor classification. Neurooncology. 2021;23:1087–99.

    CAS  Google Scholar 

  139. Rosas-Alonso R, Colmenarejo-Fernandez J, Pernia O, Burdiel M, Rodriguez-Antolin C, Losantos-Garcia I, et al. Evaluation of the clinical use of MGMT methylation in extracellular vesicle-based liquid biopsy as a tool for glioblastoma patient management. Sci Rep. 2024;14:11398.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  140. Degli Esposti C, Iadarola B, Maestri S, Beltrami C, Lavezzari D, Morini M, et al. Exosomes from plasma of neuroblastoma patients contain double stranded DNA reflecting the mutational status of parental tumor cells. J Mol Sci. 2021;22:3667.

    Article  Google Scholar 

  141. Lucchetti D, Zurlo IV, Colella F, Ricciardi-Tenore C, Di Salvatore M, Tortora G et al. Mutational status of plasma exosomal KRAS predicts outcome in patients with metastatic colorectal cancer. Sci Rep. 2021;11:22686.

  142. Galbiati S, Damin F, Brambilla D, Ferraro L, Soriani N, Ferretti AM, et al. Small EVs-associated DNA as complementary biomarker to circulating tumor DNA in plasma of metastatic colorectal cancer patients. Pharmaceuticals. 2021;14:128.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Li X, Wang Q, Wang R. Roles of exosome genomic DNA in colorectal cancer. Front Pharmacol. 2022;13:923232.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Choi J, Cho HY, Jeon J, Kim KA, Han YD, Ahn JB, et al. Detection of circulating KRAS mutant DNA in extracellular vesicles using droplet digital PCR in patients with colon cancer. Front Oncol. 2022;12:1067210.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Nakai M, Yamada T, Sekiya K, Sato A, Hankyo M, Kuriyama S, et al. Use of liquid biopsy to detect PIK3CA mutation in metastatic breast cancer. J Nippon Med Sch. 2022;89:66–71.

    Article  CAS  PubMed  Google Scholar 

  146. Andreeva OE, Shchegolev YY, Scherbakov AM, Mikhaevich EI, Sorokin DV, Gudkova MV, et al. Secretion of mutant DNA and mRNA by the exosomes of breast cancer cells. Molecules. 2021;26:2499.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Nguyen B, Meehan K, Pereira MR, Mirzai B, Lim SH, Leslie C, et al. A comparative study of extracellular vesicle-associated and cell-free DNA and RNA for HPV detection in oropharyngeal squamous cell carcinoma. Sci Rep. 2020;10:6083.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. Keserű JS, Soltész B, Lukács J, Márton É, Szilágyi-Bónizs M, Penyige A, et al. Detection of cell-free, exosomal and whole blood mitochondrial DNA copy number in plasma or whole blood of patients with serous epithelial ovarian cancer. J Biotechnol. 2019;298:76–81.

    Article  PubMed  Google Scholar 

  149. Wang L, Li Y, Guan X, Zhao J, Shen L, Liu J. Exosomal double-stranded DNA as a biomarker for the diagnosis and preoperative assessment of pheochromocytoma and paraganglioma. Mol Cancer. 2018;17:128.

    Article  PubMed  PubMed Central  Google Scholar 

  150. Zocco D, Bernardi S, Novelli M, Astrua C, Fava P, Zarovni N, et al. Isolation of extracellular vesicles improves the detection of mutant DNA from plasma of metastatic melanoma patients. Sci Rep. 2020;10:15745.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Li Y, Guo X, Guo S, Wang Y, Chen L, Liu Y, et al. Next generation sequencing-based analysis of mitochondrial DNA characteristics in plasma extracellular vesicles of patients with hepatocellular carcinoma. Oncol Lett. 2020;20:2820–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Cambier L, Stachelek K, Triska M, Jubran R, Huang M, Li W, et al. Extracellular vesicle-associated repetitive element DNAs as candidate osteosarcoma biomarkers. Sci Rep. 2021;11:94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Rucci C, de Simone G, Salathia S, Casadidio C, Censi R, Bordoni L. Exploring mitochondrialDNA copy number in circulatingcell-free DNA and extracellular vesicles across cardiovascular health status: a prospectivecase–control pilot study. FASEB J. 2024;38:e23672.

    Article  CAS  PubMed  Google Scholar 

  154. Song C, Sun Y, Chen Y, Shen Y, Lei H, Mao W, et al. Differential diagnosis of pulmonary nodules and prediction of invasive adenocarcinoma using extracellular vesicle DNA. Clin Translational Med. 2024;14:e1582.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This work was supported by grants from the National Natural Science Foundation of China (21904101), the Research Fund of Zhongnan Hospital of Wuhan University (SWYBK01-03, ZLYNXM20200213) and the Fundamental Research Funds for the Central Universities (2042022dx0003).

Author information

Authors and Affiliations

  1. Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Zhongnan Hospital of Wuhan University, Wuhan, China

    Shan Guo, Danni Shan, Yu Xiao & Kaiyu Qian

  2. Center for Disease Control and Prevention of Hubei Province, Wuhan, China

    Xin Wang

  3. Department of Urology, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China

    Yu Xiao, Lingao Ju & Gang Wang

  4. Human Genetic Resources Preservation Center, Wuhan University, Wuhan, China

    Lingao Ju

  5. Euler Technology, ZGC Life Sciences Park, Beijing, China

    Yi Zhang

  6. Center for Quantitative Biology, School of Life Sciences, Peking University, Beijing, China

    Yi Zhang

  7. Wuhan Research Center for Infectious Diseases and Cancer, Chinese Academy of Medical Sciences, Wuhan, China

    Kaiyu Qian

Authors
  1. Shan Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Danni Shan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yu Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lingao Ju
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yi Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kaiyu Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.G, X.W and D.S drafted this review and designed the tables and figures; L.J and G.W provided some valuable suggestions; Y.X, Y.Z and K.Q provided guidance and revised this manuscript. All the authors read and approved the final manuscript.

Corresponding authors

Correspondence to Shan Guo or Kaiyu Qian.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Guo, S., Wang, X., Shan, D. et al. The detection, biological function, and liquid biopsy application of extracellular vesicle-associated DNA. Biomark Res 12, 123 (2024). https://doi.org/10.1186/s40364-024-00661-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40364-024-00661-2

Keywords

Biomarker Research

ISSN: 2050-7771

Contact us