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

This review summarizes and discusses advances in EV-associated DNA (EV-DNA) research, 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.
Published in Chemistry and Biomedical Research
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Extracellular vesicles (EVs) are particles that are released from cells and delimited by a lipid bilayer membrane [1]. 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 into the extracellular space through the exocytosis pathway (i.e., exosomes with a diameter of 50–150 nm). 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 [2-4].

EVs are present in various tissues (e.g., the brain, melanoma, adipose, and liver) and body fluids (e.g., plasma, urine, breast milk, ascites, saliva, cerebrospinal fluid, and bile). The lipid membrane of EVs can protect their cargoes against degradation, particularly for nucleic acids. Compared with EV-associated 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, double-strand DNA or chromatin DNA, and mtDNA have been detected inside and/or outside EVs enriched from in vitro cultured cells and biofluids such as plasma, serum, urine, gastric juice, saliva, pleural effusion, and lymphatic drainage [5-8]. In this review, we summarize and discuss EV-DNA research advances: (1) EV-DNA detection at the population-EV and single-EV levels; (2) EV-DNA-associated biological functions, including serving as an additional gene material contributing to changes in gene expression and/or the phenotype of recipient cells and as a signal molecule activating cytoplasmic DNA-sensing pathways (e.g., cGAS-STING and the AIM2 inflammasome) in recipient cells; (3) EV-DNA-based liquid biopsy application in cancer and noncancerous diseases. This review provides potential directions or guidance for future EV-DNA investigations.

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.

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.

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.

Dr. Kaiyu Qian and Dr. Shan Guo are the corresponding authors of this paper.

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.
2.    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.
3.    Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020; 367.
4.    Marar C, Starich B, Wirtz D. Extracellular vesicles in immunomodulation and tumor progression. Nat Immunol. 2021; 22: 560-70.
5.    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.
6. 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-E9075.
7.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.
8.    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.

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