Circulating extracellular vesicle DNA (EV-DNA) and cell-free DNA in ovarian cancer: pathological relevance and diagnostic applications

Abstract

Ovarian cancer is one of the leading causes of gynecologic cancer-related mortality in women. However, a significant proportion of ovarian cancer cases are only detected at an advanced stage (III or IV) and are complicated to treat because of metastasis to the peritoneum. This challenge is compounded by vague symptoms and insufficient screening methods for early ovarian cancer detection. A promising solution is liquid biopsy, where the presence of biomarkers (proteins, lipids, and nucleic acids) associated with cancer is identified in the blood circulation. This approach facilitates the real-time monitoring of cancer progression and treatment effects in a non-invasive manner. This contrasts with traditional tumor biopsy, where only a small portion of the tumor is sampled, serial sampling of the tumor is impractical, or sometimes, tumor biopsy is not feasible. This review discusses the cell-free and extracellular vesicle components in blood, highlighting their DNA as a target in liquid biopsies for cancer diagnostics, with a specific emphasis on ovarian cancer. It also underscores the need for further research into the biological underpinnings and functional roles of these DNA fragments to integrate them into multi-omics approaches for detailed insights into tumor biology and treatment resistance in ovarian cancer.

Keywords

Ovarian cancer / extracellular vesicle-DNA / cell-free DNA / liquid biopsy / nanotechnologies / cancer diagnostics / multi-omics / treatment resistance

References

[1] Jayson GC,Kitchener HC.Ovarian cancer.Lancet2014;384:1376-88

[2] Bray F,Soerjomataram I,Torre LA.Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin2018;68:394-424

[3] Cancer Australia. Ovarian cancer statistics in Australia. Available from: https://www.canceraustralia.gov.au/cancer-types/ovarian-cancer/ovarian-cancer-statistics-australia. [Last accessed on 5 Jan 2025]

[4] Siegel RL,Jemal A.Cancer statistics, 2020.CA Cancer J Clin2020;70:7-30

[5] Mazidimoradi A,Allahqoli L.The global, regional and national epidemiology, incidence, mortality, and burden of ovarian cancer.Health Sci Rep2022;5:e936 PMCID:PMC9682200

[6] Mandel P.[Nuclear acids in human blood plasma].C R Seances Soc Biol Fil1948;142:241-3

[7] Ivanov M,Butler T,Mileyko V.Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation.BMC Genomics2015;16 Suppl 13:S1 PMCID:PMC4686799

[8] Dorayappan KDP,Cohn DE.The biological significance and clinical applications of exosomes in ovarian cancer.Gynecol Oncol2016;142:199-205 PMCID:PMC4917458

[9] Boukouris S.Exosomes in bodily fluids are a highly stable resource of disease biomarkers.Proteomics Clin Appl2015;9:358-67 PMCID:PMC5502131

[10] Kahlert C,Protopopov A.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 Chem2014;289:3869-75 PMCID:PMC3924256

[11] Chetty VK,Anchan S.Efficient small extracellular vesicles (EV) isolation method and evaluation of EV-associated DNA role in cell-cell communication in cancer.Cancers2022;14:2068 PMCID:PMC9099953

[12] Katsman E,Martignano F.Detecting cell-of-origin and cancer-specific methylation features of cell-free DNA from Nanopore sequencing.Genome Biol2022;23:158 PMCID:PMC9283844

[13] Hirahata T,Quraish AU,Naz M.Liquid biopsy: a distinctive approach to the diagnosis and prognosis of cancer.Cancer Inform2022;21:11769351221076062 PMCID:PMC8832574

[14] Sebire E,Bhattacharya S,Wood R.The implementation and impact of non-invasive prenatal testing (NIPT) for Down’s syndrome into antenatal screening programmes: a systematic review and meta-analysis.PLoS One2024;19:e0298643 PMCID:PMC11098470

[15] Romero R.A profile of Dennis Lo, DM, DPhil, FRCP, FRCPath, FRS.Am J Obstet Gynecol2018;218:371-8 PMCID:PMC5987209

[16] Lo YM,Chamberlain PF.Presence of fetal DNA in maternal plasma and serum.Lancet1997;350:485-7

[17] Jamshidi A,Klein EA.Evaluation of cell-free DNA approaches for multi-cancer early detection.Cancer Cell2022;40:1537-49.e12

[18] Basharat S. Emerging multi-cancer early detection technologies: CADTH horizon scan. Ottawa (ON); 2022. Available from: https://www.ncbi.nlm.nih.gov/books/NBK598992/. [Last accessed on 5 Jan 2025]

[19] Tsering T,Wu T,Burnier JV.Extracellular vesicle-associated DNA: ten years since its discovery in human blood.Cell Death Dis2024;15:668 PMCID:PMC11393322

[20] Song P,Yan YH.Limitations and opportunities of technologies for the analysis of cell-free DNA in cancer diagnostics.Nat Biomed Eng2022;6:232-45 PMCID:PMC9336539

[21] Sakamoto Y,Suzuki A.A new era of long-read sequencing for cancer genomics.J Hum Genet2020;65:3-10 PMCID:PMC6892365

[22] Zhang Y,Li Y.Advanced extracellular vesicle bioinformatic nanomaterials: from enrichment, decoding to clinical diagnostics.J Nanobiotechnology2023;21:366 PMCID:PMC10557264

[23] Jia Y,Ma T.Small extracellular vesicles isolation and separation: current techniques, pending questions and clinical applications.Theranostics2022;12:6548-75 PMCID:PMC9516236

[24] Balaj L,Dai L.Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences.Nat Commun2011;2:180 PMCID:PMC3040683

[25] Lázaro-Ibáñez E,Visakorpi T.Different gDNA content in the subpopulations of prostate cancer extracellular vesicles: apoptotic bodies, microvesicles, and exosomes.Prostate2014;74:1379-90 PMCID:PMC4312964

[26] Thakur BK,Becker A.Double-stranded DNA in exosomes: a novel biomarker in cancer detection.Cell Res2014;24:766-9 PMCID:PMC4042169

[27] Lázaro-Ibáñez E,Shelke GV.DNA analysis of low- and high-density fractions defines heterogeneous subpopulations of small extracellular vesicles based on their DNA cargo and topology.J Extracell Vesicles2019;8:1656993 PMCID:PMC6719264

[28] Baysa A,Kondratov K.Release of mitochondrial and nuclear DNA during on-pump heart surgery: kinetics and relation to extracellular vesicles.J Cardiovasc Transl Res2019;12:184-92

[29] Vagner T,Minciacchi VR.Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma.J Extracell Vesicles2018;7:1505403 PMCID:PMC6084494

[30] Qian Y,Yu Y.Characterization and functional analysis of extrachromosomal circular DNA discovered from circulating extracellular vesicles in liver failure.Clin Transl Med2024;14:e70059 PMCID:PMC11479749

[31] Guescini M,Stocchi V.Astrocytes and Glioblastoma cells release exosomes carrying mtDNA.J Neural Transm2010;117:1-4

[32] Tsilioni I.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 Neuroinflammation2018;15:239 PMCID:PMC6112123

[33] Takenaga K,Nagase H.Intercellular transfer of mitochondrial DNA carrying metastasis-enhancing pathogenic mutations from high- to low-metastatic tumor cells and stromal cells via extracellular vesicles.BMC Mol Cell Biol2021;22:52 PMCID:PMC8496074

[34] Jin Y,Wang Z.DNA in serum extracellular vesicles is stable under different storage conditions.BMC Cancer2016;16:753 PMCID:PMC5035490

[35] Yokoi A,Oliphint PA.Mechanisms of nuclear content loading to exosomes.Sci Adv2019;5:eaax8849 PMCID:PMC6867874

[36] Liu H,Xue C,Chen C.Analysis of extracellular vesicle DNA at the single-vesicle level by nano-flow cytometry.J Extracell Vesicles2022;11:e12206 PMCID:PMC8977970

[37] Takahashi A,Nagao K.Exosomes maintain cellular homeostasis by excreting harmful DNA from cells.Nat Commun2017;8:15287 PMCID:PMC5440838

[38] Reis-Sobreiro M,Novitskaya T.Emerin deregulation links nuclear shape instability to metastatic potential.Cancer Res2018;78:6086-97

[39] Lian Q,Yan S.Chemotherapy-induced intestinal inflammatory responses are mediated by exosome secretion of double-strand DNA via AIM2 inflammasome activation.Cell Res2017;27:784-800 PMCID:PMC5518874

[40] Jeppesen DK,Franklin JL.Reassessment of exosome composition.Cell2019;177:428-45.e18 PMCID:PMC6664447

[41] Seo W,He Y.ALDH2 deficiency promotes alcohol-associated liver cancer by activating oncogenic pathways via oxidized DNA-enriched extracellular vesicles.J Hepatol2019;71:1000-11 PMCID:PMC6801025

[42] Elzanowska J,García-Peláez B.Defining optimal conditions for tumor extracellular vesicle DNA extraction for mutation profiling.Cancers2022;14:3258 PMCID:PMC9265681

[43] Ding T,Zeng X.Influential factors on urine EV DNA methylation detection and its diagnostic potential in prostate cancer.Front Genet2024;15:1338468 PMCID:PMC10909848

[44] Nguyen B,Pereira MR.A comparative study of extracellular vesicle-associated and cell-free DNA and RNA for HPV detection in oropharyngeal squamous cell carcinoma.Sci Rep2020;10:6083 PMCID:PMC7142128

[45] Nguyen B,Semple T.Low-coverage whole-genome sequencing of extracellular vesicle-associated DNA in patients with metastatic cancer.Sci Rep2021;11:4016 PMCID:PMC7889887

[46] Li Y,Guo S.Next generation sequencing-based analysis of mitochondrial DNA characteristics in plasma extracellular vesicles of patients with hepatocellular carcinoma.Oncol Lett2020;20:2820-8 PMCID:PMC7400774

[47] Ruhen O,Clark ME.Comparison of circulating tumour DNA and extracellular vesicle DNA by low-pass whole-genome sequencing reveals molecular drivers of disease in a breast cancer patient.Biomedicines2020;9:14 PMCID:PMC7823926

[48] Bernard V,San Lucas FA.Circulating nucleic acids are associated with outcomes of patients with pancreatic cancer.Gastroenterology2019;156:108-18.e4 PMCID:PMC6434712

[49] Maire CL,Kaulich K.Genome-wide methylation profiling of glioblastoma cell-derived extracellular vesicle DNA allows tumor classification.Neuro Oncol2021;23:1087-99 PMCID:PMC8673443

[50] Németh A,Sódar BW.Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA.Sci Rep2017;7:8202 PMCID:PMC5557920

[51] Galbiati S,Brambilla D.Small EVs-associated DNA as complementary biomarker to circulating tumor DNA in plasma of metastatic colorectal cancer patients.Pharmaceuticals2021;14:128 PMCID:PMC7915475

[52] Hagey DW,Görgens A.Extracellular vesicles are the primary source of blood-borne tumour-derived mutant KRAS DNA early in pancreatic cancer.J Extracell Vesicles2021;10:e12142 PMCID:PMC8485184

[53] Zavridou M,Bournakis E,Tserpeli V.Prognostic significance of gene expression and DNA methylation markers in circulating tumor cells and paired plasma derived exosomes in metastatic castration resistant prostate cancer.Cancers2021;13:780 PMCID:PMC7918693

[54] Lee SE,Hur JY.Genomic profiling of extracellular vesicle-derived DNA from bronchoalveolar lavage fluid of patients with lung adenocarcinoma.Transl Lung Cancer Res2021;10:104-16 PMCID:PMC7867756

[55] Thakur K,Feldstein-Davydova S,Hershkovitz D.Extracellular vesicle-derived DNA vs. CfDNA as a biomarker for the detection of colon cancer.Genes2021;12:1171 PMCID:PMC8393490

[56] Demuth C,Johansen JS.Measuring KRAS mutations in circulating tumor DNA by droplet digital PCR and next-generation sequencing.Transl Oncol2018;11:1220-4 PMCID:PMC6085225

[57] Wang Y,Bollas A,Au KF.Nanopore sequencing technology, bioinformatics and applications.Nat Biotechnol2021;39:1348-65 PMCID:PMC8988251

[58] Guanzon D,Ma C,Liew YJ.Comparing methylation levels assayed in GC-rich regions with current and emerging methods.BMC Genomics2024;25:741 PMCID:PMC11289974

[59] Lau BT,Schauer M.Single-molecule methylation profiles of cell-free DNA in cancer with nanopore sequencing.Genome Med2023;15:33 PMCID:PMC10155347

[60] van der Pol Y,Evander N.Real-time analysis of the cancer genome and fragmentome from plasma and urine cell-free DNA using nanopore sequencing.EMBO Mol Med2023;15:e17282 PMCID:PMC10701599

[61] Zhao L,Ma S.Molecular profiles of serum-derived extracellular vesicles in high-grade serous ovarian cancer.Cancers2022;14:3589 PMCID:PMC9330879

[62] Werner B, Yuwono N, Duggan J, et al.; INOVATe Investigators. Cell-free DNA is abundant in ascites and represents a liquid biopsy of ovarian cancer.Gynecol Oncol2021;162:720-7

[63] Keserű JS,Lukács J.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 Biotechnol2019;298:76-81

[64] Gagliardi S,Di Corato R.Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells.Cell Commun Signal2024;22:165 PMCID:PMC10916030

[65] Zhao H,Yan G.Inflammation and tumor progression: signaling pathways and targeted intervention.Signal Transduct Target Ther2021;6:263 PMCID:PMC8273155

[66] Min L,Bao H.Advanced nanotechnologies for extracellular vesicle-based liquid biopsy.Adv Sci2021;8:e2102789 PMCID:PMC8529441

[67] Yuana Y,Grootemaat AE.Handling and storage of human body fluids for analysis of extracellular vesicles.J Extracell Vesicles2015;4:29260 PMCID:PMC4643195

[68] Xu R,Zhu HJ,Simpson RJ.Extracellular vesicle isolation and characterization: toward clinical application.J Clin Invest2016;126:1152-62 PMCID:PMC4811150

[69] Shin H,Labuz JM.High-yield isolation of extracellular vesicles using aqueous two-phase system.Sci Rep2015;5:13103 PMCID:PMC4536486

[70] Böing AN,Grootemaat AE,Sturk A.Single-step isolation of extracellular vesicles by size-exclusion chromatography.J Extracell Vesicles2014;3:23430 PMCID:PMC4159761

[71] Karimi N,Jang SC.Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins.Cell Mol Life Sci2018;75:2873-86 PMCID:PMC6021463

[72] Wei R,Kong G.Combination of size-exclusion chromatography and ultracentrifugation improves the proteomic profiling of plasma-derived small extracellular vesicles.Biol Proced Online2020;22:12 PMCID:PMC7313174

[73] Johnsen KB,Andresen TL.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 Cancer2019;1871:109-16

[74] van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles.Nat Rev Mol Cell Biol2018;19:213-28

[75] Ostenfeld MS,Jeppesen DK.miRNA profiling of circulating EpCAM⁺ extracellular vesicles: promising biomarkers of colorectal cancer.J Extracell Vesicles2016;5:31488 PMCID:PMC5005366

[76] Wang W,Wang S.Recent progress in isolation and detection of extracellular vesicles for cancer diagnostics.Adv Healthc Mater2018;7:e1800484

[77] Théry C,Aikawa E.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 Vesicles2018;7:1535750 PMCID:PMC6322352

[78] Clarke-Pearson DL.Clinical practice. Screening for ovarian cancer.N Engl J Med2009;361:170-7

[79] Wei SU,Zhang B.The diagnostic value of serum HE4 and CA-125 and ROMA index in ovarian cancer.Biomed Rep2016;5:41-4 PMCID:PMC4906902

[80] Drapkin R,Lin Y.Human epididymis protein 4 (HE4) is a secreted glycoprotein that is overexpressed by serous and endometrioid ovarian carcinomas.Cancer Res2005;65:2162-9

[81] Rani S,Kaur J,Punia RS.Osteopontin as a tumor marker in ovarian cancer.J Midlife Health2022;13:200-5 PMCID:PMC10025823

[82] Hirsch M, Duffy J, Davis CJ, Plana MN, Khan KS; International Collaboration to Harmonise Outcomes and Measures for Endometriosis. Diagnostic accuracy of cancer antigen 125 for endometriosis: a systematic review and meta-analysis.BJOG2016;123:1761-8

[83] Skates SJ,Horick NK.Large prospective study of ovarian cancer screening in high-risk women: CA125 cut-point defined by menopausal status.Cancer Prev Res2011;4:1401-8 PMCID:PMC3172691

[84] Jacobs IJ.Progress and challenges in screening for early detection of ovarian cancer.Mol Cell Proteomics2004;3:355-66

[85] Rani S,Nair S.Extracellular vesicles as mediators of cell-cell communication in ovarian cancer and beyond - a lipids focus.Cytokine Growth Factor Rev2023;73:52-68

[86] Wendler F,Simon T,Stebbing J.Extracellular vesicles swarm the cancer microenvironment: from tumor-stroma communication to drug intervention.Oncogene2017;36:877-84

[87] Godbole N,Carrion F.Extracellular vesicle miRNAs from three-dimensional ovarian cancer in vitro models and their implication in overall cancer survival.Heliyon2025;11:e42188 PMCID:PMC11872480

[88] Tresse E,Sew WQG.Mitochondrial DNA damage triggers spread of Parkinson’s disease-like pathology.Mol Psychiatry2023;28:4902-14 PMCID:PMC10914608

[89] Liaw A,Ivanovski S.The relevance of DNA methylation and histone modification in periodontitis: a scoping review.Cells2022;11:3211 PMCID:PMC9601099

[90] Han P,Salomon C.Detection of salivary small extracellular vesicles associated inflammatory cytokines gene methylation in gingivitis.Int J Mol Sci2020;21:5273 PMCID:PMC7432462

[91] Han P,Salomon C.Salivary outer membrane vesicles and DNA methylation of small extracellular vesicles as biomarkers for periodontal status: a pilot study.Int J Mol Sci2021;22:2423 PMCID:PMC7957785

[92] Han P,Ivanovski S.The emerging role of small extracellular vesicles in saliva and gingival crevicular fluid as diagnostics for periodontitis.J Periodontal Res2022;57:219-31

[93] Wang J,Cutler J,Lee RS.Microbial- and host immune cell-derived extracellular vesicles in the pathogenesis and therapy of periodontitis: a narrative review.J Periodontal Res2024;59:1115-29 PMCID:PMC11626692

[94] Rucci C,Salathia S,Censi R.Exploring mitochondrial DNA copy number in circulating cell-free DNA and extracellular vesicles across cardiovascular health status: a prospective case-control pilot study.FASEB J2024;38:e23672

[95] García-Silva S,Sánchez-Redondo S.Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAF^V600E mutation.J Exp Med2019;216:1061-70 PMCID:PMC6504207

[96] Yakubovich EI,Evtushenko VI.Principles and problems of exosome isolation from biological fluids.Biochem Suppl Ser A Membr Cell Biol2022;16:115-26 PMCID:PMC9202659

[97] Yuana Y,Grootemaat A,Nieuwland R.Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation.J Extracell Vesicles2014;3:23262 PMCID:PMC4090368

[98] Chen J,Zhang T.Review on strategies and technologies for exosome isolation and purification.Front Bioeng Biotechnol2021;9:811971 PMCID:PMC8766409

[99] Elzanowska J,Costa-Silva B.DNA in extracellular vesicles: biological and clinical aspects.Mol Oncol2021;15:1701-14 PMCID:PMC8169445

[100] Herrero C,Pérez-Fentes D.Extracellular vesicles’ genetic cargo as noninvasive biomarkers in cancer: a pilot study using ExoGAG technology.Biomedicines2023;11:404 PMCID:PMC9953104

[101] Thierry AR,Gahan PB,Stroun M.Origins, structures, and functions of circulating DNA in oncology.Cancer Metastasis Rev2016;35:347-76 PMCID:PMC5035665

[102] Stroun M,Maurice P,Lederrey C.Neoplastic characteristics of the DNA found in the plasma of cancer patients.Oncology1989;46:318-22

[103] Vasioukhin V,Maurice P,Lederrey C.Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia.Br J Haematol1994;86:774-9

[104] Sorenson GD,Valone FH,Bzik DJ.Soluble normal and mutated DNA sequences from single-copy genes in human blood.Cancer Epidemiol Biomarkers Prev1994;3:67-71

[105] Barbosa A,Pinto P,Teixeira MR.Potential clinical applications of circulating cell-free DNA in ovarian cancer patients.Expert Rev Mol Med2018;20:e6

[106] Jahr S,Englisch S.DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells.Cancer Res2001;61:1659-65

[107] Mattox AK,Wang Y.The origin of highly elevated cell-free DNA in healthy individuals and patients with pancreatic, colorectal, lung, or ovarian cancer.Cancer Discov2023;13:2166-79 PMCID:PMC10592331

[108] LaFramboise WA,Gallo PH.Cell-free DNA, tumor molecular concordance, and clinical correlates of patients with cancer treated in a large community health care network.J Mol Diagn2025;27:882-98 PMCID:PMC12489365

[109] Davidson BA,Reed SC.An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.Commun Biol2024;7:441 PMCID:PMC11006667

[110] Stejskal P,Srovnal J,van ’t Veer LJ.Circulating tumor nucleic acids: biology, release mechanisms, and clinical relevance.Mol Cancer2023;22:15 PMCID:PMC9862574

[111] Trevisi E,Colombo I.Clinical relevance of circulating tumor DNA in ovarian cancer: current issues and future opportunities.Explor Target Antitumor Ther2024;5:627-40 PMCID:PMC11220313

[112] Guo XM,Matsuo K,Salhia B.Circulating cell-free DNA methylation profiles in the early detection of ovarian cancer: a scoping review of the literature.Cancers2021;13:838 PMCID:PMC7923044

[113] Buckley DN,Gooden G.OvaPrint-A cell-free DNA methylation liquid biopsy for the risk assessment of high-grade serous ovarian cancer.Clin Cancer Res2023;29:5196-206 PMCID:PMC10722131

[114] Lu H,Wang J.Detection of ovarian cancer using plasma cell-free DNA methylomes.Clin Epigenetics2022;14:74 PMCID:PMC9185905

[115] Zhou H,Liu Q.Can circulating cell free DNA be a promising marker in ovarian cancer? - a genome-scale profiling study in a single institution.J Ovarian Res2023;16:11 PMCID:PMC9840311

[116] Kamat AA,Urbauer D.Plasma cell-free DNA in ovarian cancer: an independent prognostic biomarker.Cancer2010;116:1918-25 PMCID:PMC2854845

[117] Bronkhorst AJ,Holdenrieder S.The emerging role of cell-free DNA as a molecular marker for cancer management.Biomol Detect Quantif2019;17:100087 PMCID:PMC6425120

[118] Breitbach S,Helmig S.Direct quantification of cell-free, circulating DNA from unpurified plasma.PLoS One2014;9:e87838 PMCID:PMC3940427

[119] Stroun M,Lyautey J,Maurice PA.Isolation and characterization of DNA from the plasma of cancer patients.Eur J Cancer Clin Oncol1987;23:707-12

[120] Spinelli M,Keller I.Hypertensive disorders of pregnancy share common cfDNA methylation profiles.Sci Rep2022;12:19837 PMCID:PMC9674847

[121] Janku F,Pereira DY.A novel method for liquid-phase extraction of cell-free DNA for detection of circulating tumor DNA.Sci Rep2021;11:19653 PMCID:PMC8490367

[122] Bronkhorst AJ,Holdenrieder S.Comparison of methods for the isolation of cell-free DNA from cell culture supernatant.Tumour Biol2020;42:1010428320916314

[123] Lucena-Aguilar G,Barberán-Aceituno C,López-Guerrero JA.DNA source selection for downstream applications based on DNA quality indicators analysis.Biopreserv Biobank2016;14:264-70 PMCID:PMC4991598

[124] Bartels S,Hasemeier B,Kreipe H.Molecular analysis of circulating cell-free DNA from lung cancer patients in routine laboratory practice: a cross-platform comparison of three different molecular methods for mutation detection.J Mol Diagn2017;19:722-32

[125] Neuberger EWI,Ehlert T.Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients.Sci Rep2021;11:13581 PMCID:PMC8245561

[126] Alcaide M,Hillman J.Evaluating the quantity, quality and size distribution of cell-free DNA by multiplex droplet digital PCR.Sci Rep2020;10:12564 PMCID:PMC7387491

[127] van Ginkel JH, Huibers MMH, van Es RJJ, de Bree R, Willems SM. Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients.BMC Cancer2017;17:428 PMCID:PMC5477260

[128] Zhao Y,Hsieh K.Multiplex digital methylation-specific PCR for noninvasive screening of lung cancer.Adv Sci2023;10:e2206518 PMCID:PMC10238189

[129] Zhang Q,Yang Q.A multiplex methylation-specific PCR assay for the detection of early-stage ovarian cancer using cell-free serum DNA.Gynecol Oncol2013;130:132-9

[130] Alborelli I,Jermann P.Cell-free DNA analysis in healthy individuals by next-generation sequencing: a proof of concept and technical validation study.Cell Death Dis2019;10:534 PMCID:PMC6624284

[131] Esposito Abate R,Maiello MR.Next generation sequencing-based profiling of cell free DNA in patients with advanced non-small cell lung cancer: advantages and pitfalls.Cancers2020;12:3804 PMCID:PMC7766403

[132] Singh RR.Next-generation sequencing in high-sensitive detection of mutations in tumors: challenges, advances, and applications.J Mol Diagn2020;22:994-1007

[133] Jackson PE,Friesen MD.Specific p53 mutations detected in plasma and tumors of hepatocellular carcinoma patients by electrospray ionization mass spectrometry.Cancer Res2001;61:33-5

[134] Tsang JCH,Chow KCK.Mass spectrometry-based detection of hemoglobin E mutation by allele-specific base extension reaction.Clin Chem2007;53:2205-9

[135] Chiappetta C,Leopizzi M,Della Rocca C.Use of a new generation of capillary electrophoresis to quantify circulating free DNA in non-small cell lung cancer.Clin Chim Acta2013;425:93-6

[136] Covas Lage LA,Reichert CO.Circulating cell-free DNA (ccfDNA) concentrations measured by capillary electrophoresis: high cost-effectiveness in discriminating DLBCL patients and associations with high tumor burden.Blood2023;142:6085

[137] Medina JE,Lof P.Early detection of ovarian cancer using cell-free DNA fragmentomes and protein biomarkers.Cancer Discov2025;15:105-18 PMCID:PMC11726017

[138] Mazzone PJ,Carey J.Clinical validation of a cell-free DNA fragmentome assay for augmentation of lung cancer early detection.Cancer Discov2024;14:2224-42 PMCID:PMC11528203

[139] Leighl NB,Raymond VM.Clinical utility of comprehensive cell-free DNA analysis to identify genomic biomarkers in patients with newly diagnosed metastatic non-small cell lung cancer.Clin Cancer Res2019;25:4691-700

[140] Pritchett MA,Patel M.Prospective clinical validation of the InVisionFirst-lung circulating tumor DNA assay for molecular profiling of patients with advanced nonsquamous non-small-cell lung cancer.JCO Precis Oncol2019;3:1-15 PMCID:PMC7450945

[141] Woodhouse R,Hughes J.Clinical and analytical validation of FoundationOne Liquid CDx, a novel 324-Gene cfDNA-based comprehensive genomic profiling assay for cancers of solid tumor origin.PLoS One2020;15:e0237802 PMCID:PMC7518588

[142] García-Pardo M,Li JJN,Leighl NB.Integrating circulating-free DNA (cfDNA) analysis into clinical practice: opportunities and challenges.Br J Cancer2022;127:592-602 PMCID:PMC9381753

[143] Taylor ML,Amrhein KD,Wang Y.Nanomaterials for molecular detection and analysis of extracellular vesicles.Nanomaterials2023;13:524 PMCID:PMC9920192

[144] Ye S,Song S.Nanostructures and nanotechnologies for the detection of extracellular vesicle.Adv Biol2023;7:e2200201

[145] Picciolini S,Bedoni M.Advances in the field of micro- and nanotechnologies applied to extracellular vesicle research: take-home message from ISEV2021.Micromachines2021;12:1563 PMCID:PMC8707249

[146] Shao H,Castro CM,Weissleder R.New technologies for analysis of extracellular vesicles.Chem Rev2018;118:1917-50 PMCID:PMC6029891

[147] Chiang CY.Toward characterizing extracellular vesicles at a single-particle level.J Biomed Sci2019;26:9 PMCID:PMC6332877

[148] Yang L,Rathnam C.Harnessing the therapeutic potential of extracellular vesicles for biomedical applications using multifunctional magnetic nanomaterials.Small2022;18:e2104783 PMCID:PMC9344859

[149] Zhang Y,Liu H.Exosomes: biogenesis, biologic function and clinical potential.Cell Biosci2019;9:19 PMCID:PMC6377728

[150] Liu F,Mani V.The exosome total isolation chip.ACS Nano2017;11:10712-23 PMCID:PMC5983373

[151] Dong X,Zheng L.Efficient isolation and sensitive quantification of extracellular vesicles based on an integrated ExoID-Chip using photonic crystals.Lab Chip2019;19:2897-904

[152] Woo HK,Park J.Exodisc for rapid, size-selective, and efficient isolation and analysis of nanoscale extracellular vesicles from biological samples.ACS Nano2017;11:1360-70

[153] Zhang Y,Lou D.High-efficiency separation of extracellular vesicles from lipoproteins in plasma by agarose gel electrophoresis.Anal Chem2020;92:7493-9

[154] Jeong H,Cho S,Park J.Analysis of extracellular vesicles using coffee ring.ACS Appl Mater Interfaces2018;10:22877-82

[155] Jeong H,Yi J.Size-based analysis of extracellular vesicles using sequential transfer of an evaporating droplet.Lab Chip2019;19:3326-36

[156] Sitar S,Pahovnik D.Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation.Anal Chem2015;87:9225-33

[157] Petersen KE,White T.Exosome isolation: cyclical electrical field flow fractionation in low-ionic-strength fluids.Anal Chem2018;90:12783-90

[158] Zhang H.Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization.Nat Protoc2019;14:1027-53 PMCID:PMC6733524

[159] Wunsch BH,Gifford SM.Nanoscale lateral displacement arrays for the separation of exosomes and colloids down to 20 nm.Nat Nanotechnol2016;11:936-40

[160] Smith JT,Dogra N.Integrated nanoscale deterministic lateral displacement arrays for separation of extracellular vesicles from clinically-relevant volumes of biological samples.Lab Chip2018;18:3913-25

[161] Hattori Y,Yasui T,Baba Y.Micro- and nanopillar chips for continuous separation of extracellular vesicles.Anal Chem2019;91:6514-21

[162] Ibsen SD,Lewis JM.Rapid isolation and detection of exosomes and associated biomarkers from plasma.ACS Nano2017;11:6641-51

[163] Moore JH,Su YH.Conductance-based biophysical distinction and microfluidic enrichment of nanovesicles derived from pancreatic tumor cells of varying invasiveness.Anal Chem2019;91:10424-31

[164] Wu M,Wang Z.Isolation of exosomes from whole blood by integrating acoustics and microfluidics.Proc Natl Acad Sci U S A2017;114:10584-9 PMCID:PMC5635903

[165] Lee K,Weissleder R.Acoustic purification of extracellular microvesicles.ACS Nano2015;9:2321-7 PMCID:PMC4373978

[166] Zhao J,Li Y.Thermophoretic detection of exosomal microRNAs by nanoflares.J Am Chem Soc2020;142:4996-5001

[167] Liu C,Tian F.Low-cost thermophoretic profiling of extracellular-vesicle surface proteins for the early detection and classification of cancers.Nat Biomed Eng2019;3:183-93

[168] Huang M,Wang T.Homogeneous, low-volume, efficient, and sensitive quantitation of circulating exosomal PD-L1 for cancer diagnosis and immunotherapy response prediction.Angew Chem Int Ed Engl2020;59:4800-5

[169] Liu C,Li B.Single-exosome-counting immunoassays for cancer diagnostics.Nano Lett2018;18:4226-32

[170] Gao X,Ding F.Rapid detection of exosomal microRNAs using virus-mimicking fusogenic vesicles.Angew Chem Int Ed Engl2019;58:8719-23

[171] Wan Y,Liu X.Rapid magnetic isolation of extracellular vesicles via lipid-based nanoprobes.Nat Biomed Eng2017;1:0058 PMCID:PMC5618714

[172] Wan Y,He HZ.Enrichment of extracellular vesicles with lipid nanoprobe functionalized nanostructured silica.Lab Chip2019;19:2346-55 PMCID:PMC6669184

[173] Kang YT,Palacios-Rolston C.Isolation and profiling of circulating tumor-associated exosomes using extracellular vesicular lipid-protein binding affinity based microfluidic device.Small2019;15:e1903600 PMCID:PMC6951813

[174] Nakai W,Diez D.A novel affinity-based method for the isolation of highly purified extracellular vesicles.Sci Rep2016;6:33935 PMCID:PMC5034288

[175] Xu H,Zuo P,Ye BC.Magnetic-based microfluidic device for on-chip isolation and detection of tumor-derived exosomes.Anal Chem2018;90:13451-8

[176] Stapane L,Hincke MT.The glycoproteins EDIL3 and MFGE8 regulate vesicle-mediated eggshell calcification in a new model for avian biomineralization.J Biol Chem2019;294:14526-45 PMCID:PMC6779430

[177] Gao F,Xia C.A novel strategy for facile serum exosome isolation based on specific interactions between phospholipid bilayers and TiO₂.Chem Sci2019;10:1579-88 PMCID:PMC6369439

[178] Jeong S,Pathania D,Weissleder R.Integrated magneto-electrochemical sensor for exosome analysis.ACS Nano2016;10:1802-9 PMCID:PMC4802494

[179] Zhang P,Zeng Y.Multiplexed immunophenotyping of circulating exosomes on nano-engineered ExoProfile chip towards early diagnosis of cancer.Chem Sci2019;10:5495-504 PMCID:PMC6544119

[180] Zhu L,Cui J.Label-free quantitative detection of tumor-derived exosomes through surface plasmon resonance imaging.Anal Chem2014;86:8857-64 PMCID:PMC4151789

[181] Jin D,Zhang Y.ExoAPP: exosome-oriented, aptamer nanoprobe-enabled surface proteins profiling and detection.Anal Chem2018;90:14402-11

[182] Zhang K,Wu S,Shi J.Rapid capture and nondestructive release of extracellular vesicles using aptamer-based magnetic isolation.ACS Sens2019;4:1245-51

[183] Zhao Z,Gamero-Kubota P.Microfluidic on-demand engineering of exosomes towards cancer immunotherapy.Lab Chip2019;19:1877-86 PMCID:PMC6520140

[184] Fang X,Adkins GB.Highly efficient exosome isolation and protein analysis by an integrated nanomaterial-based platform.Anal Chem2018;90:2787-95 PMCID:PMC5820131

[185] Zhang P,Zeng Y.Ultrasensitive microfluidic analysis of circulating exosomes using a nanostructured graphene oxide/polydopamine coating.Lab Chip2016;16:3033-42 PMCID:PMC4970962

[186] Wang Z,Fine D.Ciliated micropillars for the microfluidic-based isolation of nanoscale lipid vesicles.Lab Chip2013;13:2879-82 PMCID:PMC3740541

[187] Zhang P,Gardashova G.Molecular and functional extracellular vesicle analysis using nanopatterned microchips monitors tumor progression and metastasis.Sci Transl Med2020;12:eaaz2878 PMCID:PMC8024111

[188] Poellmann MJ,Bu J,Kimple RJ.Immunoavidity-based capture of tumor exosomes using poly(amidoamine) dendrimer surfaces.Nano Lett2020;20:5686-92 PMCID:PMC12180921

[189] Dong J,Sun N.Coupling nanostructured microchips with covalent chemistry enables purification of sarcoma-derived extracellular vesicles for downstream functional studies.Adv Funct Mater2020;30:2003237 PMCID:PMC8248519

[190] Sun N,Kim M.Covalent chemistry-mediated multimarker purification of circulating tumor cells enables noninvasive detection of molecular signatures of hepatocellular carcinoma.Adv Mater Technol2021;6:2001056 PMCID:PMC8240468

[191] Zhang P,He M.Ultrasensitive detection of circulating exosomes with a 3D-nanopatterned microfluidic chip.Nat Biomed Eng2019;3:438-51 PMCID:PMC6556143

[192] Mori K,Morishige T.A pretreatment-free, polymer-based platform prepared by molecular imprinting and post-imprinting modifications for sensing intact exosomes.Angew Chem Int Ed Engl2019;58:1612-5

[193] Takeuchi T,Sunayama H.Antibody-conjugated signaling nanocavities fabricated by dynamic molding for detecting cancers using small extracellular vesicle markers from tears.J Am Chem Soc2020;142:6617-24

[194] Oh HJ,Park H,Hwang DW.Graphene-oxide quenching-based molecular beacon imaging of exosome-mediated transfer of neurogenic miR-193a on microfluidic platform.Biosens Bioelectron2019;126:647-56

[195] Tayebi M,Yang HY.A MoS₂-MWCNT based fluorometric nanosensor for exosome detection and quantification.Nanoscale Adv2019;1:2866-72 PMCID:PMC9419881

[196] Cho S,Rhee WJ.Simultaneous multiplexed detection of exosomal microRNAs and surface proteins for prostate cancer diagnosis.Biosens Bioelectron2019;146:111749

[197] Lee JH,Choi HK,Lee KB.Functional nanoarrays for investigating stem cell fate and function.Nanoscale2020;12:9306-26 PMCID:PMC7671654

[198] Rodrigues M,Ning B,Hu TY.Rapid lipid-based approach for normalization of quantum-dot-detected biomarker expression on extracellular vesicles in complex biological samples.Nano Lett2019;19:7623-31 PMCID:PMC8162763

[199] Pang X,Gao K.Visible light-driven self-powered device based on a straddling nano-heterojunction and bio-application for the quantitation of exosomal RNA.ACS Nano2019;13:1817-27 PMCID:PMC6613566

[200] Lyu Y,Huang J,Miao Y.Near-infrared afterglow semiconducting nano-polycomplexes for the multiplex differentiation of cancer exosomes.Angew Chem Int Ed Engl2019;58:4983-7

[201] Fan Y,Zhao M.High-sensitive and multiplex biosensing assay of NSCLC-derived exosomes via different recognition sites based on SPRi array.Biosens Bioelectron2020;154:112066

[202] Yang Q,Hu L.An integrative microfluidic device for isolation and ultrasensitive detection of lung cancer-specific exosomes from patient urine.Biosens Bioelectron2020;163:112290

[203] Wang Q,Yang X.Direct quantification of cancerous exosomes via surface plasmon resonance with dual gold nanoparticle-assisted signal amplification.Biosens Bioelectron2019;135:129-36

[204] Lee J,Heo Y.Enhanced paper-based ELISA for simultaneous EVs/exosome isolation and detection using streptavidin agarose-based immobilization.Analyst2019;145:157-64

[205] Zhang H,Zhang Q,Liu Y.Ti₃C₂ MXenes nanosheets catalyzed highly efficient electrogenerated chemiluminescence biosensor for the detection of exosomes.Biosens Bioelectron2019;124-125:184-90

[206] Zhang H,Wang F,Wang H.In situ formation of gold nanoparticles decorated Ti₃C₂ MXenes nanoprobe for highly sensitive electrogenerated chemiluminescence detection of exosomes and their surface proteins.Anal Chem2020;92:5546-53

[207] Sun Y,Jiang X.Assembly of black phosphorus nanosheets and MOF to form functional hybrid thin-film for precise protein capture, dual-signal and intrinsic self-calibration sensing of specific cancer-derived exosomes.Anal Chem2020;92:2866-75

[208] Jiang Q,Wang L,Zhong W.Rapid enrichment and detection of extracellular vesicles enabled by CuS-enclosed microgels.Anal Chem2019;91:15951-8 PMCID:PMC7417204

[209] Chen J,Lu Y.Isolation and visible detection of tumor-derived exosomes from plasma.Anal Chem2018;90:14207-15

[210] Boriachek K,Palma C.Avoiding pre-isolation step in exosome analysis: direct isolation and sensitive detection of exosomes using gold-loaded nanoporous ferric oxide nanozymes.Anal Chem2019;91:3827-34

[211] Huang R,Xia Y.A sensitive aptasensor based on a hemin/G-quadruplex-assisted signal amplification strategy for electrochemical detection of gastric cancer exosomes.Small2019;15:e1900735

[212] Wu D,Jin D,Zhang ZY.Dual-aptamer modified graphene field-effect transistor nanosensor for label-free and specific detection of hepatocellular carcinoma-derived microvesicles.Anal Chem2020;92:4006-15

[213] Wu D,Jin D.Microvesicle detection by a reduced graphene oxide field-effect transistor biosensor based on a membrane biotinylation strategy.Analyst2019;144:6055-63

[214] Cheng HL,Kuo WC.Detecting miRNA biomarkers from extracellular vesicles for cardiovascular disease with a microfluidic system.Lab Chip2018;18:2917-25

[215] Zhu F,Ding Q.2D magnetic MoS₂-Fe₃O₄ hybrid nanostructures for ultrasensitive exosome detection in GMR sensor.Biosens Bioelectron2020;147:111787

[216] Wang C,Jin D.AuNP-amplified surface acoustic wave sensor for the quantification of exosomes.ACS Sens2020;5:362-9

[217] Suthar J,Hoogenboom BW,Guldin S.Acoustic immunosensing of exosomes using a quartz crystal microbalance with dissipation monitoring.Anal Chem2020;92:4082-93 PMCID:PMC7145312

[218] Zheng H,Wang X,Gao M.Deconstruction of heterogeneity of size-dependent exosome subpopulations from human urine by profiling N-glycoproteomics and phosphoproteomics simultaneously.Anal Chem2020;92:9239-46

[219] He D,Chan HN.Molecular-recognition-based DNA nanodevices for enhancing the direct visualization and quantification of single vesicles of tumor exosomes in plasma microsamples.Anal Chem2019;91:2768-75

[220] Chen C,Liu Y.Profiling of exosomal biomarkers for accurate cancer identification: combining DNA-PAINT with machine- learning-based classification.Small2019;15:e1901014

[221] Ridolfi A,Montis C.AFM-based high-throughput nanomechanical screening of single extracellular vesicles.Anal Chem2020;92:10274-82

[222] Tian Y,Gong M.Protein profiling and sizing of extracellular vesicles from colorectal cancer patients via flow cytometry.ACS Nano2018;12:671-80

[223] Wu D,Shen X.Profiling surface proteins on individual exosomes using a proximity barcoding assay.Nat Commun2019;10:3854 PMCID:PMC6710248

[224] Pallares RM,Su X.Sensing of circulating cancer biomarkers with metal nanoparticles.Nanoscale2019;11:22152-71

[225] Heitzer E,Geigl JB.Circulating tumor DNA as a liquid biopsy for cancer.Clin Chem2015;61:112-23

[226] Pallares RM,Ru TH,Lu Y.A plasmonic nanosensor with inverse sensitivity for circulating cell-free DNA quantification.Chem Commun2015;51:14524-7

[227] Pallares RM,Su X.Tunable plasmonic colorimetric assay with inverse sensitivity for extracellular DNA quantification.Chem Commun2018;54:11260-3

[228] Ito H,Hasegawa Y.Silver nanoscale hexagonal column chips for detecting cell-free DNA and circulating nucleosomes in cancer patients.Sci Rep2015;5:10455 PMCID:PMC4440038

[229] Ito H,Hasegawa K.Use of surface-enhanced Raman scattering for detection of cancer-related serum-constituents in gastrointestinal cancer patients.Nanomedicine2014;10:599-608

[230] Huang H,Wang Y.Nanoparticulate cell-free DNA scavenger for treating inflammatory bone loss in periodontitis.Nat Commun2022;13:5925 PMCID:PMC9546917