Extracellular vesicles as a promising biomarker resource in liquid biopsy for cancer

Abstract

Liquid biopsy is a minimally invasive biopsy method that uses molecules in body fluids as biomarkers, and it has attracted attention as a new cancer therapy tool. Liquid biopsy has considerable clinical application potential, such as in early diagnosis, pathological condition monitoring, and tailored treatment development based on cancer biology and the predicted treatment response of individual patients. Extracellular vesicles (EVs) are lipid membranous vesicles released from almost all cell types, and they represent a novel liquid biopsy resource. EVs carry complex molecular cargoes, such as proteins, RNAs [e.g., mRNA and noncoding RNAs (microRNA, transfer RNA, circular RNA and long noncoding RNA)], and DNA fragments; these cargoes are delivered to recipient cells and serve as a cell-to-cell communication system. The molecular contents of EVs largely reflect the cell of origin and thus show cell-type specificity. In particular, cancer-derived EVs contain cancer-specific molecules expressed in parental cancer cells. Therefore, analysis of cancer-derived EVs might indicate the presence and nature of cancer. High-speed analytical technologies, such as mass spectrometry and high-throughput sequencing, have generated large data sets for EV cargoes that can be used to identify many candidate EV-associated biomarkers. Here, we will discuss the challenges and prospects of EV-based liquid biopsy compared to other biological resources (e.g., circulating tumor cells and cell-free DNA) and summarize the novel studies that have identified the remarkable potential of EVs as a cancer biomarker.

Keywords

Extracellular vesicles / liquid biopsy / cancer biomarker / microRNA

References

[1] Doyle LM.Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis.Cells2019;8:727 PMCID:PMC6678302

[2] Siravegna G,Siena S.Integrating Liquid Biopsies into the Management of Cancer.Nat Rev Clin Oncol2017;14:531-48

[3] Ashley EA.Towards Precision Medicine.Nat Rev Genet2016;17:507-22

[4] Hamburg MA.The path to personalized medicine.N Engl J Med2010;363:301-4

[5] Siravegna G,Venesio T.How liquid biopsies can change clinical practice in oncology.Ann Oncol2019;30:1580-90

[6] Bettegowda C,Leary RJ.Detection of circulating tumor DNA in early- and late-stage human malignancies.Sci Transl Med2014;6:224ra24 PMCID:PMC4017867

[7] Cheng F,Qian C.Circulating tumor DNA: a promising biomarker in the liquid biopsy of cancer.Oncotarget 2016;7:48832-41 PMCID:PMC5217053

[8] Roy S,Jones PS.Extracellular vesicles: the growth as diagnostics and therapeutics; a Survey.J Extracell Vesicles2018;7:1438720 PMCID:PMC5827771

[9] Yáñez-Mó M,Andreu Z.Biological properties of extracellular vesicles and their physiological functions.J Extracell Vesicles2015;4:27066 PMCID:PMC4433489

[10] Li Y,Bao C.Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.Cell Res2015;25:981-4 PMCID:PMC4528056

[11] Keller S,Rupp AK,Altevogt P.Body fluid derived exosomes as a novel template for clinical diagnostics.J Transl Med2011;9:86 PMCID:PMC3118335

[12] Krebs MG,Carter L.Molecular analysis of circulating tumour cells - biology and biomarkers.Nat Rev Clin Oncol2014;11:129-44

[13] Diaz LA Jr.Liquid biopsies: genotyping circulating tumor DNA.J Clin Oncol2014;32:579-86 PMCID:PMC4820760

[14] Cristofanilli M,Ellis MJ.Circulating tumor cells, disease progression, and survival in metastatic breast cancer.N Engl J Med2004;351:781-91

[15] de Bono JS,Montgomery RB.Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer.Clin Cancer Res2008;14:6302-9

[16] Danila DC,Gignac GA.Circulating tumor cell number and prognosis in progressive castration-resistant prostate cancer.Clin Cancer Res2007;13:7053-8

[17] Heller G,Kheoh T.Circulating Tumor Cell Number as a Response Measure of Prolonged Survival for Metastatic Castration-Resistant Prostate Cancer: A Comparison with Prostate-Specific Antigen across Five Randomized Phase III Clinical Trials.J Clin Oncol 2018;36:572-80 PMCID:PMC5815402

[18] Goldkorn A,Quinn DI.Circulating tumor cell counts are prognostic of overall survival in SWOG S0421: a phase III trial of docetaxel with or without atrasentan for metastatic castration-resistant prostate cancer.J Clin Oncol2014;32:1136-42 PMCID:PMC3970171

[19] Scher HI,Molina A.Circulating tumor cell biomarker panel as an individual-level surrogate for survival in metastatic castration-resistant prostate cancer.J Clin Oncol2015;33:1348-55 PMCID:PMC4397279

[20] Allard WJ,Miller MC.Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.Clin Cancer Res2004;10:6897-904

[21] Alix-Panabières C.Circulating tumor cells: liquid biopsy of cancer.Clin Chem2013;59:110-8

[22] Qin J,Hunsley BA,Fernando MR.Stabilization of circulating tumor cells in blood using a collection device with a preservative reagent.Cancer Cell Int2014;14:23 PMCID:PMC3995911

[23] Alix-Panabières C.Challenges in circulating tumour cell research.Nat Rev Cancer2014;14:623-31

[24] Allard WJ.CCR 20th anniversary commentary: paving the way for circulating tumor cells.Clin Cancer Res 2015;21:2883-5

[25] Markou A,Sourvinou I.Direct comparison of metastasis-related miRNAs expression levels in circulating tumor cells, corresponding plasma, and primary tumors of breast cancer patients.Clin Chem2016;62:1002-11

[26] Antonarakis ES,Luber B.Clinical Significance of Androgen Receptor Splice Variant-7 mRNA Detection in Circulating Tumor Cells of Men With Metastatic Castration-Resistant Prostate Cancer Treated With First- and Second-Line Abiraterone and Enzalutamide.J Clin Oncol2017;35:2149-56 PMCID:PMC5493048

[27] Sinkala E,Renier C.Profiling protein expression in circulating tumour cells using microfluidic western blotting.Nat Commun2017;8:14622 PMCID:PMC5376644

[28] Armbrecht L,Szczerba BM,Aceto N.Quantification of Protein Secretion from Circulating Tumor Cells in Microfluidic Chambers.Adv Sci (Weinh)2020;7:1903237 PMCID:PMC7284199

[29] Zill OA,Fairclough SR.The Landscape of Actionable Genomic Alterations in Cell-Free Circulating Tumor DNA from 21,807 Advanced Cancer Patients.Clin Cancer Res2018;24:3528-38

[30] Chun FK,Lange I.Circulating tumour-associated plasma DNA represents an independent and informative predictor of prostate cancer.BJU Int2006;98:544-8

[31] Altimari A,Benedettini E.Diagnostic role of circulating free plasma DNA detection in patients with localized prostate cancer.Am J Clin Pathol2008;129:756-62

[32] Bergsmedh A,Henriksson M.Horizontal transfer of oncogenes by uptake of apoptotic bodies.Proc Natl Acad Sci U S A2001;98:6407-11 PMCID:PMC33481

[33] Trejo-Becerril C,Taja-Chayeb L.Cancer progression mediated by horizontal gene transfer in an in vivo model.PLoS One2012;7:e52754 PMCID:PMC3532306

[34] Mahon KL,Devaney J.PRIMe consortiumMethylated Glutathione S-transferase 1 (mGSTP1) is a potential plasma free DNA epigenetic marker of prognosis and response to chemotherapy in castrate-resistant prostate cancer.Br J Cancer2014;111:1802-9 PMCID:PMC4453725

[35] Azad AA,Wyatt AW.Androgen Receptor Gene Aberrations in Circulating Cell-Free DNA: Biomarkers of Therapeutic Resistance in Castration-Resistant Prostate Cancer.Clin Cancer Res2015;21:2315-24

[36] Lallous N,Awrey S.Functional analysis of androgen receptor mutations that confer anti-androgen resistance identified in circulating cell-free DNA from prostate cancer patients.Genome Biol2016;17:10 PMCID:PMC4729137

[37] Salvi S,Conteduca V.Circulating cell-free AR and CYP17A1 copy number variations may associate with outcome of metastatic castration-resistant prostate cancer patients treated with abiraterone.Br J Cancer2015;112:1717-24 PMCID:PMC4430717

[38] Carreira S,Goodall J.Tumor clone dynamics in lethal prostate cancer.Sci Transl Med2014;6:254ra125 PMCID:PMC4422178

[39] Diehl F,Choti MA.Circulating mutant DNA to assess tumor dynamics.Nat Med2008;14:985-90 PMCID:PMC2820391

[40] Leung F,Diamandis EP.Circulating tumor DNA as a cancer biomarker: fact or fiction?.Clin Chem2016;62:1054-60 PMCID:PMC5326709

[41] Sozzi G,Conte D.Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays.J Natl Cancer Inst2005;97:1848-50

[42] Gormally E,Vineis P.Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance.Mutat Res2007;635:105-17

[43] Cai X,Zhan Q.Accessing genetic information with liquid biopsies.Trends Genet2015;31:564-75

[44] Schwarzenbach H,Pantel K.Cell-free nucleic acids as biomarkers in cancer patients.Nat Rev Cancer2011;11:426-37

[45] Gould SJ.As we wait: coping with an imperfect nomenclature for extracellular vesicles.J Extracell Vesicles2013;2:20389 PMCID:PMC3760635

[46] György B,Pásztói M.Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.Cell Mol Life Sci2011;68:2667-88 PMCID:PMC3142546

[47] 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

[48] Witwer KW.Extracellular vesicles or exosomes?.J Extracell Vesicles2019;8:1648167 PMCID:PMC6711079

[49] György B,Pásztói M.Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.Cell Mol Life Sci2011;68:2667-88 PMCID:PMC3142546

[50] Raposo G.Extracellular vesicles: exosomes, microvesicles, and friends.J Cell Biol2013;200:373-83 PMCID:PMC3575529

[51] Kowal J,Colombo M.Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.Proc Natl Acad Sci U S A2016;113:E968-77 PMCID:PMC4776515

[52] Pan B.Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: Selective externalization of the receptor.Cell1983;33:967-78

[53] Johnstone RM,Hammond JR,Turbide C.Vesicle formation during reticulocyte maturation. association of plasma membrane activities with released vesicles (exosomes).J Biol Chem1987;262;9412-20

[54] Raposo G,Stoorvogel W.B lymphocytes secrete antigen-presenting vesicles.J Exp Med1996;183:1161-72 PMCID:PMC2192324

[55] Valadi H,Bossios A,Lee JJ.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol2007;9:654-9

[56] Aebersold R.Mass-spectrometric exploration of proteome structure and function.Nature2016;537:347-55

[57] Reuter JA,Snyder MP.High-throughput sequencing technologies.Mol Cell2015;58:586-97 PMCID:PMC4494749

[58] Welch JL,Margolick JB.Effect of prolonged freezing of semen on exosome recovery and biologic activity.Sci Rep2017;7:45034 PMCID:PMC5364471

[59] Tovar-Camargo OA,Goel A.Exosomal microRNA biomarkers: emerging frontiers in colorectal and other human cancers.Expert Rev Mol Diagn2016;16:553-67 PMCID:PMC4935983

[60] Li P,Lee SH,Gao Z.Progress in exosome isolation techniques.Theranostics 2017;7:789-804 PMCID:PMC5327650

[61] Nordin JZ,Vader P.Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties.Nanomedicine2015;11:879-83

[62] Gupta S,Arora V.An improvised one-step sucrose cushion ultracentrifugation method for exosome isolation from culture supernatants of mesenchymal stem cells.Stem Cell Res Ther2018;9:180 PMCID:PMC6033286

[63] Van Deun J,Sormunen R.The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.J Extracell Vesicles2014;3:24858 PMCID:PMC4169610

[64] Coumans FAW,Buzas EI.Methodological guidelines to study extracellular vesicles.Circ Res2017;120:1632-48

[65] Helwa I,Drewry MD.A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents.PLoS One2017;12:e0170628 PMCID:PMC5256994

[66] Clayton A,Navabi H.Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry.J Immunol Methods2001;247:163-74

[67] Théry C,Raposo G.Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr Protoc Cell Biol2006;Chapter 3:Unit 3.22

[68] Brennan K,FitzGerald SP.A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum.Sci Rep2020;10:1039 PMCID:PMC6978318

[69] Liangsupree T,Riekkola ML.Modern isolation and separation techniques for extracellular vesicles.J Chromatogr A2021;1636:461773

[70] An M,Zhu J.Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum.J Proteome Res2018;17:3599-605 PMCID:PMC6292670

[71] Koh YQ,Vaswani K,Mitchell MD.Exosome enrichment by ultracentrifugation and size exclusion chromatography.Front Biosci (Landmark Ed)2018;23:865-74

[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] Simons M.Exosomes - vesicular carriers for intercellular communication.Curr Opin Cell Biol 2009;21:575-81

[74] Mathivanan S,Simpson RJ.Exosomes: extracellular organelles important in intercellular communication.J Proteomics2010;73:1907-20

[75] Kowal J,Théry C.Biogenesis and secretion of exosomes.Curr Opin Cell Biol2014;29:116-25

[76] Clayton A,Buzas EI.Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles.J Extracell Vesicles2019;8:1647027 PMCID:PMC6711123

[77] Jørgensen M,Pedersen S,Kristensen SR.Extracellular Vesicle (EV) Array: microarray capturing of exosomes and other extracellular vesicles for multiplexed phenotyping.J Extracell Vesicles2013;2:20920 PMCID:PMC3760630

[78] Jakobsen KR,Bæk R,Sorensen BS.Exosomal proteins as potential diagnostic markers in advanced non-small cell lung carcinoma.J Extracell Vesicles2015;4:26659 PMCID:PMC4348413

[79] Shao H,Balaj L.Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy.Nat Med2012;18:1835-40 PMCID:PMC3518564

[80] Kravets VG,Jalil R.Singular phase nano-optics in plasmonic metamaterials for label-free single-molecule detection.Nat Mater2013;12:304-9

[81] Im H,Park YI.Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor.Nat Biotechnol2014;32:490-5 PMCID:PMC4356947

[82] Yoshioka Y,Konishi Y.Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen.Nat Commun2014;5:3591 PMCID:PMC3988821

[83] Zhao Z,Zeng Y.A microfluidic ExoSearch chip for multiplexed exosome detection towards blood-based ovarian cancer diagnosis.Lab Chip2016;16:489-96 PMCID:PMC4729647

[84] Mitchell PJ,Staffurth J.Can urinary exosomes act as treatment response markers in prostate cancer?.J Transl Med2009;7:4 PMCID:PMC2631476

[85] Logozzi M,Iessi E.Increased PSA expression on prostate cancer exosomes in in vitro condition and in cancer patients.Cancer Lett2017;403:318-29

[86] Huber V,Iero M.Human colorectal cancer cells induce T-cell death through release of proapoptotic microvesicles: role in immune escape.Gastroenterology2005;128:1796-804

[87] Khan S,Valenzuela MM.Plasma-derived exosomal survivin, a plausible biomarker for early detection of prostate cancer.PLoS One2012;7:e46737 PMCID:PMC3473028

[88] Khan S,Turay D.Early diagnostic value of survivin and its alternative splice variants in breast cancer.BMC Cancer2014;14:176 PMCID:PMC3995700

[89] Melo SA,Kahlert C.Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.Nature2015;523:177-82 PMCID:PMC4825698

[90] Book: ISEV2017.J Extracell Vesicles2017;6:1310414 PMCID:PMC5637785

[91] Giampieri R,Occhipinti G.Clinical impact of different exosomes' protein expression in pancreatic ductal carcinoma patients treated with standard first line palliative chemotherapy.PLoS One2019;14:e0215990 PMCID:PMC6497273

[92] Ning K,Sun X.UCH-L1-containing exosomes mediate chemotherapeutic resistance transfer in breast cancer.J Surg Oncol2017;115:932-40

[93] Lv MM,Chen WX.Exosomes mediate drug resistance transfer in MCF-7 breast cancer cells and a probable mechanism is delivery of P-glycoprotein.Tumour Biol2014;35:10773-9

[94] Zhang FF,Zhao QN.Microvesicles mediate transfer of P-glycoprotein to paclitaxel-sensitive A2780 human ovarian cancer cells, conferring paclitaxel-resistance.Eur J Pharmacol2014;738:83-90

[95] Corcoran C,O'Brien K.Docetaxel-resistance in prostate cancer: evaluating associated phenotypic changes and potential for resistance transfer via exosomes.PLoS One2012;7:e50999 PMCID:PMC3519481

[96] Ciravolo V,Ghedini GC.Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy.J Cell Physiol2012;227:658-67

[97] Martinez VG,Salimu J.Resistance to HER2-targeted anti-cancer drugs is associated with immune evasion in cancer cells and their derived extracellular vesicles.Oncoimmunology2017;6:e1362530 PMCID:PMC5706614

[98] Chen L.Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future.J Clin Invest2015;125:3384-91 PMCID:PMC4588282

[99] Ribas A,Daud A.Association of pembrolizumab with tumor response and survival among patients with advanced melanoma.JAMA2016;315:1600-9

[100] Zaretsky JM,Shin DS.Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma.N Engl J Med2016;375:819-29 PMCID:PMC5007206

[101] Chen G,Zhang W.Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response.Nature2018;560:382-6 PMCID:PMC6095740

[102] Cao L,Hu J.Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications.Biosens Bioelectron 2017;90:459-74

[103] Ratajczak J,Kucia M.Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery.Leukemia2006;20:847-56

[104] Kosaka N,Yoshioka Y,Matsuki Y.Secretory mechanisms and intercellular transfer of microRNAs in living cells.J Biol Chem2010;285:17442-52 PMCID:PMC2878508

[105] Pegtel DM,Thorley-Lawson DA.Functional delivery of viral miRNAs via exosomes.Proc Natl Acad Sci U S A2010;107:6328-33 PMCID:PMC2851954

[106] Zhang Y,Chen X.Secreted monocytic miR-150 enhances targeted endothelial cell migration.Mol Cell2010;39:133-44

[107] Zhu L,Gong Y.Exosomal tRNA-derived small RNA as a promising biomarker for cancer diagnosis.Mol Cancer2019;18:74 PMCID:PMC6444574

[108] Zhang PF,Huang XY.Cancer cell-derived exosomal circUHRF1 induces natural killer cell exhaustion and may cause resistance to anti-PD1 therapy in hepatocellular carcinoma.Mol Cancer2020;19:110 PMCID:PMC7320583

[109] Gusachenko ON,Vlassov VV.Nucleic acids in exosomes: disease markers and intercellular communication molecules.Biochem 2013;78:1-7

[110] Bullock MD,Kanlikilicer-Unaldi P.Exosomal non-coding RNAs: diagnostic, prognostic and therapeutic applications in cancer.Noncoding RNA 2015;1:53-68 PMCID:PMC5932539

[111] Mitchell PS,Kroh EM.Circulating microRNAs as stable blood-based markers for cancer detection.Proc Natl Acad Sci U S A2008;105:10513-8 PMCID:PMC2492472

[112] Taylor DD.MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.Gynecol Oncol2008;110:13-21

[113] Rabinowits G,Day JM,Kloecker GH.Exosomal microRNA: a diagnostic marker for lung cancer.Clin Lung Cancer2009;10:42-6

[114] Takeshita N,Mori M.Serum microRNA expression profile: miR-1246 as a novel diagnostic and prognostic biomarker for oesophageal squamous cell carcinoma.Br J Cancer2013;108:644-52 PMCID:PMC3593570

[115] Nilsson J,Nordstrand A.Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer.Br J Cancer2009;100:1603-7 PMCID:PMC2696767

[116] Foj L,Serra M.Exosomal and Non-Exosomal Urinary miRNAs in Prostate Cancer Detection and Prognosis.Prostate2017;77:573-83

[117] Yasui T,Ito S.Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires.Sci Adv2017;3:e1701133 PMCID:PMC5744465

[118] McKiernan J,O'Neill V.A Novel Urine Exosome Gene Expression Assay to Predict High-grade Prostate Cancer at Initial Biopsy.JAMA Oncol2016;2:882-9

[119] Huang X,Liang M.Exosomal miR-1290 and miR-375 as prognostic markers in castration-resistant prostate cancer.Eur Urol2015;67:33-41 PMCID:PMC4252606

[120] Tanaka Y,Kinoshita K.Clinical impact of serum exosomal microRNA-21 as a clinical biomarker in human esophageal squamous cell carcinoma.Cancer2013;119:1159-67

[121] Matsumura T,Iinuma H.Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer.Br J Cancer2015;113:275-81 PMCID:PMC4506387

[122] Işın M,Özgür E.Exosomal lncRNA-p21 levels may help to distinguish prostate cancer from benign disease.Front Genet2015;6:168 PMCID:PMC4422020

[123] Lin LY,Zeng Q.Tumor-originated exosomal LncUEGC1 as a circulating biomarker for early-stage gastric cancer.Mol Cancer2018;17:84 PMCID:PMC5978993

[124] Zhao R,Zhang X.Exosomal long noncoding RNA HOTTIP as potential novel diagnostic and prognostic biomarker test for gastric cancer.Mol Cancer2018;17:68 PMCID:PMC6389063

[125] Lee YR,Tak WY.Circulating exosomal noncoding RNAs as prognostic biomarkers in human hepatocellular carcinoma.Int J Cancer2019;144:1444-52

[126] Chen X,Wei WS.PRMT5 Circular RNA Promotes Metastasis of Urothelial Carcinoma of the Bladder through Sponging miR-30c to Induce Epithelial-Mesenchymal Transition.Clin Cancer Res2018;24:6319-30

[127] Ogata-Kawata H,Kurioka D.Circulating exosomal microRNAs as biomarkers of colon cancer.PLoS One2014;9:e92921 PMCID:PMC3976275

[128] Svedman FC,Bottai M.Extracellular microvesicle microRNAs as predictive biomarkers for targeted therapy in metastastic cutaneous malignant melanoma.PLoS One2018;13:e0206942 PMCID:PMC6219796

[129] Tang S,Tang Y,Zou T.Overexpression of serum exosomal HOTAIR is correlated with poor survival and poor response to chemotherapy in breast cancer patients.J Biosci2019;44

[130] Dong H,Chen R.Exosome-mediated transfer of lncRNASNHG14 promotes trastuzumab chemoresistance in breast cancer.Int J Oncol2018;53:1013-26 PMCID:PMC6065402

[131] Mikamori M,Eguchi H.MicroRNA-155 Controls Exosome Synthesis and Promotes Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma.Sci Rep2017;7:42339 PMCID:PMC5309735

[132] Del Re M,Crucitta S.The Detection of Androgen Receptor Splice Variant 7 in Plasma-derived Exosomal RNA Strongly Predicts Resistance to Hormonal Therapy in Metastatic Prostate Cancer Patients.Eur Urol2017;71:680-7

[133] Woo HK,Ku JY.Urine-based liquid biopsy: non-invasive and sensitive AR-V7 detection in urinary EVs from patients with prostate cancer.Lab Chip2018;19:87-97

[134] Del Re M,Pasquini G.PD-L1 mRNA expression in plasma-derived exosomes is associated with response to anti-PD-1 antibodies in melanoma and NSCLC.Br J Cancer2018;118:820-4 PMCID:PMC5886129

[135] Ma X,Hua D.Essential role for TrpC5-containing extracellular vesicles in breast cancer with chemotherapeutic resistance.Proc Natl Acad Sci U S A2014;111:6389-94 PMCID:PMC4035923

[136] Yang SJ,Li J.Predictive role of GSTP1-containing exosomes in chemotherapy-resistant breast cancer.Gene2017;623:5-14

[137] Shao H,Lee K.Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma.Nat Commun2015;6:6999 PMCID:PMC4430127

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

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

[140] Thakur BK,Becker A.Double-stranded DNA in exosomes: a novel biomarker in cancer detection.Cell Res 2014;24:766-9 PMCID:PMC4042169

[141] 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

[142] Kahlert C.Liquid Biopsy: Is There an Advantage to Analyzing Circulating Exosomal DNA Compared to cfDNA or Are They the Same?.Cancer Res2019;79:2462-5

[143] Allenson K,San Lucas FA.High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients.Ann Oncol2017;28:741-7 PMCID:PMC5834026

[144] Bernard V,San Lucas FA.Circulating Nucleic Acids Are Associated With Outcomes of Patients With Pancreatic Cancer.Gastroenterology2019;156:108-118.e4 PMCID:PMC6434712

[145] Castellanos-Rizaldos E,Tadigotla V.Exosome-Based Detection of.EGFR24:2944-50

[146] Krug AK,Karlovich C.Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma.Ann Oncol2018;29:2143

[147] Lee DH,Park S.Urinary Exosomal and cell-free DNA Detects Somatic Mutation and Copy Number Alteration in Urothelial Carcinoma of Bladder.Sci Rep2018;8:14707 PMCID:PMC6168539

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

[149] Fischer S,Speiseder T.Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles.PLoS One2016;11:e0163665 PMCID:PMC5042424

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

[151] Mandel P.Les Acides Nucleiques Du Plasma Sanguin Chez l’ Homme.C R Seances Soc Biol Fil1948;142:241-3. (in French)

[152] Schwarzenbach H,Calin G.Data Normalization Strategies for MicroRNA Quantification.Clin Chem2015;61:1333-42 PMCID:PMC4890630

[153] Vandesompele J,Pattyn F.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.Genome Biol2002;3:RESEARCH0034 PMCID:PMC126239

[154] Mathivanan S.ExoCarta: A compendium of exosomal proteins and RNA.Proteomics2009;9:4997-5000

[155] Nguyen HQ,Kim Y.Platelet Factor 4 as a Novel Exosome Marker in MALDI-MS Analysis of Exosomes from Human Serum.Anal Chem2019;91:13297-305

[156] Lu Q,Allison R.Identification of extracellular delta-catenin accumulation for prostate cancer detection.Prostate2009;69:411-8 PMCID:PMC2633034

[157] Kharaziha P,Rutishauser D.Molecular profiling of prostate cancer derived exosomes may reveal a predictive signature for response to docetaxel.Oncotarget2015;6:21740-54 PMCID:PMC4673300

[158] Biggs CN,Al-Zahrani AA.Prostate extracellular vesicles in patient plasma as a liquid biopsy platform for prostate cancer using nanoscale flow cytometry.Oncotarget2016;7:8839-49 PMCID:PMC4891008

[159] Smalley DM,Nelson K.Isolation and identification of potential urinary microparticle biomarkers of bladder cancer.J Proteome Res2008;7:2088-96

[160] Chen CL,Tang P.Comparative and targeted proteomic analyses of urinary microparticles from bladder cancer and hernia patients.J Proteome Res2012;11:5611-29

[161] Lin SY,Wu HC.Proteome Profiling of Urinary Exosomes Identifies Alpha 1-Antitrypsin and H2B1K as Diagnostic and Prognostic Biomarkers for Urothelial Carcinoma.Sci Rep2016;6:34446 PMCID:PMC5043375

[162] Silvers CR,Messing EM,Lee YF.Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer.Oncotarget2017;8:91199-208 PMCID:PMC5710916

[163] Silvers CR,Wu CH,Messing EM.Identification of extracellular vesicle-borne periostin as a feature of muscle-invasive bladder cancer.Oncotarget2016;7:23335-45 PMCID:PMC5029630

[164] Beckham CJ,Yin PN.Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression.J Urol2014;192:583-92

[165] Welton JL,Giles PJ.Proteomics analysis of bladder cancer exosomes.Mol Cell Proteomics2010;9:1324-38 PMCID:PMC2877990

[166] Raimondo F,Corbetta S.Differential protein profiling of renal cell carcinoma urinary exosomes.Mol Biosyst2013;9:1220-33

[167] Wang X,Bu J.Exosomal protein CD82 as a diagnostic biomarker for precision medicine for breast cancer.Mol Carcinog2019;58:674-85

[168] Rupp AK,Keller S.Loss of EpCAM expression in breast cancer derived serum exosomes: role of proteolytic cleavage.Gynecol Oncol2011;122:437-46

[169] Wang T,Lu TX.Increasing circulating exosomes-carrying TRPC5 predicts chemoresistance in metastatic breast cancer patients.Cancer Sci2017;108:448-54 PMCID:PMC5378269

[170] Szajnik M,Lach M.Exosomes in Plasma of Patients with Ovarian Carcinoma: Potential Biomarkers of Tumor Progression and Response to Therapy.Gynecol Obstet (Sunnyvale)2013;Suppl 4:3 PMCID:PMC3899646

[171] Li J,Tsai-Turton M,Roden RB.Claudin-containing exosomes in the peripheral circulation of women with ovarian cancer.BMC Cancer2009;9:244 PMCID:PMC2719664

[172] Tang MKS,Ip PP.Soluble E-cadherin promotes tumor angiogenesis and localizes to exosome surface.Nat Commun2018;9:2270 PMCID:PMC5995921

[173] Graves LE,Navari JR,Stack MS.Proinvasive properties of ovarian cancer ascites-derived membrane vesicles.Cancer Res2004;64:7045-9

[174] Keller S,Marmé F.Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes.Cancer Lett2009;278:73-81

[175] Frampton AE,López-Jiménez E.Glypican-1 is enriched in circulating-exosomes in pancreatic cancer and correlates with tumor burden.Oncotarget2018;9:19006-13 PMCID:PMC5922373

[176] Kimura H,Harada T.CKAP4, a DKK1 Receptor, Is a Biomarker in Exosomes Derived from Pancreatic Cancer and a Molecular Target for Therapy.Clin Cancer Res2019;25:1936-47

[177] Costa-Silva B,Ocean AJ.Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.Nat Cell Biol2015;17:816-26 PMCID:PMC5769922

[178] Yokoyama S,Yamaguchi S.Clinical implications of carcinoembryonic antigen distribution in serum exosomal fraction-Measurement by ELISA.PLoS One2017;12:e0183337 PMCID:PMC5560664

[179] Choi DS,Jang SC.Proteomic analysis of microvesicles derived from human colorectal cancer ascites.Proteomics2011;11:2745-51

[180] Baran J,Weglarczyk K.Circulating tumour-derived microvesicles in plasma of gastric cancer patients.Cancer Immunol Immunother2010;59:841-50

[181] Yamashita T,Kanasaki S.Epidermal growth factor receptor localized to exosome membranes as a possible biomarker for lung cancer diagnosis.Pharmazie 2013;68;969-73

[182] Niu L,Wang N,Song X.Tumor-derived exosomal proteins as diagnostic biomarkers in non-small cell lung cancer.Cancer Sci2019;110:433-42 PMCID:PMC6317937

[183] Sandfeld-Paulsen B,Bæk R.Exosomal proteins as prognostic biomarkers in non-small cell lung cancer.Mol Oncol2016;10:1595-602 PMCID:PMC5423137

[184] Li Y,Qiu F.Proteomic identification of exosomal LRG1: a potential urinary biomarker for detecting NSCLC.Electrophoresis2011;32:1976-83

[185] Logozzi M,Lugini L.High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients.PLoS One2009;4:e5219 PMCID:PMC2667632

[186] Peinado H,Lavotshkin S.Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.Nat Med2012;18:883-91 PMCID:PMC3645291

[187] Bryant RJ,Catto JW.Changes in circulating microRNA levels associated with prostate cancer.Br J Cancer2012;106:768-74 PMCID:PMC3322952

[188] Xu Y,An T,Huang Y.MiR-145 detection in urinary extracellular vesicles increase diagnostic efficiency of prostate cancer based on hydrostatic filtration dialysis method.Prostate2017;77:1167-75

[189] Koppers-Lalic D,de Menezes R.Noninvasive prostate cancer detection by measuring miRNA variants (isomiRs) in urine extracellular vesicles.Oncotarget2016;7:22566-78 PMCID:PMC5008382

[190] Wani S,Mavuduru RS,Bhatia A.Urinary-exosomal miR-2909: A novel pathognomonic trait of prostate cancer severity.J Biotechnol2017;259:135-9

[191] Rodríguez M,Hessvik NP.Identification of non-invasive miRNAs biomarkers for prostate cancer by deep sequencing analysis of urinary exosomes.Mol Cancer2017;16:1-6 PMCID:PMC5629793

[192] Endzeliņš E,Melne V.Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients.BMC Cancer2017;17:730 PMCID:PMC5679326

[193] Li Z,Wang J.Exosomal microRNA-141 is upregulated in the serum of prostate cancer patients.Onco Targets Ther2016;9:139-48 PMCID:PMC4706124

[194] Hessvik NP,Llorente A.Exosomal miRNAs as Biomarkers for Prostate Cancer.Front Genet2013;4:36 PMCID:PMC3604630

[195] Moltzahn F,Baehner L.Microfluidic-based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in the sera of prostate cancer patients.Cancer Res2011;71:550-60 PMCID:PMC3022112

[196] Lodes MJ,Suciu D,Kumar A.Detection of cancer with serum miRNAs on an oligonucleotide microarray.PLoS One2009;4:e6229 PMCID:PMC2704963

[197] Bhagirath D,Bucay N.microRNA-1246 Is an Exosomal Biomarker for Aggressive Prostate Cancer.Cancer Res2018;78:1833-44 PMCID:PMC5890910

[198] Wang YH,Wang BC.Tumor-Derived Exosomal Long Noncoding RNAs as Promising Diagnostic Biomarkers for Prostate Cancer.Cell Physiol Biochem2018;46:532-45

[199] De Long J,Humphrey J.A non-invasive miRNA based assay to detect bladder cancer in cell-free urine.Am J Transl Res2015;7:2500-9 PMCID:PMC4697726

[200] Armstrong DA,Seigne JD,Marsit CJ.MicroRNA molecular profiling from matched tumor and bio-fluids in bladder cancer.Mol Cancer2015;14:194 PMCID:PMC4650939

[201] Andreu Z,Redruello A.Extracellular vesicles as a source for non-invasive biomarkers in bladder cancer progression.Eur J Pharm Sci2017;98:70-9

[202] Baumgart S,Edelmann P.MP78-05 INVASION-ASSOCIATED MIRNAS S AS POSSIBLE DIAGNOSTIC BIOMARKERS OF MUSCLE INVASIVE BLADDER CANCER IN TUMOR TISSUES AND URINARY EXOSOMES.J Urol2018;199

[203] Berrondo C,Kucherov V.Expression of the Long Non-Coding RNA HOTAIR Correlates with Disease Progression in Bladder Cancer and Is Contained in Bladder Cancer Patient Urinary Exosomes.PLoS One2016;11:e0147236 PMCID:PMC4723257

[204] Zhang S,Wang L.Evaluation of serum exosomal LncRNA-based biomarker panel for diagnosis and recurrence prediction of bladder cancer.J Cell Mol Med2019;23:1396-405 PMCID:PMC6349164

[205] Xue M,Xiang A.Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1.Mol Cancer2017;16:143 PMCID:PMC5574139

[206] Zhang W,Su Y.MicroRNAs in Serum Exosomes as Potential Biomarkers in Clear-cell Renal Cell Carcinoma.Eur Urol Focus2018;4:412-9

[207] Wang X,Chen C.Serum exosomal miR-210 as a potential biomarker for clear cell renal cell carcinoma.J Cell Biochem2018:1492-502

[208] Butz H,Ding Q.Exosomal MicroRNAs Are Diagnostic Biomarkers and Can Mediate Cell-Cell Communication in Renal Cell Carcinoma.Eur Urol Focus2016;2:210-8

[209] Fujii N,Ueno K.Extracellular MiR-224 as a prognostic marker for clear cell renal cell carcinoma.Oncotarget 2017;8:109877-88 PMCID:PMC5746350

[210] Du M,Tian Y.Plasma exosomal miRNAs-based prognosis in metastatic kidney cancer.Oncotarget2017;8:63703-14 PMCID:PMC5609954

[211] De Palma G,Curci C.The Three-Gene Signature in Urinary Extracellular Vesicles from Patients with Clear Cell Renal Cell Carcinoma.J Cancer2016;7:1960-7 PMCID:PMC5118659

[212] Hannafon BN,Calloway CL.Plasma exosome microRNAs are indicative of breast cancer.Breast Cancer Res2016;18:90 PMCID:PMC5016889

[213] Zhou W,Min Y.Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis.Cancer Cell2014;25:501-15 PMCID:PMC4016197

[214] Rodríguez-Martínez A,Ortega FG.Exosomal miRNA profile as complementary tool in the diagnostic and prediction of treatment response in localized breast cancer under neoadjuvant chemotherapy.Breast Cancer Res2019;21:21 PMCID:PMC6366103

[215] Santos JC,Sarian LO,Ribeiro ML.Exosome-mediated breast cancer chemoresistance via miR-155 transfer.Sci Rep2018;8:829 PMCID:PMC5770414

[216] Gregory PA,Paterson EL.The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1.Nat Cell Biol2008;10:593-601

[217] Pigati L,Iyengar R.Selective release of microRNA species from normal and malignant mammary epithelial cells.PLoS One2010;5:e13515 PMCID:PMC2958125

[218] Kanlikilicer P,Denizli M.Exosomal miRNA confers chemo resistance via targeting Cav1/p-gp/M2-type macrophage axis in ovarian cancer.EBioMedicine2018;38:100-12 PMCID:PMC6306310

[219] Que R,Chen J.Analysis of serum exosomal microRNAs and clinicopathologic features of patients with pancreatic adenocarcinoma.World J Surg Oncol2013;11:219 PMCID:PMC3766671

[220] Lau C,Chia D.Role of pancreatic cancer-derived exosomes in salivary biomarker development.J Biol Chem2013;288:26888-97 PMCID:PMC3772238

[221] Tian XP,Jin XH.Acidic Microenvironment Up-Regulates Exosomal miR-21 and miR-10b in Early-Stage Hepatocellular Carcinoma to Promote Cancer Cell Proliferation and Metastasis.Theranostics2019;9:1965-79 PMCID:PMC6485281

[222] Xie M,Jing X.Exosomal circSHKBP1 promotes gastric cancer progression via regulating the miR-582-3p/HUR/VEGF axis and suppressing HSP90 degradation.Mol Cancer2020;19:112 PMCID:PMC7322843

[223] Liu C,Shen J.Serum exosomal miR-4772-3p is a predictor of tumor recurrence in stage II and III colon cancer.Oncotarget2016;7:76250-60 PMCID:PMC5342811

[224] Bjørnetrø T,Meltzer S.An experimental strategy unveiling exosomal microRNAs 486-5p, 181a-5p and 30d-5p from hypoxic tumour cells as circulating indicators of high-risk rectal cancer.J Extracell Vesicles2019;8:1567219 PMCID:PMC6352936

[225] Cazzoli R,Di Nicola M.microRNAs derived from circulating exosomes as noninvasive biomarkers for screening and diagnosing lung cancer.J Thorac Oncol2013;8:1156-62 PMCID:PMC4123222

[226] Silva J,Zaballos Á.Vesicle-related microRNAs in plasma of nonsmall cell lung cancer patients and correlation with survival.Eur Respir J2011;37:617-23

[227] Aushev VN,Laktionov KK.Comparisons of microRNA patterns in plasma before and after tumor removal reveal new biomarkers of lung squamous cell carcinoma.PLoS One2013;8:e78649 PMCID:PMC3793941

[228] Wei F,Zhou T.Exosomes derived from gemcitabine-resistant cells transfer malignant phenotypic traits via delivery of miRNA-222-3p.Mol Cancer2017;16:132 PMCID:PMC5526308

[229] Yuwen DL,Liu J,Shu YQ.MiR-146a-5p level in serum exosomes predicts therapeutic effect of cisplatin in non-small cell lung cancer.Eur Rev Med Pharmacol Sci2017;21:2650-8

[230] Shi L,Yang J,Zhang S.MiR-21 protected human glioblastoma U87MG cells from chemotherapeutic drug temozolomide induced apoptosis by decreasing Bax/Bcl-2 ratio and caspase-3 activity.Brain Res2010;1352:255-64

[231] Akers JC,Kim R.MiR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF): a platform for glioblastoma biomarker development.PLoS One2013;8:e78115 PMCID:PMC3804457

[232] Manterola L,Gállego Pérez-Larraya J.A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool.Neuro Oncol2014;16:520-7 PMCID:PMC3956347