Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

At a glance: The STEP trials

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.
ADVANCED SEARCH
Log in
Top
Journal of Translational Medicine
Published in: Journal of Translational Medicine 1/2018

Open Access 01-12-2018 | Research

Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy

Authors: Bijaya Malla, Daniel M. Aebersold, Alan Dal Pra

Published in: Journal of Translational Medicine | Issue 1/2018

Login to get access

Abstract

Background

Circulating exosomes from prostate cancer (PCa) patients undergoing radiotherapy are attractive candidate biomarkers for monitoring treatment response. Multiple workflows for isolation and content characterization of exosomes in biofluids have been attempted. We report a protocol to isolate and characterize exosomal miRNAs content and assess radiation-induced changes.

Methods

In this pilot study, we performed targeted exosomal miRNA profiling of 25 serum samples obtained from PCa patients with intermediate- and high-risk disease treated with curative radiotherapy (RT), and controls. Post-treatment blood samples were collected at least 28 days after radiation therapy as a paired follow-up sample. The complete workflow consisted of two phases: I) filtration and polyethylene glycol salt precipitation phase which enriched particles below 200 nm in size followed by characterization using electron microscopy, and II) flow cytometry. Finally, miRNA expression analysis between untreated and treated patient samples was performed using RNA extraction kit, and qRT-PCR.

Results

In our preliminary data, 1 ml of serum from PCa patients showed higher exosomal concentration (3.68E+10) compared to controls (6.07E+08). The overall expression of exosomes after RT was found to be higher compared to untreated samples; the median value changed from 3.68E+10 to 5.40E+10; p = 0.52. Using electron microscopy, we were able to visualize cup-shaped vesicles with morphology and size compatible with exosomes. The bead-based flow cytometry showed positivity for exosomal tetraspanins surface markers CD63 and CD9. All five miRNAs (hsa-let-7a-5p, hsa-miR-141-3p, hsa-miR-145-5p, hsa-miR-21-5p, hsa-miR-99b-5p) have been identified in exosomes. Despite overall changes in hsa-let-7a-5p expression after radiation, the difference was significant only in the high-risk group (p = 0.037). In addition, the radiation response to hsa-miR-21-5p was elevated in the high-risk group compared to the intermediate group (p = 0.036).

Conclusions

Herewith, we demonstrated a protocol for isolation of serum exosomes and exosomal miRNA amplification. The recovery of exosomal miRNAs and their differential expression after radiation treatment suggests promising biomarker potential that requires further investigation in larger patient cohorts.
Appendix
Available only for authorised users
Literature
1.
2.
Murthy V, Rishi A, Gupta S, Kannan S, Mahantshetty U, Tongaonkar H, Bakshi G, Prabhash K, Bhanushali P, Shinde B, et al. Clinical impact of prostate specific antigen (PSA) inter-assay variability on management of prostate cancer. Clin Biochem. 2016;49:79–84.CrossRefPubMed
3.
Di Meo A, Bartlett J, Cheng Y, Pasic MD, Yousef GM. Liquid biopsy: a step forward towards precision medicine in urologic malignancies. Mol Cancer. 2017;16:80.CrossRefPubMedPubMedCentral
4.
Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles: artefacts no more. Trends Cell Biol. 2009;19:43–51.CrossRefPubMed
5.
Malla B, Zaugg K, Vassella E, Aebersold DM, Dal Pra A. Exosomes and exosomal microRNAs in prostate cancer radiation therapy. Int J Radiat Oncol Biol Phys. 2017;98:982–95.CrossRefPubMed
6.
Endzelins E, Berger A, Melne V, Bajo-Santos C, Sobolevska K, Abols A, Rodriguez M, Santare D, Rudnickiha A, Lietuvietis V, et al. Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients. BMC Cancer. 2017;17:730.CrossRefPubMedPubMedCentral
7.
Fabris L, Ceder Y, Chinnaiyan AM, Jenster GW, Sorensen KD, Tomlins S, Visakorpi T, Calin GA. The potential of microRNAs as prostate cancer biomarkers. Eur Urol. 2016;70:312–22.CrossRefPubMedPubMedCentral
8.
9.
Gandellini P, Rancati T, Valdagni R, Zaffaroni N. miRNAs in tumor radiation response: bystanders or participants? Trends Mol Med. 2014;20:529–39.CrossRefPubMed
10.
Hatano K, Kumar B, Zhang Y, Coulter JB, Hedayati M, Mears B, Ni X, Kudrolli TA, Chowdhury WH, Rodriguez R, et al. A functional screen identifies miRNAs that inhibit DNA repair and sensitize prostate cancer cells to ionizing radiation. Nucleic Acids Res. 2015;43:4075–86.CrossRefPubMedPubMedCentral
11.
Al-Mayah AH, Irons SL, Pink RC, Carter DR, Kadhim MA. Possible role of exosomes containing RNA in mediating non targeted effect of ionizing radiation. Radiat Res. 2012;177:539–45.CrossRefPubMed
12.
Suh SO, Chen Y, Zaman MS, Hirata H, Yamamura S, Shahryari V, Liu J, Tabatabai ZL, Kakar S, Deng G, et al. MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer. Carcinogenesis. 2011;32:772–8.CrossRefPubMedPubMedCentral
13.
Fuse M, Nohata N, Kojima S, Sakamoto S, Chiyomaru T, Kawakami K, Enokida H, Nakagawa M, Naya Y, Ichikawa T, Seki N. Restoration of miR-145 expression suppresses cell proliferation, migration and invasion in prostate cancer by targeting FSCN1. Int J Oncol. 2011;38:1093–101.PubMed
14.
Dal Pra A, Locke JA, Borst G, Supiot S, Bristow RG. Mechanistic insights into molecular targeting and combined modality therapy for aggressive, localized prostate cancer. Front Oncol. 2016;6:24.
15.
Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014;3:26913.CrossRefPubMed
16.
Witwer KW, Buzas EI, Bemis LT, Bora A, Lasser C, Lotvall J, Nolte-’t Hoen EN, Piper MG, Sivaraman S, Skog J, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013;2:20360.CrossRef
17.
Mohler J, Bahnson RR, Boston B, Busby JE, D’Amico A, Eastham JA, Enke CA, George D, Horwitz EM, Huben RP, et al. NCCN clinical practice guidelines in oncology: prostate cancer. J Natl Compr Cancer Netw. 2010;8:162–200.CrossRef
18.
19.
Vestad B, Llorente A, Neurauter A, Phuyal S, Kierulf B, Kierulf P, Skotland T, Sandvig K, Haug KBF, Ovstebo R. Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. J Extracell Vesicles. 2017;6:1344087.CrossRefPubMedPubMedCentral
20.
Lasser C, Eldh M, Lotvall J. Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study. J Vis Exp. 2012;59:e3037.
21.
van der Vlist EJ, Nolte-’t Hoen EN, Stoorvogel W, Arkesteijn GJ, Wauben MH. Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry. Nat Protoc. 2012;7:1311–26.CrossRefPubMed
22.
Kroh EM, Parkin RK, Mitchell PS, Tewari M. Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods. 2010;50:298–301.CrossRefPubMedPubMedCentral
23.
Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles. 2014;3:24858.CrossRef
24.
25.
Li M, Rai AJ, DeCastro GJ, Zeringer E, Barta T, Magdaleno S, Setterquist R, Vlassov AV. An optimized procedure for exosome isolation and analysis using serum samples: application to cancer biomarker discovery. Methods. 2015;87:26–30.CrossRefPubMed
26.
Zeringer E, Li M, Barta T, Schageman J, Pedersen KW, Neurauter A, Magdaleno S, Setterquist R, Vlassov AV. Methods for the extraction and RNA profiling of exosomes. World J Methodol. 2013;3:11–8.CrossRefPubMedPubMedCentral
27.
Dal Pra A, Souhami L. Prostate cancer radiation therapy: a physician’s perspective. Phys Med. 2016;32:438–45.CrossRef
28.
Helwa I, Cai J, Drewry MD, Zimmerman A, Dinkins MB, Khaled ML, Seremwe M, Dismuke WM, Bieberich E, Stamer WD, et al. A comparative study of serum exosome isolation using differential ultracentrifugation and three commercial reagents. PLoS ONE. 2017;12:e0170628.CrossRefPubMedPubMedCentral
29.
Ouyang Y, Li D, Pater JL, Levine M. The importance of temporal effects in evaluating the prognostic impact of joint ERPR expression in premenopausal women with node-positive breast cancer. Breast Cancer Res Treat. 2005;92:115–23.CrossRefPubMed
30.
Tu H, Sun L, Dong X, Gong Y, Xu Q, Jing J, Long Q, Flanders WD, Bostick RM, Yuan Y. Temporal changes in serum biomarkers and risk for progression of gastric precancerous lesions: a longitudinal study. Int J Cancer. 2015;136:425–34.CrossRefPubMed
31.
Lewis GD, Metcalf TG. Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples. Appl Environ Microbiol. 1988;54:1983–8.PubMedPubMedCentral
32.
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Moller A. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles. 2015;4:27031.CrossRefPubMed
33.
Niu Z, Pang RTK, Liu W, Li Q, Cheng R, Yeung WSB. Polymer-based precipitation preserves biological activities of extracellular vesicles from an endometrial cell line. PLoS ONE. 2017;12:e0186534.CrossRefPubMedPubMedCentral
34.
Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods. 2012;56:293–304.CrossRefPubMed
35.
Tang YT, Huang YY, Zheng L, Qin SH, Xu XP, An TX, Xu Y, Wu YS, Hu XM, Ping BH, Wang Q. Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum. Int J Mol Med. 2017;40:834–44.CrossRefPubMedPubMedCentral
36.
Gerritzen MJH, Martens DE, Wijffels RH, Stork M. High throughput nanoparticle tracking analysis for monitoring outer membrane vesicle production. J Extracell Vesicles. 2017;6:1333883.CrossRefPubMedPubMedCentral
37.
Tong M, Brown OS, Stone PR, Cree LM, Chamley LW. Flow speed alters the apparent size and concentration of particles measured using NanoSight nanoparticle tracking analysis. Placenta. 2016;38:29–32.CrossRefPubMed
38.
Gould SJ, Raposo G. As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles. 2013;2:20389.CrossRef
39.
Mizutani K, Terazawa R, Kameyama K, Kato T, Horie K, Tsuchiya T, Seike K, Ehara H, Fujita Y, Kawakami K, et al. Isolation of prostate cancer-related exosomes. Anticancer Res. 2014;34:3419–23.PubMed
40.
Yamashita T, Takahashi Y, Nishikawa M, Takakura Y. Effect of exosome isolation methods on physicochemical properties of exosomes and clearance of exosomes from the blood circulation. Eur J Pharm Biopharm. 2016;98:1–8.CrossRefPubMed
41.
Gardiner C, Ferreira YJ, Dragovic RA, Redman CW, Sargent IL. Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J Extracell Vesicles. 2013;2:19671.CrossRef
42.
Willms E, Johansson HJ, Mager I, Lee Y, Blomberg KE, Sadik M, Alaarg A, Smith CI, Lehtio J, El Andaloussi S, et al. Cells release subpopulations of exosomes with distinct molecular and biological properties. Sci Rep. 2016;6:22519.CrossRefPubMedPubMedCentral
43.
Kowal J, Arras G, Colombo M, Jouve M, Morath JP, Primdal-Bengtson B, Dingli F, Loew D, Tkach M, Thery C. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci USA. 2016;113:E968–77.CrossRefPubMed
44.
Pospichalova V, Svoboda J, Dave Z, Kotrbova A, Kaiser K, Klemova D, Ilkovics L, Hampl A, Crha I, Jandakova E, et al. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer. J Extracell Vesicles. 2015;4:25530.CrossRefPubMed
45.
Nolte-’t Hoen EN, van der Vlist EJ, Aalberts M, Mertens HC, Bosch BJ, Bartelink W, Mastrobattista E, van Gaal EV, Stoorvogel W, Arkesteijn GJ, Wauben MH. Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles. Nanomedicine. 2012;8:712–20.CrossRefPubMed
46.
Channavajjhala SK, Rossato M, Morandini F, Castagna A, Pizzolo F, Bazzoni F, Olivieri O. Optimizing the purification and analysis of miRNAs from urinary exosomes. Clin Chem Lab Med. 2014;52:345–54.CrossRefPubMed
47.
Garcia-Elias A, Alloza L, Puigdecanet E, Nonell L, Tajes M, Curado J, Enjuanes C, Diaz O, Bruguera J, Marti-Almor J, et al. Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs. Sci Rep. 2017;7:7725.CrossRefPubMedPubMedCentral
48.
Baudrimont A, Voegeli S, Viloria EC, Stritt F, Lenon M, Wada T, Jaquet V, Becskei A. Multiplexed gene control reveals rapid mRNA turnover. Sci Adv. 2017;3:e1700006.CrossRefPubMedPubMedCentral
49.
Tiberio P, Callari M, Angeloni V, Daidone MG, Appierto V. Challenges in using circulating miRNAs as cancer biomarkers. Biomed Res Int. 2015;2015:731479.CrossRefPubMedPubMedCentral
50.
Zhao L, Yu J, Wang J, Li H, Che J, Cao B. Isolation and Identification of miRNAs in exosomes derived from serum of colon cancer patients. J Cancer. 2017;8:1145–52.CrossRefPubMedPubMedCentral
51.
Vigneron N, Meryet-Figuiere M, Guttin A, Issartel JP, Lambert B, Briand M, Louis MH, Vernon M, Lebailly P, Lecluse Y, et al. Towards a new standardized method for circulating miRNAs profiling in clinical studies: interest of the exogenous normalization to improve miRNA signature accuracy. Mol Oncol. 2016;10:981–92.CrossRefPubMedPubMedCentral
52.
Blondal T, Jensby Nielsen S, Baker A, Andreasen D, Mouritzen P, Wrang Teilum M, Dahlsveen IK. Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods. 2013;59:S1–6.CrossRefPubMed
53.
Zeka F, Mestdagh P, Vandesompele J. RT-qPCR-based quantification of small non-coding RNAs. Methods Mol Biol. 2015;1296:85–102.CrossRefPubMed
54.
55.
Torres A, Torres K, Wdowiak P, Paszkowski T, Maciejewski R. Selection and validation of endogenous controls for microRNA expression studies in endometrioid endometrial cancer tissues. Gynecol Oncol. 2013;130:588–94.CrossRefPubMed
56.
Gee HE, Buffa FM, Camps C, Ramachandran A, Leek R, Taylor M, Patil M, Sheldon H, Betts G, Homer J, et al. The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis. Br J Cancer. 2011;104:1168–77.CrossRefPubMedPubMedCentral
57.
Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F, Speleman F, Vandesompele J. A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol. 2009;10:R64.CrossRefPubMedPubMedCentral
58.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 2001;25:402–8.CrossRefPubMed
59.
Zedan AH, Blavnsfeldt SG, Hansen TF, Nielsen BS, Marcussen N, Pleckaitis M, Osther PJS, Sorensen FB. Heterogeneity of miRNA expression in localized prostate cancer with clinicopathological correlations. PLoS ONE. 2017;12:e0179113.CrossRefPubMedPubMedCentral
60.
Cochetti G, Poli G, Guelfi G, Boni A, Egidi MG, Mearini E. Different levels of serum microRNAs in prostate cancer and benign prostatic hyperplasia: evaluation of potential diagnostic and prognostic role. Onco Targets Ther. 2016;9:7545–53.CrossRefPubMedPubMedCentral
61.
62.
Simone NL, Soule BP, Ly D, Saleh AD, Savage JE, Degraff W, Cook J, Harris CC, Gius D, Mitchell JB. Ionizing radiation-induced oxidative stress alters miRNA expression. PLoS ONE. 2009;4:e6377.CrossRefPubMedPubMedCentral
63.
McDonald MK, Capasso KE, Ajit SK. Purification and microRNA profiling of exosomes derived from blood and culture media. J Vis Exp. 2013;76:e50294.
64.
Nikolic I, Elsworth B, Dodson E, Wu SZ, Gould CM, Mestdagh P, Marshall GM, Horvath LG, Simpson KJ, Swarbrick A. Discovering cancer vulnerabilities using high-throughput micro-RNA screening. Nucleic Acids Res. 2017;45:12657–70.CrossRefPubMedPubMedCentral
65.
Pernot E, Hall J, Baatout S, Benotmane MA, Blanchardon E, Bouffler S, El Saghire H, Gomolka M, Guertler A, Harms-Ringdahl M, et al. Ionizing radiation biomarkers for potential use in epidemiological studies. Mutat Res. 2012;751:258–86.CrossRefPubMed
66.
Boutros PC, Fraser M, Harding NJ, de Borja R, Trudel D, Lalonde E, Meng A, Hennings-Yeomans PH, McPherson A, Sabelnykova VY, et al. Spatial genomic heterogeneity within localized, multifocal prostate cancer. Nat Genet. 2015;47:736–45.CrossRefPubMed
67.
Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci USA. 2011;108:5003–8.CrossRefPubMed
Metadata
Title
Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy
Authors
Bijaya Malla
Daniel M. Aebersold
Alan Dal Pra
Publication date
01-12-2018
Publisher
BioMed Central
Published in
Journal of Translational Medicine / Issue 1/2018
Electronic ISSN: 1479-5876
DOI
https://doi.org/10.1186/s12967-018-1592-6

Other articles of this Issue 1/2018

Journal of Translational Medicine 1/2018 Go to the issue

Research

The dimensionality of fatigue in Parkinson’s disease

Research

Deregulated microRNA and mRNA expression profiles in the peripheral blood of patients with Marfan syndrome

Correction

Correction to: First results on survival from a large Phase 3 clinical trial of an autologous dendritic cell vaccine in newly diagnosed glioblastoma

Research

Evaluation of the prognostic value of paraoxonase 1 in the recurrence and metastasis of hepatocellular carcinoma and establishment of a liver-specific predictive model of survival

Research

Knockdown of KLF5 suppresses hypoxia-induced resistance to cisplatin in NSCLC cells by regulating HIF-1α-dependent glycolysis through inactivation of the PI3K/Akt/mTOR pathway

Research

Real-world use of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers/β-blocks in Chinese patients before acute myocardial infarction occurs: patient characteristics and hospital follow-up
Obesity Clinical Trial Summary

At a glance: The STEP trials

Read more

A round-up of the STEP phase 3 clinical trials evaluating semaglutide for weight loss in people with overweight or obesity.

Developed by: Springer Medicine

Highlights from the ACC 2024 Congress

Year in Review: Pediatric cardiology

Pediatric Cardiology Video

Watch Dr. Anne Marie Valente present the last year's highlights in pediatric and congenital heart disease in the official ACC.24 Year in Review session.

Year in Review: Pulmonary vascular disease

Cardiopulmonary Comorbidity Video

The last year's highlights in pulmonary vascular disease are presented by Dr. Jane Leopold in this official video from ACC.24.

Year in Review: Valvular heart disease

Valvular Heart Disease Video

Watch Prof. William Zoghbi present the last year's highlights in valvular heart disease from the official ACC.24 Year in Review session.

Year in Review: Heart failure and cardiomyopathies

Heart Failure Video

Watch this official video from ACC.24. Dr. Biykem Bozkurt discuss last year's major advances in heart failure and cardiomyopathies.

ACC 2024 Congress Coverage

Heart Failure Video

Year in Review: Heart failure and cardiomyopathies

Watch now

Watch this official video from ACC.24. Dr. Biykem Bozkurt discuss last year's major advances in heart failure and cardiomyopathies.

Semaglutide benefits people with type 2 diabetes and obesity-related heart failure

16-04-2024 Type 2 Diabetes News

Incentivised to exercise

11-04-2024 Lifestyle Modifications News

New intervention for STEMI-related cardiogenic shock

10-04-2024 Myocardial Infarction News

» View more highlights on the ACC 2024 congress hub page

Image Credits