Urinary Extracellular Vesicles as Potential Biomarkers for Urologic Cancers: An Overview of Current Methods and Advances
Abstract
:Simple Summary
Abstract
1. Overview
2. Urinary Extracellular Vesicles
2.1. uEV Isolation Methods
2.1.1. Density-Based Methods
- Ultracentrifugation
- Density Gradient Centrifugation
2.1.2. Size-Based Techniques
- Ultrafiltration (UF)
- Size Exclusion Chromatography (SEC)
- Polymer Precipitation
2.1.3. Microfluidic-Based Strategies
2.2. Characterization Methods
3. uEVs as Biomarkers in The Three Urologic Cancers: miRNA and Protein Markers
3.1. Prostate Cancer
3.1.1. Protein Biomarkers in PCa
3.1.2. miRNA Biomarkers in PCa
3.2. Bladder Cancer
3.2.1. Protein Biomarkers in BlCa
3.2.2. miRNA Biomarkers in BlCa
3.3. Kidney Cancer
3.3.1. Protein Biomarkers in RCC
3.3.2. miRNA Biomarkers in RCC
4. Limitations of Urine as a Biofluid
5. Discussion
6. Concluding Remarks and Future Perspectives
Author Contributions
Funding
Conflicts of Interest
References
- Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Cancer J. Clin. 2018, 68, 394–424. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ferlay, J.; Ervik, M.; Lam, F.; Colombet, M.; Mery, L.; Piñeros, M.; Znaor, A.; Soerjomataram, I.; Bray, F. Available online: https://gco.iarc.fr/today (accessed on 21 December 2020).
- Verma, S.; Bhavsar, A.S.; Donovan, J. MR imaging-guided prostate biopsy techniques. Magn. Reson. Imaging Clin. N. Am. 2014, 22, 135–144. [Google Scholar] [CrossRef] [PubMed]
- DeGeorge, K.C.; Holt, H.R.; Hodges, S.C. Bladder Cancer: Diagnosis and Treatment. Am. Fam. Phys. 2017, 96, 507–514. [Google Scholar]
- Blecher, G.; McDermott, K.; Challacombe, B. Renal cancer. Surg. Oxf. Int. Ed. 2019, 37, 508–512. [Google Scholar] [CrossRef]
- Dy, G.W.; Gore, J.L.; Forouzanfar, M.H.; Naghavi, M.; Fitzmaurice, C. Global Burden of Urologic Cancers, 1990–2013. Eur. Urol. 2017, 71, 437–446. [Google Scholar] [CrossRef] [PubMed]
- Constâncio, V.; Barros-Silva, D.; Jerónimo, C.; Henrique, R. Known epigenetic biomarkers for prostate cancer detection and management: Exploring the potential of blood-based liquid biopsies. Expert Rev. Mol. Diagn. 2019, 19, 367–375. [Google Scholar] [CrossRef]
- Gorin, M.A.; Verdone, J.E.; van der Toom, E.; Bivalacqua, T.J.; Allaf, M.E.; Pienta, K.J. Circulating tumour cells as biomarkers of prostate, bladder, and kidney cancer. Nat. Rev. Urol. 2017, 14, 90–97. [Google Scholar] [CrossRef]
- Truong, M.; Yang, B.; Jarrard, D.F. Toward the detection of prostate cancer in urine: A critical analysis. J. Urol. 2013, 189, 422–429. [Google Scholar] [CrossRef] [Green Version]
- Hattori, S.; Kojima, K.; Minoshima, K.; Yamaha, M.; Horie, M.; Sawamura, T.; Kikuchi, A.; Deguchi, T. Detection of bladder cancer by measuring CD44v6 expression in urine with real-time quantitative reverse transcription polymerase chain reaction. Urology 2014, 83, 1443.e9–1443.e15. [Google Scholar] [CrossRef]
- Kistler, A.D.; Serra, A.L.; Siwy, J.; Poster, D.; Krauer, F.; Torres, V.E.; Mrug, M.; Grantham, J.J.; Bae, K.T.; Bost, J.E.; et al. Urinary Proteomic Biomarkers for Diagnosis and Risk Stratification of Autosomal Dominant Polycystic Kidney Disease: A Multicentric Study. PLoS ONE 2013, 8, e53016. [Google Scholar] [CrossRef]
- Konoshenko, M.Y.; Lekchnov, E.A.; Vlassov, A.V.; Laktionov, P.P. Isolation of Extracellular Vesicles: General Methodologies and Latest Trends. Biomed Res. Int. 2018, 2018. [Google Scholar] [CrossRef]
- Pisitkun, T.; Shen, R.F.; Knepper, M.A. Identification and proteomic profiling of exosomes in human urine. Proc. Natl. Acad. Sci. USA 2004, 101, 13368–13373. [Google Scholar] [CrossRef] [Green Version]
- Gonzales, P.; Pisitkun, T.; Knepper, M.A. Urinary exosomes: Is there a future? Nephrol. Dial. Transplant. 2008, 23, 1799–1801. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Doyle, L.M.; Wang, M.Z. Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis. Cells 2019, 8, 727. [Google Scholar] [CrossRef] [Green Version]
- Théry, C.; Witwer, K.W.; Aikawa, E.; Alcaraz, M.J.; Anderson, J.D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.; Atkin-Smith, G.K.; et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 2018, 7, 1535750. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ruivo, C.F.; Adem, B.; Silva, M.; Melo, S.A. The biology of cancer exosomes: Insights and new perspectives. Cancer Res. 2017, 77, 6480–6488. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Junker, K.; Heinzelmann, J.; Beckham, C.; Ochiya, T.; Jenster, G. Extracellular Vesicles and Their Role in Urologic Malignancies. Eur. Urol. 2016, 70, 323–331. [Google Scholar] [CrossRef] [PubMed]
- Liang, L.-G.; Kong, M.-Q.; Zhou, S.; Sheng, Y.-F.; Wang, P.; Yu, T.; Inci, F.; Kuo, W.P.; Li, L.-J.; Demirci, U.; et al. An integrated double-filtration microfluidic device for isolation, enrichment and quantification of urinary extracellular vesicles for detection of bladder cancer. Sci. Rep. 2017, 7, 1–10. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Katsuda, T.; Kosaka, N.; Ochiya, T. The roles of extracellular vesicles in cancer biology: Toward the development of novel cancer biomarkers. Proteomics 2014, 14, 412–425. [Google Scholar] [CrossRef]
- Valadi, H.; Ekström, K.; Bossios, A.; Sjöstrand, M.; Lee, J.J.; Lötvall, J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 2007, 9, 654–659. [Google Scholar] [CrossRef] [Green Version]
- Antimisiaris, S.G.; Mourtas, S.; Marazioti, A. Exosomes and Exosome-Inspired Vesicles for Targeted Drug Delivery. Pharmaceutics 2018, 10, 218. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Vázquez-Ríos, A.J.; Molina-Crespo, Á.; Bouzo, B.L.; López-López, R.; Moreno-Bueno, G.; de la Fuente, M. Exosome-mimetic nanoplatforms for targeted cancer drug delivery. J. Nanobiotechnol. 2019, 17, 1–15. [Google Scholar] [CrossRef] [Green Version]
- Gerlach, J.Q.; Krüger, A.; Gallogly, S.; Hanley, S.A.; Hogan, M.C.; Ward, C.J.; Joshi, L.; Griffin, M.D. Surface Glycosylation Profiles of Urine Extracellular Vesicles. PLoS ONE 2013, 8, e74801. [Google Scholar] [CrossRef]
- Van Deun, J.; Mestdagh, P.; Agostinis, P.; Akay, Ö.; Anand, S.; Anckaert, J.; Martinez, Z.A.; Baetens, T.; Beghein, E.; Bertier, L.; et al. EV-TRACK: Transparent reporting and centralizing knowledge in extracellular vesicle research. Nat. Methods 2017, 14, 228–232. [Google Scholar] [CrossRef] [PubMed]
- Fernández-Llama, P.; Khositseth, S.; Gonzales, P.A.; Star, R.A.; Pisitkun, T.; Knepper, M.A. Tamm-Horsfall protein and urinary exosome isolation. Kidney Int. 2010, 77, 736–742. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rood, I.M.; Deegens, J.K.J.; Merchant, M.L.; Tamboer, W.P.M.; Wilkey, D.W.; Wetzels, J.F.M.; Klein, J.B. Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome. Kidney Int. 2010, 78, 810–816. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Alvarez, M.L.; Khosroheidari, M.; Kanchi Ravi, R.; Distefano, J.K. Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. Kidney Int. 2012, 82, 1024–1032. [Google Scholar] [CrossRef] [Green Version]
- Channavajjhala, S.K.; 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, 345–354. [Google Scholar] [CrossRef]
- Royo, F.; Zuñiga-Garcia, P.; Sanchez-Mosquera, P.; Egia, A.; Perez, A.; Loizaga, A.; Arceo, R.; Lacasa, I.; Rabade, A.; Arrieta, E.; et al. Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples. J. Extracell. Vesicles 2016, 5, 29497. [Google Scholar] [CrossRef]
- Wachalska, M.; Koppers-Lalic, D.; van Eijndhoven, M.; Pegtel, M.; Geldof, A.A.; Lipinska, A.D.; van Moorselaar, R.J.; Bijnsdorp, I.V. Protein Complexes in Urine Interfere with Extracellular Vesicle Biomarker Studies. J. Circ. Biomark. 2016, 5, 4. [Google Scholar] [CrossRef]
- Gheinani, A.H.; Vögeli, M.; Baumgartner, U.; Vassella, E.; Draeger, A.; Burkhard, F.C.; Monastyrskaya, K. Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery. Sci. Rep. 2018, 8, 1–17. [Google Scholar] [CrossRef] [PubMed]
- He, L.; Zhu, D.; Wang, J.; Wu, X. A highly efficient method for isolating urinary exosomes. Int. J. Mol. Med. 2019, 43, 83–90. [Google Scholar] [CrossRef] [Green Version]
- Cho, S.; Yang, H.C.; Rhee, W.J. Development and comparative analysis of human urine exosome isolation strategies. Process Biochem. 2020, 88, 197–203. [Google Scholar] [CrossRef]
- Dhondt, B.; Geeurickx, E.; Tulkens, J.; Van Deun, J.; Vergauwen, G.; Lippens, L.; Miinalainen, I.; Rappu, P.; Heino, J.; Ost, P.; et al. Unravelling the proteomic landscape of extracellular vesicles in prostate cancer by density-based fractionation of urine. J. Extracell. Vesicles 2020, 9, 1736935. [Google Scholar] [CrossRef]
- Zhou, M.; Weber, S.R.; Zhao, Y.; Chen, H.; Sundstrom, J.M. Methods for exosome isolation and characterization. In Exosomes; Elsevier Inc.: Amsterdam, The Netherlands, 2020; pp. 23–38. ISBN 9780128160534. [Google Scholar]
- Zhang, Z.; Wang, C.; Li, T.; Liu, Z.; Li, L. Comparison of ultracentrifugation and density gradient separation methods for isolating Tca8113 human tongue cancer cell line-derived exosomes. Oncol. Lett. 2014, 8, 1701–1706. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tauro, B.J.; Greening, D.W.; Mathias, R.A.; Ji, H.; Mathivanan, S.; Scott, A.M.; Simpson, R.J. 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. [Google Scholar] [CrossRef]
- Cheruvanky, A.; Zhou, H.; Pisitkun, T.; Kopp, J.B.; Knepper, M.A.; Yuen, P.S.T.; Star, R.A. Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am. J. Physiol. Ren. Physiol. 2007, 292, F1657–F1661. [Google Scholar] [CrossRef] [Green Version]
- Merchant, M.L.; Powell, D.W.; Wilkey, D.W.; Cummins, T.D.; Deegens, J.K.; Rood, I.M.; McAfee, K.J.; Fleischer, C.; Klein, E.; Klein, J.B. Microfiltration isolation of human urinary exosomes for characterization by MS. Proteom. Clin. Appl. 2010, 4, 84–96. [Google Scholar] [CrossRef] [PubMed]
- Lozano-Ramos, I.; Bancu, I.; Oliveira-Tercero, A.; Armengol, M.P.; Menezes-Neto, A.; Del Portillo, H.A.; Lauzurica-Valdemoros, R.; Borràs, F.E. Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples. J. Extracell. Vesicles 2015, 4, 27369. [Google Scholar] [CrossRef] [Green Version]
- Nawaz, M.; Camussi, G.; Valadi, H.; Nazarenko, I.; Ekström, K.; Wang, X.; Principe, S.; Shah, N.; Ashraf, N.M.; Fatima, F.; et al. The emerging role of extracellular vesicles as biomarkers for urogenital cancers. Nat. Rev. Urol. 2014, 11, 688–701. [Google Scholar] [CrossRef]
- Gholizadeh, S.; Shehata Draz, M.; Zarghooni, M.; Sanati-Nezhad, A.; Ghavami, S.; Shafiee, H.; Akbari, M. Microfluidic approaches for isolation, detection, and characterization of extracellular vesicles: Current status and future directions. Biosens. Bioelectron. 2017, 91, 588–605. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hou, R.; Li, Y.; Sui, Z.; Yuan, H.; Yang, K.; Liang, Z.; Zhang, L.; Zhang, Y. Advances in exosome isolation methods and their applications in proteomic analysis of biological samples. Anal. Bioanal. Chem. 2019, 411, 5351–5361. [Google Scholar] [CrossRef] [PubMed]
- Kanwar, S.S.; Dunlay, C.J.; Simeone, D.M.; Nagrath, S. Microfluidic device (ExoChip) for on-chip isolation, quantification and characterization of circulating exosomes. Lab Chip 2014, 14, 1891–1900. [Google Scholar] [CrossRef] [PubMed]
- Liu, F.; Vermesh, O.; Mani, V.; Ge, T.J.; Madsen, S.J.; Sabour, A.; Hsu, E.-C.; Gowrishankar, G.; Kanada, M.; Jokerst, J.V.; et al. The Exosome Total Isolation Chip. ACS Nano 2017, 11, 10712–10723. [Google Scholar] [CrossRef] [PubMed]
- Hisey, C.L.; Dorayappan, K.D.P.; Cohn, D.E.; Selvendiran, K.; Hansford, D.J. Microfluidic affinity separation chip for selective capture and release of label-free ovarian cancer exosomes. Lab Chip 2018, 18, 3144–3153. [Google Scholar] [CrossRef]
- He, M.; Crow, J.; Roth, M.; Zeng, Y.; Godwin, A.K. Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology. Lab Chip 2014, 14, 3773–3780. [Google Scholar] [CrossRef] [Green Version]
- Dorayappan, K.D.P.; Gardner, M.L.; Hisey, C.L.; Zingarelli, R.A.; Smith, B.Q.; Lightfoot, M.D.S.; Gogna, R.; Flannery, M.M.; Hays, J.; Hansford, D.J.; et al. A microfluidic chip enables isolation of exosomes and establishment of their protein profiles and associated signaling pathways in ovarian cancer. Cancer Res. 2019, 79, 3503–3513. [Google Scholar] [CrossRef] [PubMed]
- Zhang, P.; Zhou, X.; He, M.; Shang, Y.; Tetlow, A.L.; Godwin, A.K.; Zeng, Y. Ultrasensitive detection of circulating exosomes with a 3D-nanopatterned microfluidic chip. Nat. Biomed. Eng. 2019, 3, 438–451. [Google Scholar] [CrossRef]
- Woo, H.-K.; Sunkara, V.; Park, J.; Kim, T.-H.; Han, J.-R.; Kim, C.-J.; Choi, H.-I.; Kim, Y.-K.; Cho, Y.-K. Exodisc for Rapid, Size-Selective, and Efficient Isolation and Analysis of Nanoscale Extracellular Vesicles from Biological Samples. ACS Nano 2017, 11, 1360–1370. [Google Scholar] [CrossRef]
- Rupert, D.L.M.; Claudio, V.; Lässer, C.; Bally, M. Methods for the physical characterization and quantification of extracellular vesicles in biological samples. Biochim. Biophys. Acta Gen. Subj. 2017, 1861, 3164–3179. [Google Scholar] [CrossRef]
- Cizmar, P.; Yuana, Y. Detection and Characterization of Extracellular Vesicles by Transmission and Cryo-Transmission Electron Microscopy. Methods Mol. Biol. 2017, 1660, 221–232. [Google Scholar] [CrossRef]
- Yuana, Y.; Koning, R.I.; Kuil, M.E.; Rensen, P.C.N.; Koster, A.J.; Bertina, R.M.; Osanto, S. Cryo-electron microscopy of extracellular vesicles in fresh plasma. J. Extracell. Vesicles 2013, 2, 21494. [Google Scholar] [CrossRef] [PubMed]
- Dragovic, R.A.; Gardiner, C.; Brooks, A.S.; Tannetta, D.S.; Ferguson, D.J.P.; Hole, P.; Carr, B.; Redman, C.W.G.; Harris, A.L.; Dobson, P.J.; et al. Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine 2011, 7, 780–788. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- McNicholas, K.; Li, J.Y.; Michael, M.Z.; Gleadle, J.M. Albuminuria is not associated with elevated urinary vesicle concentration but can confound nanoparticle tracking analysis. Nephrology 2017, 22, 854–863. [Google Scholar] [CrossRef] [PubMed]
- Hartjes, T.A.; Mytnyk, S.; Jenster, G.W.; van Steijn, V.; van Royen, M.E. Extracellular vesicle quantification and characterization: Common methods and emerging approaches. Bioengineering 2019, 6, 7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Colombo, M.; Raposo, G.; Théry, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 2014, 30, 255–289. [Google Scholar] [CrossRef]
- Wu, Z.; Zhang, Z.; Xia, W.; Cai, J.; Li, Y.; Wu, S. Extracellular vesicles in urologic malignancies—Implementations for future cancer care. Cell Prolif. 2019, 52, e12659. [Google Scholar] [CrossRef] [Green Version]
- Lee, J.; McKinney, K.Q.; Pavlopoulos, A.J.; Niu, M.; Kang, J.W.; Oh, J.W.; Kim, K.P.; Hwang, S. Altered proteome of extracellular vesicles derived from bladder cancer patients urine. Mol. Cells 2018, 41, 179–187. [Google Scholar] [CrossRef]
- Mohler, J.L.; Armstrong, A.J.; Bahnson, R.R.; D’Amico, A.V.; Davis, B.J.; Eastham, J.A.; Enke, C.A.; Farrington, T.A.; Higano, C.S.; Horwitz, E.M.; et al. Prostate Cancer, Version 1.2016. J. Natl. Compr. Cancer Netw. 2016, 14, 19–30. [Google Scholar] [CrossRef] [Green Version]
- Parker, C.; Castro, E.; Fizazi, K.; Heidenreich, A.; Ost, P.; Procopio, G.; Tombal, B.; Gillessen, S. Prostate cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†. Ann. Oncol. 2020, 26, v69–v77. [Google Scholar] [CrossRef]
- Carlsson, S.V.; Vickers, A.J. Screening for Prostate Cancer. Med. Clin. N. Am. 2020. [Google Scholar] [CrossRef]
- Roddam, A.W.; Duffy, M.J.; Hamdy, F.C.; Ward, A.M.; Patnick, J.; Price, C.P.; Rimmer, J.; Sturgeon, C.; White, P.; Allen, N.E. Use of prostate-specific antigen (PSA) isoforms for the detection of prostate cancer in men with a PSA level of 2-10 ng/mL: Systematic review and meta-analysis. Eur. Urol. 2005, 48, 386–389. [Google Scholar] [CrossRef] [PubMed]
- Miyahira, A.K.; Sharp, A.; Ellis, L.; Jones, J.; Kaochar, S.; Larman, H.B.; Quigley, D.A.; Ye, H.; Simons, J.W.; Pienta, K.J.; et al. Prostate cancer research: The next generation; report from the 2019 Coffey-Holden Prostate Cancer Academy Meeting. Prostate 2020, 80, 113–132. [Google Scholar] [CrossRef] [PubMed]
- Overbye, A.; Skotland, T.; Koehler, C.J.; Thiede, B.; Seierstad, T.; Berge, V.; Sandvig, K.; Llorente, A. Identification of prostate cancer biomarkers in urinary exosomes. Oncotarget 2015, 6, 30357–30376. [Google Scholar] [CrossRef] [Green Version]
- Welton, J.L.; Brennan, P.; Gurney, M.; Webber, J.P.; Spary, L.K.; Carton, D.G.; Falcón-Pérez, J.M.; Walton, S.P.; Mason, M.D.; Tabi, Z.; et al. Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array. J. Extracell. Vesicles 2016, 5, 31209. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fujita, K.; Kume, H.; Matsuzaki, K.; Kawashima, A.; Ujike, T.; Nagahara, A.; Uemura, M.; Miyagawa, Y.; Tomonaga, T.; Nonomura, N. Proteomic analysis of urinary extracellular vesicles from high Gleason score prostate cancer. Sci. Rep. 2017, 7, 1–9. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sequeiros, T.; Rigau, M.; Chiva, C.; Montes, M.; Garcia-Grau, I.; Garcia, M.; Diaz, S.; Celma, A.; Bijnsdorp, I.; Campos, A.; et al. Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer. Oncotarget 2017, 8, 4960–4976. [Google Scholar] [CrossRef] [Green Version]
- Wang, L.; Skotland, T.; Berge, V.; Sandvig, K.; Llorente, A. Exosomal proteins as prostate cancer biomarkers in urine: From mass spectrometry discovery to immunoassay-based validation. Eur. J. Pharm. Sci. 2017, 98, 80–85. [Google Scholar] [CrossRef]
- Koppers-lalic, D.; Hackenberg, M.; de Menezes, R.; Misovic, B.; Wachalska, M.; Geldof, A.; Zini, N.; de Reijke, T.; Wurdinger, T.; Vis, A.; et al. Non—Invasive prostate cancer detection by measuring miRNA variants (isomiRs) in urine extracellular vesicles. Oncotarget 2016, 7, 22566. [Google Scholar] [CrossRef] [Green Version]
- Bryzgunova, O.E.; Zaripov, M.M.; Skvortsova, T.E.; Lekchnov, E.A.; Grigor’eva, A.E.; Zaporozhchenko, I.A.; Morozkin, E.S.; Ryabchikova, E.I.; Yurchenko, Y.B.; Voitsitskiy, V.E.; et al. Comparative study of extracellular vesicles from the urine of healthy individuals and prostate cancer patients. PLoS ONE 2016, 11, e0157566. [Google Scholar] [CrossRef]
- Xu, Y.; Qin, S.; An, T.; Tang, Y.; Huang, Y.; Zheng, L. MiR-145 detection in urinary extracellular vesicles increase diagnostic efficiency of prostate cancer based on hydrostatic filtration dialysis method. Prostate 2017, 77, 1167–1175. [Google Scholar] [CrossRef] [PubMed]
- Wani, S.; Kaul, D.; Mavuduru, R.S.; Kakkar, N.; Bhatia, A. Urinary-exosomal miR-2909: A novel pathognomonic trait of prostate cancer severity. J. Biotechnol. 2017, 259, 135–139. [Google Scholar] [CrossRef]
- Rodríguez, M.; Bajo-Santos, C.; Hessvik, N.P.; Lorenz, S.; Fromm, B.; Berge, V.; Sandvig, K.; Line, A.; Llorente, A. Identification of non-invasive miRNAs biomarkers for prostate cancer by deep sequencing analysis of urinary exosomes. Mol. Cancer 2017, 16, 4–9. [Google Scholar] [CrossRef] [PubMed]
- Foj, L.; Ferrer, F.; Serra, M.; Arévalo, A.; Gavagnach, M.; Giménez, N.; Filella, X. Exosomal and Non-Exosomal Urinary miRNAs in Prostate Cancer Detection and Prognosis. Prostate 2017, 77, 573–583. [Google Scholar] [CrossRef] [PubMed]
- Bryzgunova, O.E.; Zaporozhchenko, I.A.; Lekchnov, E.A.; Amelina, E.V.; Konoshenko, M.Y.; Yarmoschuk, S.V.; Pashkovskaya, O.A.; Zheravin, A.A.; Pak, S.V.; Rykova, E.Y.; et al. Data analysis algorithm for the development of extracellular miRNA-based diagnostic systems for prostate cancer. PLoS ONE 2019, 14, e0215003. [Google Scholar] [CrossRef] [Green Version]
- Davey, M.; Benzina, S.; Savoie, M.; Breault, G.; Ghosh, A.; Ouellette, R.J. Affinity Captured Urinary Extracellular Vesicles Provide mRNA and miRNA Biomarkers for Improved Accuracy of Prostate Cancer Detection: A Pilot Study. Int. J. Mol. Sci. 2020, 21, 8330. [Google Scholar] [CrossRef] [PubMed]
- Isharwal, S.; Konety, B. Non-muscle invasive bladder cancer risk stratification. Indian J. Urol. 2015, 31, 289–296. [Google Scholar] [CrossRef] [PubMed]
- Nuhn, P.; May, M.; Sun, M.; Fritsche, H.-M.; Brookman-May, S.; Buchner, A.; Bolenz, C.; Moritz, R.; Herrmann, E.; Burger, M.; et al. External Validation of Postoperative Nomograms for Prediction of All-Cause Mortality, Cancer-Specific Mortality, and Recurrence in Patients With Urothelial Carcinoma of the Bladder. Eur. Urol. 2012, 61, 58–64. [Google Scholar] [CrossRef] [PubMed]
- Ogawa, K.; Shimizu, Y.; Uketa, S.; Utsunomiya, N.; Kanamaru, S. Prognosis of patients with muscle invasive bladder cancer who are intolerable to receive any anti-cancer treatment. Cancer Treat. Res. Commun. 2020, 24, 100195. [Google Scholar] [CrossRef]
- Mossanen, M.; Gore, J.L. The burden of bladder cancer care: Direct and indirect costs. Curr. Opin. Urol. 2014, 24, 487–491. [Google Scholar] [CrossRef]
- Babjuk, M.; Böhle, A.; Burger, M.; Capoun, O.; Cohen, D.; Compérat, E.M.; Hernández, V.; Kaasinen, E.; Palou, J.; Rouprêt, M.; et al. EAU Guidelines on Non–Muscle-invasive Urothelial Carcinoma of the Bladder: Update 2016. Eur. Urol. 2017, 71, 447–461. [Google Scholar] [CrossRef] [PubMed]
- De Oliveira, M.C.; Caires, H.R.; Oliveira, M.J.; Fraga, A.; Vasconcelos, M.H.; Ribeiro, R. Urinary biomarkers in bladder cancer: Where do we stand and potential role of extracellular vesicles. Cancers 2020, 12, 1400. [Google Scholar] [CrossRef]
- Smalley, D.M.; Sheman, N.E.; Nelson, K.; Theodorescu, D. Isolation and identification of potential urinary microparticle biomarkers of bladder cancer. J. Proteome Res. 2008, 7, 2088–2096. [Google Scholar] [CrossRef] [PubMed]
- Chen, C.L.; Lai, Y.F.; Tang, P.; Chien, K.Y.; Yu, J.S.; Tsai, C.H.; Chen, H.W.; Wu, C.C.; Chung, T.; Hsu, C.W.; et al. Comparative and targeted proteomic analyses of urinary microparticles from bladder cancer and hernia patients. J. Proteome Res. 2012, 11, 5611–5629. [Google Scholar] [CrossRef] [PubMed]
- Andreu, Z.; Otta Oshiro, R.; Redruello, A.; López-Martín, S.; Gutiérrez-Vázquez, C.; Morato, E.; Marina, A.I.; Olivier Gómez, C.; Yáñez-Mó, M. Extracellular vesicles as a source for non-invasive biomarkers in bladder cancer progression. Eur. J. Pharm. Sci. 2017, 98, 70–79. [Google Scholar] [CrossRef] [PubMed]
- Lin, S.Y.; Chang, C.H.; Wu, H.C.; Lin, C.C.; Chang, K.P.; Yang, C.R.; Huang, C.P.; Hsu, W.H.; Chang, C.T.; Chen, C.J. Proteome profiling of urinary exosomes identifies alpha 1-antitrypsin and H2B1K as diagnostic and prognostic biomarkers for urothelial carcinoma. Sci. Rep. 2016, 6, 1–12. [Google Scholar] [CrossRef]
- Silvers, C.R.; Miyamoto, H.; Messing, E.M.; Netto, G.J.; Lee, Y.F. Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. Oncotarget 2017, 8, 91199–91208. [Google Scholar] [CrossRef] [PubMed]
- Hiltbrunner, S.; Mints, M.; Eldh, M.; Rosenblatt, R.; Holmström, B.; Alamdari, F.; Johansson, M.; Veerman, R.E.; Winqvist, O.; Sherif, A.; et al. Urinary Exosomes from Bladder Cancer Patients Show a Residual Cancer Phenotype despite Complete Pathological Downstaging. Sci. Rep. 2020, 10, 1–9. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Armstrong, D.A.; Green, B.B.; Seigne, J.D.; Schned, A.R.; Marsit, C.J. MicroRNA molecular profiling from matched tumor and bio-fluids in bladder cancer. Mol. Cancer 2015, 14, 1–9. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- De Long, J.; Sullivan, T.B.; Humphrey, J.; Logvinenko, T.; Summerhayes, K.A.; Kozinn, S.; Harty, N.; Summerhayes, I.C.; Libertino, J.A.; Holway, A.H.; et al. A non-invasive miRNA based assay to detect bladder cancer in cell-free urine. Am. J. Transl. Res. 2015, 7, 2500–2509. [Google Scholar]
- Matsuzaki, K.; Fujita, K.; Jingushi, K.; Kawashima, A.; Ujike, T.; Nagahara, A.; Ueda, Y.; Tanigawa, G.; Yoshioka, I.; Ueda, K.; et al. MiR-21-5p in urinary extracellular vesicles is a novel biomarker of urothelial carcinoma. Oncotarget 2017, 8, 24668–24678. [Google Scholar] [CrossRef] [Green Version]
- Baumgart, S.; Meschkat, P.; Edelmann, P.; Heinzelmann, J.; Pryalukhin, A.; Bohle, R.; Heinzelbecker, J.; Stöckle, M.; Junker, K. MicroRNAs in tumor samples and urinary extracellular vesicles as a putative diagnostic tool for muscle-invasive bladder cancer. J. Cancer Res. Clin. Oncol. 2019, 145, 2725–2736. [Google Scholar] [CrossRef]
- Qin, Z.; Xu, Q.; Hu, H.; Yu, L.; Zeng, S. Extracellular Vesicles in Renal Cell Carcinoma: Multifaceted Roles and Potential Applications Identified by Experimental and Computational Methods. Front. Oncol. 2020, 10, 724. [Google Scholar] [CrossRef]
- Padala, S.A.; Barsouk, A.; Thandra, K.C.; Saginala, K.; Mohammed, A.; Vakiti, A.; Rawla, P.; Barsouk, A. Epidemiology of Renal Cell Carcinoma. World J. Oncol. 2020, 11, 79–87. [Google Scholar] [CrossRef]
- Marconi, L.; Dabestani, S.; Lam, T.B.; Hofmann, F.; Stewart, F.; Norrie, J.; Bex, A.; Bensalah, K.; Canfield, S.E.; Hora, M.; et al. Systematic Review and Meta-analysis of Diagnostic Accuracy of Percutaneous Renal Tumour Biopsy. Eur. Urol. 2016, 69, 660–673. [Google Scholar] [CrossRef]
- Lakshminarayanan, H.; Rutishauser, D.; Schraml, P.; Moch, H.; Bolck, H.A. Liquid Biopsies in Renal Cell Carcinoma—Recent Advances and Promising New Technologies for the Early Detection of Metastatic Disease. Front. Oncol. 2020, 10, 2302. [Google Scholar] [CrossRef] [PubMed]
- Linxweiler, J.; Junker, K. Extracellular vesicles in urological malignancies: An update. Nat. Rev. Urol. 2020, 17, 11–27. [Google Scholar] [CrossRef] [PubMed]
- Hsieh, J.J.; Purdue, M.P.; Signoretti, S.; Swanton, C.; Albiges, L.; Schmidinger, M.; Heng, D.Y.; Larkin, J.; Ficarra, V. Renal cell carcinoma. Nat. Rev. Dis. Prim. 2017, 3, 1–19. [Google Scholar] [CrossRef]
- Janzen, N.K.; Kim, H.L.; Figlin, R.A.; Belldegrun, A.S. Surveillance after radical or partial nephrectomy for localized renal cell carcinoma and management of recurrent disease. Urol. Clin. N. Am. 2003, 30, 843–852. [Google Scholar] [CrossRef]
- Craven, R.A.; Vasudev, N.S.; Banks, R.E. Proteomics and the search for biomarkers for renal cancer. Clin. Biochem. 2013, 46, 456–465. [Google Scholar] [CrossRef] [PubMed]
- Salih, M.; Zietse, R.; Hoorn, E.J. Urinary extracellular vesicles and the kidney: Biomarkers and beyond. Am. J. Physiol. Ren. Physiol. 2014, 306, F1251–F1259. [Google Scholar] [CrossRef] [Green Version]
- Raimondo, F.; Morosi, L.; Corbetta, S.; Chinello, C.; Brambilla, P.; Della Mina, P.; Villa, A.; Albo, G.; Battaglia, C.; Bosari, S.; et al. Differential protein profiling of renal cell carcinoma urinary exosomes. Mol. Biosyst. 2013, 9, 1220–1233. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Butz, H.; Nofech-Mozes, R.; Ding, Q.; Khella, H.W.Z.; Szabó, P.M.; Jewett, M.; Finelli, A.; Lee, J.; Ordon, M.; Stewart, R.; et al. Exosomal MicroRNAs Are Diagnostic Biomarkers and Can Mediate Cell-Cell Communication in Renal Cell Carcinoma. Eur. Urol. Focus 2016, 2, 210–218. [Google Scholar] [CrossRef] [PubMed]
- Song, S.; Long, M.; Yu, G.; Cheng, Y.; Yang, Q.; Liu, J.; Wang, Y.; Sheng, J.; Wang, L.; Wang, Z.; et al. Urinary exosome miR-30c-5p as a biomarker of clear cell renal cell carcinoma that inhibits progression by targeting HSPA5. J. Cell. Mol. Med. 2019, 23, 6755–6765. [Google Scholar] [CrossRef] [PubMed]
- Zhou, H.; Yuen, P.S.T.; Pisitkun, T.; Gonzales, P.A.; Yasuda, H.; Dear, J.W.; Gross, P.; Knepper, M.A.; Star, R.A. Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int. 2006, 69, 1471–1476. [Google Scholar] [CrossRef] [Green Version]
- Raimondo, F.; Chinello, C.; Stella, M.; Santorelli, L.; Magni, F.; Pitto, M. Effects of Hematuria on the Proteomic Profile of Urinary Extracellular Vesicles: Technical Challenges. J. Proteome Res. 2018, 17, 2572–2580. [Google Scholar] [CrossRef]
- Cheng, L.; Sun, X.; Scicluna, B.J.; Coleman, B.M.; Hill, A.F. Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine. Kidney Int. 2014, 86, 433–444. [Google Scholar] [CrossRef] [Green Version]
Study | Urine Source (n) | Type of Urine/Pre-Treatment | Isolation Methods | Description | Characterization |
---|---|---|---|---|---|
[26] | Healthy (10) | First-void urine and 12 h collection | UC | Centrifuged (17,000× g, 10 min, 37 °C); IS; centrifuged (17,000× g, 10 min, 37 °C); centrifuged (200,000× g, 1 h, 37 °C) | TEM WB: Alix, TSG101, CD9, HSP70, AQP2 |
PI | UC + DTT (200,000× g) | Described as above; DTT | |||
* UC + DTT (17,000× g) | Centrifuged and IS, as described for UC; DTT; centrifuged (17,000× g, 10 min, 37 °C); centrifuged (200,000× g, 1 h, 37 °C) | ||||
[27] | Healthy (NS) IMN (NS) FSG (NS) | NS | UC | Centrifuged (200,000× g, 110 min); IS | TEM WB: AQP2, neprilysin, PODXL and albumin |
UC + DTT | Described as UC; DTT; centrifuged (200,000× g, 110 min) | ||||
Filtered; SC (17,000× g, 15 min) | UF | Vivaspin 20NC (100 kDa MWCO); centrifuged (3000× g); pre-heated Laemmli buffer | |||
* UC + SEC | Centrifuged (200,000× g, 110 min); IS; SEC column; centrifuged (3000× g), Amicon Ultra-4 (10 kDa MWCO) | ||||
[28] | Healthy (4) | First-void urine | * UC | Centrifuged (17,000× g, 10 min, 37 °C); IS; DTT; centrifuged (17,000× g, 10 min, 37 °C); centrifuged (165,000× g, 70 min, 37 °C); IS; DTT; centrifuged (165,000× g, 70 min, 37 °C) | ELISA: CD9 WB: Alix and TSG101 |
PI; different pre-process depending on the isolation method | * dUC (sucrose cushion) | Steps prior to last high-speed centrifugation as described above; 30% sucrose/D2O; centrifuged 2× (165,000× g, 1 h, 37 °C) | |||
UC + 0.22-µm filter | 1st Centrifuation, IS, DTT as described above; centrifuged (17,000× g, 10 min, 37 °C) + 0.22 µm filter; centrifuged (165,000× g, 70 min, 37 °C); IS; DTT; centrifuged (165,000× g, 70 min, 37 °C) | ||||
UF | Steps prior to last high-speed centrifuged as described in UC; Vivaspin 20 NC (100 kDa MWCO); centrifuged (3000× g, 1 h, 25 °C); concentrator | ||||
Precipitation (ExoQuick std) | Centrifuged (3000× g, 10 min, 25 °C); ExoQuick-TC (4 °C, >12 h); centrifuged (1500× g, 30 min, 25 °C) | ||||
* Precipitation (ExoQuick modified) | 1st Centrifugation; IS; DTT; 2nd centrifugation as above; ExoQuick-TC; centrifuged (10,000× g, 30 min, 25 °C) | ||||
# [24] | ADPKD (7) Healthy (7) | First-void urine | UC | Centrifuged (110,000× g, 17 °C, 2.5 h); concentrated (100 kDa filter) | IZON qNano TEM WB: CD24, AQP2 |
PI; centrifuged (1800× g, 10 min) UC, UF → −80 °C; heated to 40 °C; centrifuged (17,000× g, 20 °C, 15 min) | UF | Filtered (100 kDa MWCO); centrifuged (1500× g, 20 °C); spin device | |||
dUC | Centrifuged (4000× g); centrifuged (150,000× g, 1 h); 5–30% sucrose/D2O; centrifuged (100,000× g, 24 h); fractionation device | ||||
[29] | Healthy (NS) | First-void urine | * UF | Centrifuged (20,000× g, 20 min); Vivaspin 20 NC (100 kDa MWCO); centrifuged (3500× g, 1 h); DTT | DLS WB: AQP2 |
PI; centrifuged (3500× g, 40 min); filtered (0.22 μm); −20 °C | UC | Centrifuged (20,000× g, 20 min); centrifuged (200,000× g, 1 h); DTT; centrifuged (200,000× g, 1 h) | |||
Precipitation (ExoQuick) | ExoQuick-TC (4 °C, >12 h); centrifuged (1500× g, 30 min) | ||||
[30] | Healthy (10) | First-void urine | UC | Centrifuged (100,000× g, 90 min); ERB | TEM WB: CD9, CD10, CD63, TSG101, CD10, Alix, AQP2 and FLT1 |
Precipitation (ExoQuick) | ExoQuick-TC (4 °C, 16 h); centrifuged (1500× g, 30 min); ERB | ||||
Centrifuged (2000× g, 10 min); filtered (0.22-µm); −80 °C | TEI solution (INVITROGEN) | INVITROGEN mix (1 h, RT); centrifuged (10,000× g, 1 h); ERB | |||
* Norgen (modified) | Slurry component; centrifuged (2000× g, 2 min) | ||||
Lectin-based purification (STL) | Biotinylated STL/Streptavidin Dynabeads (1 h, RT) | ||||
[31] | Healthy (NS) | NS | * UC | Centrifuged (2000× g, 15 min and 10,000× g, 30 min); DTT; centrifuged (17,000× g, 10 min, 37 °C); centrifuged 2× (100,000× g, 1.5 h); PI | TEM WB: Alix and TSG101, CD63 |
−80 °C | Norgen (modified) | Remove debris; slurry component; centrifuged (15,000× g) | |||
TEI solution (INVITROGEN) | Remove debris; INVITROGEN mix; centrifuged (15,000× g) | ||||
[19] | BlCa (16) Healthy (8) | NS | UC | Centrifuged (100,000× g, 70 min); −80 °C | TEM DLS Fluorescence staining: CD9 |
Centrifuged (20,000× g, RT, 15 min); filtered (0.22 μm) | * Double-filtration microfluidic | Double-filtrationdevice | |||
[32] | Healthy (5) | First-void mid-stream urine | UC | Centrifuged 2× (120,000× g, 70 min) | TEM NTA WB: CD63, CD9, TSG101 and CD81 |
PEG | PEG solution (4 °C, 12 h); centrifuged (1000× g, 30 min) | ||||
Vortexed; SC (200× g, 20 min, 2×, and 16,000× g, 20 min); 4 °C | Concentration + SEC | Vacuum filtration; centrifuged (4000× g, 30 min); Sepharose CL-2B | |||
* UC + SEC (+ F + DTT + PI) | PI; SC; DTT; vortex; centrifuged (16,000× g, 20 min); filtered (0.22 μm); centrifuged (120,000× g, 70 min); Sepharose CL-2B | ||||
PEG + SEC | PEG solution (4 °C, 12 h); centrifuged (1000× g, 30 min); Sepharose CL-2B | ||||
[33] | Healthy (NS) | First-void, afternoon and evening urine | UC | Centrifuged (200,000× g, 60 min) | TEM NTA WB: CD63 and Hsp70 |
−80°C; SC (2000× g, 30 and 60 min, 17,000× g) | * OUF (F + ExoQuick) | Filtered (0.22 µm); centrifuged (3000 g, 30 min); dialysis membrane (10,000 kDa MWCO); ExoQuick-TC (30 min, 4 °C); centrifuged (15,279 g, 2 min) | |||
[34] | Healthy (3) | First-void urine | Precipitation (ExoQuick std) | ExoQuick-TC (1:4 ratio, 4 °C, 12 h); centrifuged (1500× g, 30 min, 4 °C) | NTA WB: CD63 TEM |
SC (300× g, 10 min and 3000× g, 20 min, 4 °C and 17,000× g, 20 min, 4 °C); DTT; filtered (0.22 μm) | Precipitation (ExoQuick modified) (MEQ) | ExoQuick-TC (3:7 ratio, 4 °C, 12 h); centrifuged (10,000× g, 30 min, 4 °C) | |||
Precipitation PEG6000 (PE6) | PEG 6000 (4 °C, 12 h); centrifuged (4000× g, 60 min) | ||||
UC | Centrifuged (200,000× g, 75 min) | ||||
UF | Amicon Ultra-15 Filter (10 and 100 kDa MWCO); centrifuged (4000× g, 10 min) | ||||
* SEC + MEQ or UF | Concentrated (qEV size exclusion columns) | ||||
[35] | BHP (12) | Second-void urine | UC | Centrifuged (30 min, 2000× g, 4 °C and 45 min, 12,000× g, 4 °C); centrifuged (110,000× g, 2 h); filtered (0.22 µm); centrifuged (110,000× g, 70 min); −80 °C | TEM NTA WB: Alix, CD9, Flotillin-1 |
Concentrated (10kDa filter); tris buffer | Precipitation (ExoQuick) | Concentrated (10 kDa filter); ExoQuick-TC (4 °C, >12h); centrifuged 2× (1500× g, 30 and 5 min) | |||
SEC | Concentrated (10 kDa filter); Sepharose CL-2B; | ||||
* Bottom-up Optiprep (dUC) | 40% iodixanol; layered on bottom of a discontinuous bottom-up ODG; centrifuged (18 h, 100,000× g, 4 °C); ODG fractions collected from top; centrifuged (3 h, 100,000× g, 4 °C); −80 °C | ||||
Top-down Optiprep (dUC) | Concentrated (10 kDa filter); loaded on top of a discontinuous top-down ODG; processed as above |
Study | Urine Source (n)/Type of Urine | Urine Pre-Treatment | Isolation Method | Characterization | Biomarker Candidates | Biomarker Performance | Biomarker Type | ||
---|---|---|---|---|---|---|---|---|---|
AUC | SE (%) | SP (%) | |||||||
Proteins | |||||||||
[66] | PCa (15) Healthy (15) | SC (15 min, 2000× g, RT and 30 min, 10,000× g) | UC Centrifuged 2× (100,000× g, 70 min, RT); vortexed; filtered (0.22 µm); centrifuged (100,000× g, 70 min) | DLS: Mean 149 nm TEM WB: CD9, CD63 and TSG101 | ↑TM256 | 0.87 | -- | -- | Diagnosis |
Morning urine (PCa); first-void urine (healthy) | TM256 and LAMTOR1 combination | 0.94 | -- | -- | |||||
[67] | mPCa (5) Healthy (13) | SC (400× g 7 min, 20 °C and 2000× g, 15 min); vacuum filtered (0.22 µm); −80 °C | UC + SEC Centrifuged (400× g 7 min, 20 °C); vacuum filtered (0.22 µm); centrifuged (200,000× g, 2 h, 4 °C); Sepharose CL-2B | NTA: Mean 118 nm, peak 73 nm cryo-EM: ~100 nm ELISA: CD9, ApoB, THP, HAS WB: TSG101, ALIX, LAMP2, HAS | ↑Afamin, cardiotrophin-1, CDON, ARTS-1, FGF19, IL17RC, NAMPT, IL1RAPL2, CD226, IGFBP2, CCL16, TNFSF18, IGFBP5; AADC | -- | -- | -- | mPCa predictive treatment (prognosis) |
Morning urine (excluding first-void) | |||||||||
[68] | PCa (low- and high-grade) (18) Negative biopsy (11) | Centrifuged (2000× g, 30 min); −80 °C | UC Centrifuged (17,000× g, 30 min); centrifuged 2× (100,000× g, 90 min); DTT; centrifuged (100,000× g, 90 min); −80 °C; MPEX PTS solution (95 °C, 5 min); centrifuged (100,000× g, 30 min, 4 °C) | TEM WB: CD9 | ↑ FABP5 and significantly associated with GS | 0.76 (GS ≥ 6) | -- | -- | High-GS PCa |
First catch urine after DRE | 0.86 (GS ≥ 7) | 60.0 | 100 | ||||||
[69] | PCa (53) -Low-grade PCa -High-grade PCa Negative biopsy (54) | Centrifuged (2500× g, 10 min, 4 °C); PI; −80 °C | UC Centrifuged (16,500× g, 20 min); DTT; centrifuged (16,500× g, 20 min); filtered (0.2 μM); centrifuged (100,000× g, 120 min, 4 °C); centrifuged (100,000× g, 60 min, 4 °C) | TEM NTA WB: TSG101, CD81 or Rab5 | ADSV-TGM4 combination classifies benign and PCa. | 0.65 | -- | -- | Diagnosis and prognosis |
CD63-GLPK5-SPHMPSA-PAPP combination distinguish between high- and low-grade PCa | 0.70 | -- | -- | ||||||
First catch urine after DRE | |||||||||
[70] | PCa (26) Healthy (16) Morning urine | Centrifuged (15 min, RT, 2000× g) | UC Centrifuged (30 min, 10,000× g); centrifuged (100,000× g, 70min, RT); centrifuged (100,000× g, 70 min, 4 °C); filtered (0.22 µm); centrifuged (100,000× g, 70 min, 4 °C) | NP | ↑Flotillin 2 (WB) | 0.91 | 88 | 94 | Diagnosis |
(PCa); first-void urine (healthy) | ↑Flotillin 2, Parkinson protein 7 combination (ELISA) | -- | 68 | 93 | |||||
[35] | PCa (12) Prior to and three months after local treatment Healthy (12) | Concentrated (10 kDa filter device); tris buffer | Bottom-up Optiprep (dUC) 40% iodixanol; layered on bottom of a discontinuous bottom-up ODG; centrifuged (18 h, 100,000× g, 4 °C); ODG fractions collected from top; centrifuged (3 h, 100,000× g, 4 °C); −80 °C | TEM NTA: Mean 132 nm, peak 111 nm | ↑FKBP5, FAM129A, RAB27A, FASN, NEFH | -- | -- | -- | Diagnosis |
Second-void urine | |||||||||
miRNAs | |||||||||
[71] | PCa (48) Negative biopsy (26) | −80 °C; centrifuged (20,000× g, 30 min, 4 °C) | dUC Centrifuged 2× (100,000× g, 90 min, 4 °C); 30–40% sucrose gradient; centrifuged (100,000× g, 90 min) | TEM: 50–150 nm WB: TSG101 and ALIX | isomiR panel: ↑ miR-204; ↓ miR-21 and miR-375 | 0.82 | -- | -- | PCa diagnosis |
First catch urine after DRE | |||||||||
[72] | PCa (14) Healthy (20) | SC (400× g, 20 °C, 20 min and 17,000× g, 20 °C, 20 min); −20 °C | UC Centrifuged (100,000× g, 18 °C, 90 min); filtered (0.1 μm); centrifuged (100,000× g, 18 °C, 90 min) | TEM: 20–230 nm Immunogold staining: CD63, CD9 and CD24 | ↑ miR-19b | -- | 93 | 100 | PCa diagnosis |
NS | |||||||||
[73] | PCa (60) BPH (37) Healthy (24) | Centrifuged (2000× g, 30 min, 4 °C); PI; −80 °C | UC Centrifuged (17,000× g, 4 °C, 10 min); IS; DTT; centrifuged (17,000× g, 4 °C, 10 min); centrifuged 2× (200,000× g, 4 °C, 60 min) HFD Separating funnel connected with dialysis membrane (1000 kDa MWCO) | TEM NTA: <300 nm WB: TSG101, CD63, CD9, and ALIX | ↑ miR-145 Compared with BPH and healthy controls. In GS ≥ 8 tumors compared with GS ≤ 7 | 0.623 | -- | -- | PCa diagnosis and prognosis |
First-void urine | |||||||||
[74] | PCa (90) BPH (10) Untreated BlCa (60) Healthy (50) | PI; −80°C | ExiqonmiRCURYexosomeisolationkit | SEM: ~100 nm WB: CD63 | miR-2909: ↑with severity Distinguish PCa from BlCa. | -- | -- | -- | PCa diagnosis and prognosis |
NS | |||||||||
[75] | PCa (28) Healthy (19) | Centrifuged (2000× g, 15 min, RT); centrifuged (10,000× g, 30 min, RT) | UC Centrifuged (100,000× g, 70 min, RT); centrifuged (100,000× g, 70 min, 4 °C); filtered (0.22 µm); centrifuged (100,000× g, 70 min, 4 °C); −80 °C | NP | ↓ miR-196a-5p ↓ miR-501-3p | 0.92 (NGS) | 100 | 89 | PCa diagnosis |
NS | 0.72 | -- | -- | ||||||
[76] | PCa (52) Healthy (10) | SC (2000× g, 20 min, 4 °C and 2000× g, 5 min, 4 °C); QIAzol; −80 °C | UC Centrifuged (17,000× g, 45 min, 4 °C); centrifuged (200,000× g, 2 h, 4 °C); −80 °C | TEM | ↑ miR-21, miR-375 and let-7C | 0.71; 0.80; 0.68 | -- | -- | PCa diagnosis and prognosis |
Freshly voided, collected after massage | |||||||||
[77] | PCa (10) BPH (10) Healthy (10) | Centrifuged (17,000× g, 20 °C, 20 min); −20 °C | UC Centrifuged (100,000× g, 18 °C, 90 min); filtered (0.1 μm); centrifuged (100,000× g, 18 °C, 90 min) | NP | 5 miRNA pairs (miR-30a: miR-125b; miR-425: miR-331; miR-29b: miR-21; miR-191: miR-200a; miR-331: miR-106b) | 97.5% accuracy. | -- | 100 | PCa diagnosis |
NS | |||||||||
[78] | PCa (28) Negative biopsy (28) | SC (650× g, 10 min, RT and 10,000× g, 30 min); −80°C | Vn96peptide Centrifuged (17,000× g, 15 min, RT); Vn96 synthetic peptide (30× g/ mL urine); centrifuged (17,000× g, 15 min, RT) | WB: CD9, CD63, CD24, Hsp/c70, PDCD6IP | ↑ miR-375-3p and miR-574-3p panel | 0.74 | -- | -- | PCa diagnosis |
Freshly voided, collected after massage and DRE |
Study | Urine Source (n)/Type of Urine | Urine Pre-Treatment | Isolation Method(s) | Characterization | Biomarker Candidates | Biomarker Performance | Biomarker Type | ||
---|---|---|---|---|---|---|---|---|---|
AUC | SE (%) | SP (%) | |||||||
Protein | |||||||||
[85] | BlCa (2) Healthy (2) | PMSF; centrifuged (250× g, 10 min); −80 °C | UC PI; Centrifuged (250× g, 10 min) centrifuged (17,000× g, 30 min); centrifuged 2× (200,000× g, 60 min); −80 °C | NP | ↑ Resistin, GTPase NRas, EPS8L2, Mucin 4, EPS8L1, RAI3, Alpha subunit of GsGTP binding protein, EHD4EH | -- | -- | -- | BlCa diagnosis |
NS | |||||||||
[86] | BlCa (28) Hernia (12) | PI and sodium azide; centrifuged (5000× g, 30 min, 4 °C); −80 °C | UC Centrifuged (17,000× g, 30 min, 4 °C); centrifuged 2× (100,000× g, 70 min, 4 °C) | TEM: 30–100 nm WB: TSG101 and CD9 Flow Cytometry: CD9 | ↑ TACSTD2 | 0.80 | 73.6 | 76.5 | BlCa diagnosis and prognosis |
First-void urine | |||||||||
[87] | BlCa (16) (high-, low-grade) Healthy (10) | Centrifuged (3500× g, 25 min, 4 °C); filtered (0.22 μm) | UC Centrifuged 2× (100,000× g, 4 °C, 1 h) | NTA TEM WB: ERM and CD9 | ↑ ApoB (high-grade) | -- | -- | -- | BlCa diagnosis and prognosis |
First-void urine | |||||||||
[88] | BlCa (129) Healthy (62) | PI; centrifuged (1000× g, 10 min); −80 °C | UC Centrifuged (17,000× g, 10 min, 4 °C); IS; DTT; centrifuged (17,000× g, 30 min, 4 °C); centrifuged (200,000× g, 1 h, 4 °C) | TEM: 50–100 nm WB: TSG101, Alix | ↑ Alpha 1-antitrypsin | 0.74 | 50.4 | 96.9 | BlCa diagnosis and prognosis |
First-void urine | ↑ Histone H2B1K | 0.77 | 62.0 | 92.3 | |||||
[89] | BlCa pT1-pT3 (6) Healthy (6) | SC (400 g and 15,500× g); −80 °C | UC Spun; centrifuged 2× (200,000× g, 70 min); centrifuged (15,500× g) | TEM NTA: Mean 35–300 nm, peak 105 nm | ↑ HEXB, S100A4, and SND1 | -- | -- | -- | BlCa diagnosis |
Perioperative (BlCa) NS (healthy) | |||||||||
[60] | BlCa (10) Healthy (10) | −80 °C; Centrifuged (2000× g, 10 min); filtered (0.45 μm); centrifuged (30 min, 18,000× g) | UC Centrifuged (200,000× g, 16 h); centrifuged (200,000× g, 1 h); filtered (0.22 μm); PI | TEM NTA: peak ~124 nm Flow cytometry and WB: Alix TSG101, CD63, HSP70, Flotillin-1 | ↑ Mucin-1, CEACAM-5, EPS8L2, and moesin | -- | -- | -- | BlCa Diagnosis |
First-void urine | |||||||||
[90] | BlCa (13) | Spun (3000× g, 30 min); filtered (0.22 µm) | UC Centrifuged (100,000× g, 2 h); −80 °C | Flow cytometry: CD9 CD81, CD63 NTA: Peak ~155 nm TEM | ↓ SLC4A1 ↑ TPP1, TMPRSS2, FOLR1, RALB and RAB35 | -- | -- | -- | BlCa recurrence |
Perioperative urine | |||||||||
miRNA | |||||||||
[91] | BlCa (16) Low-grade NMIBC to high-grade MIBC | Centrifuged (1200 and 2500 rpm, 20 min, 20 °C); −80 °C | Norgen Urine Exosome RNA Isolation Kit | NP | ↑ miR-4454, miR-21, miR-205, miR-200C-3p, miR-29b-3p | -- | -- | -- | BlCa diagnosis |
NS | |||||||||
[92] | BlCa (TaG1, T1G3, ≥T2, CIS) (85) Healthy (35) | Centrifuged (3000 rpm, 4 °C, 15 min); −20 °C | UC Centrifuged (17,000× g, 10 min, 4 °C); centrifuged (200,000× g, 1 h, 4 °C); DTT; centrifuged (200,000 g, 1 h, 4 °C) | WB: Alix and TSG101 | ↑ miR-26a, miR-93, miR-191, and miR-940 panel | 0.89 | 88 | 78 | BlCa diagnosis |
NS | |||||||||
[87] | BlCa (34) Low- and high-grade Healthy (9) | Centrifuged (3500× g, 25 min, 4 °C); filtered (0.22 μm) | UC Centrifuged 2× (1 h, 100,000× g, 4 °C) | WB: ERM and CD9 NTA TEM | ↓ miR-375 in low-grade ↑ miR-146a high-grade | -- | -- | -- | BlCa diagnosis and prognosis |
First-void urine | |||||||||
[93] | BlCa (36) Healthy (24) | SC (2000× g, 30 min and 30 min, 17,000× g) | UC Centrifuged 2× (130,000× g, 90 min); −80 °C | NTA: <200 nm TEM ELISA: CD9 WB: CD9 and CD63 | ↑ miR-21-5p | 0.90 | 75.0 | 95.8 | BlCa diagnosis |
Voided urine (NS) | ↑ miR-155-5p, miR-15a-5p, miR-132-3p, miR-31-5p | 0.820; 0.841; 0.821; 0.821 | -- | -- | |||||
[94] | NMIBC (17) MIBC (20) | −80 °C; SC (2000× g, 4 °C, 20 min and 15,000× g, 30 min) | Total Exosome Isolation From Urine Kit | WB: CD63 and GM130 NTA: Median ~125 nm TEM: ~48 nm | ↑ miR-146b-5p and miR-155-5p in MIBC than NMIBC | -- | -- | -- | MIBC prognosis |
Perioperative urine |
Study | Urine Source (n)/ Type of Urine | Urine Pre-Treatment | Isolation Method(s) | Characterization | Biomarker Candidates | Biomarker Performance | Biomarker Type | ||
---|---|---|---|---|---|---|---|---|---|
AUC | SE (%) | SP (%) | |||||||
Proteins | |||||||||
[104] | RCC (29) Healthy (23) | –80 °C; centrifuged (10 min, 4 °C, 1000× g); PI; centrifuged (15 min, 4 °C, 17,000× g) | OptiPrep (dUC) Discontinuous OptiPrep gradient; centrifuged (100,000× g, 16 h) collected from top of gradient; centrifuged (100,000× g, 3 h) | TEM WB: CD9, TSG101 and Flotillin-1 | ↑ MMP-9, CP, PODXL, DKK4 and CAIX | 0.938; 1; 1; 0.979; 0.862 | -- | -- | RCC diagnosis |
Second morning urine samples | ↓ AQP1, EMMPRIN, CD10, Dipeptidase 1 and Syntenin-1 | 0.891; 0.879, 0.794; 0.760; 0,733 | -- | -- | |||||
miRNAs | |||||||||
[105] | ccRCC (81) Benign kidney tumors (24) Healthy (33) | Centrifuged (2000× g, 10 min, 4 °C); −80 °C | Norgen kit | NP | Combinations of miR-126-3p with miR-449a or miR-34b-5p distinguish ccRCC from controls. | 0.84; 0.79 | -- | -- | RCC diagnosis |
Preoperative urine | Combination of miR-126-3p and miR-34b-5p distinguish SRM from controls. | 0.79 | -- | -- | |||||
miR-126-3p and miR-486-5p combination differentiate benign lesions from ccRCC | 0.85 | -- | -- | ||||||
[106] | ccRCC (T1aN0M0) (70) Healthy (30) | Centrifuged (2000× g, 5 min, 4 °C); filtered (0.22 μm); −80 °C | UC Centrifuged (150,000× g, overnight, 4 °C); centrifuged (150,000× g, 4 °C, 2 h) | TEM NTA: Peak ~116 nm WB: CD81, CD63, CD9 | ↓ miR-30C-5p | 0.82 | 68.57 | 100 | RCC diagnosis |
Morning urine (NS) |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Lourenço, C.; Constâncio, V.; Henrique, R.; Carvalho, Â.; Jerónimo, C. Urinary Extracellular Vesicles as Potential Biomarkers for Urologic Cancers: An Overview of Current Methods and Advances. Cancers 2021, 13, 1529. https://doi.org/10.3390/cancers13071529
Lourenço C, Constâncio V, Henrique R, Carvalho Â, Jerónimo C. Urinary Extracellular Vesicles as Potential Biomarkers for Urologic Cancers: An Overview of Current Methods and Advances. Cancers. 2021; 13(7):1529. https://doi.org/10.3390/cancers13071529
Chicago/Turabian StyleLourenço, Catarina, Vera Constâncio, Rui Henrique, Ângela Carvalho, and Carmen Jerónimo. 2021. "Urinary Extracellular Vesicles as Potential Biomarkers for Urologic Cancers: An Overview of Current Methods and Advances" Cancers 13, no. 7: 1529. https://doi.org/10.3390/cancers13071529