Human urinary exosomes in bladder cancer patients: properties, concentrations and possible clinical application
OBJECTIVE: High grade bladder cancer is extremely aggressive. Early detection is thus an important challenge. Development of non-invasive diagnostic tools particularly using urine samples could be of importance in the diagnosis and surveillance of these patients. Exosomes are small vesicles present in the urine and have the potential to be used as biomarkers of cancer. Thus studies of the properties and concentrations of these particles in bladder cancer patients are of importance.
MATERIALS AND METHODS: The concentration of exosomes present in urine was determined by nanoparticle tracking analysis using a Nanosight LM10 unit. Clinical urine samples were routinely collected and fixed using Preservcyt. The morphology of exosomes was studied in electron micrographs and characteristic exosome markers using Western blots.
RESULTS: The exosome concentration of fixed samples stored at room temperature was constant for 48 hours and the same as fresh samples. Exosomes derived from patients presenting for a transurethral resection of their bladder tumor exhibited the exosome markers ALIX and TSG101 and also the classic cup shaped appearance in electron micrographs. The concentration of exosomes in patients presenting for transurethral resection of a bladder tumor was significantly greater than in patients presenting for check cystoscopy with no recurrence (median 77.2 compared with 38.8 × 108, P<0.001). A ROC analysis (area under the curve 77.4%) suggested that a suitable cut-off concentration of 85 × 108 is associated with a sensitivity of 43% and specificity of 91% for diagnosing bladder cancer.
CONCLUSIONS: Thus the concentration and properties of exosomes can be conveniently studied in fixed urine samples derived from bladder cancer patients. The characteristic properties of exosomes were preserved and increased numbers were found in patients presenting for transurethral resection of their tumor. With an appropriate cut-off value, urinary exosome concentrations may have utility in excluding a cancer recurrence when monitoring patients successfully treated for bladder cancer.
1. PitsitkinT, Shen R-F, Knepper MA. (2004). Identification and proteomic profiling of exosomes in human urine. Proc. Natl. Acad. Sci. USA 101: 13368-13373.
2. Théry C, Amigorena S, Raposos G, Clayton A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. In Current Protocols in Cell Biology, pp 23.22.21-23.22.29, John Wiley & Sons, Inc., New York .
3. Soo,CY, Song Y, Zheng Y, Campbell,EC, Riches AC, Gunn-Moore F, Powis SJ (2012). Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells. Immunology 136: 192-197
4. Riches A, Campbell E, Borger E, Powis S. (2014). Regulation of exosome release from mammary epithelial and breast cancer cells – A new regulatory pathway. Eur. J. Cancer 50: 1025-1034.
5. Hao S, Bai O, Li F, Yuan J, Laferte S, Xiang J. (2007).Mature dendritic cells pulsed with exosomes stimulate efficient cytotoxic T-lymphocyte responses and antitumor immunity. Immunology 120: 90-102.
6. Kosaka N, Iguchi H, Hagiwara K, Yoshioka Y, Takeshita F, Ochiya T. (2013). Neutral sphingomyelinase 2 (nSMase2)-dependent exosomal transfer of angiogenic microRNAs regulate cancer cell metastasis. J. Biol. Chem. 288: 10849-10859.
7. Peinado H, Alečković M, Lavotshkin S, Matei I, Costa-Silva B et al (2012). Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nature Medicine 18: 883-891.
8. Marleau A, Chen C-S, Joyce J, Tullis R. (2012). Exosome removal as a therapeutic adjuvant in cancer. J. Trans. Med. 10:134: 1-12.
9. Skog J, Wurdinger T, van Rijn S, Meijer DH, Gainche L et al. (2008).Glioblastoma microvesicles transport RNA and proteins that promote tumor growth and provide diagnostic biomarkers. Nat. Cell Biol. 10: 1470-1476
10. Lv LL, Cao Y, Liu H, Tang RN, Ma BC et al. (2013). Isolation and quantification of microRNAs from urinary exosomes/microvesicles for biomarker discovery. Int.J.Biol.Sci. 9, 1021-1031.
11. Huang, X, Liang M, Dittmar R, Wang L. (2013). Extracellular microRNAs in urologic malignanacies: chances and challenges. Int.J.Mol.Sci. 14: 14785-14799
12. Taylor DD, Gercel-Taylor C. (2008) Micro-RNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol. Oncol. 110: 13-21.
13. Rosell R, Wei J, Taron M.(2009). Circulating microRNA signatures of tumor-derived exosomes for early diagnosis of non-small-cell lung cancer. Clin. Lung Cancer 10: 8-9.
14. Rabinowitz G, Gercel-Taylor C, Day JM, Taylor DD, Kloecker GH. (2009). Exosomal microRNA: a diagnostic marker for lung cancer. Clin. Lung Cancer 10: 42-46 .
15. Beckham CJ, Olsen J, Yin PN, Wu CH, Ting HJ et al. (2014). Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression. J. Urology 192: 583-592.
16. Chen CC, Lai YF, Tang P, Chen KY, Yu JS et al (2012). Comparative and targeted analyses of urinary microparticles from bladder cancer and hernia patients. J. Proteome Res. 11: 5611-5629.
17. Lucocq J, Manifava M, Bi K, Roth MG, Ktistakis NT. (2001) Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin. Eur. J. Cell Biol. 80: 508-20.
18. Lucocq J. Can data provenance go the full monty? (2012) Trends Cell Biol. 22: 229-230.
19. Oosthuyzen W, Sime NE, Ivy JR, Turtle EJ, Street J et al. (2013). Quantification of human urinary exosomes by nanoparticle tracking analysis. J. Physiol. 591.23: 5833-5842
20. Gardiner C, Ferreira YJ, Dragovic RA, Redman CWG, Sargent IL. (2013). Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis. J. Extracell Vesicles 2: 1-11.
21. Sokolva V, Ludwig A, Hornung S, Rotan O, Horn PA et al. (2011) Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces 87: 146-150.
22. Akers JC, Gonda D, Kim R, Carter BS, Chen CC (2013) Biogenesis of extracellualr vesicles (EV): Exosomes, microvesicles, retrovirus-like vesicles and apoptotic bodies. J. Neurooncol 113 : 1-11.
23. Mathivanan S, Ji H, Simpson RJ (2010) Exosomes: extracellular organelles important on intercellular communication. J. Proteomics 73: 1907-1920.
24. Zheng Y, Campbell EC, Lucocq J, Riches A, Powis SP. (2012). Monitoring the Rab27 associated exosome pathway using nanoparticle tracking analysis. Exp Cell Res. 12: 1706-1713.
25. Lokeshwar VB, Habuchi T, Grossman HB, Murphy WM, Hautmann SH et al. (2005) Bladder tumor markers beyond cytology: International Consensus Panel on bladder tumor markers. Urology 66: 35-63.
26. Li Y, Zhang Y, Qiu F, Qiu Z (2011) Proteomic identification of exosomal LRG1: A potential urinary biomarker for detecting NSCLC. Electrophoresis 32: 1976-1983.
27. Mitchell PJ, Welton J, Staffurth J, Court J, Mason MD etal (2009) Can urinary exosomes act as treatment response markers in prostate cancer ? J. Transl Med 7: 4.
28. Nilsson J, Skog J, Nordstrand A, Baranov V, Mincheva-Nilsson L. et al (2009) Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. Br. J. Cancer 100:1603-1607.
29. Blackwell RH, Franzen CA, Flanigan RC, Kuo PC, Gupta GN (2014) The untapped potential of urine shed bladder cancer exosomes: biomarkers, signalling and therapeutics. Bladder 1: 1-6.
30. Franzen C, Simms PE, Van-Huis AF, Foreman KE, Kuo PC (2014) Characterisation of uptake and internalization of exosomes by blader cancer cells. Biomed Research International 2014:Article ID 619829 11:619829.
31. Yang L, Wu XH, Wang D, Luo CL, Chen LX (2013) Bladder cancer cell-derived exosomes inhibit tumor cell apoptosis and induce cell proliferation in vitro. Mol Med Rep 8:1271-1278.