Extracellular Vesicles in Biological Fluids. A Biomarker of Exposure to Cigarette Smoke and Treatment with Chemopreventive drugs


Extracellular vesicles
cigarette smoke
bronchoalveolar lavage fluid
blood serum


Extracellular vesicles (EVs) are released from cells and enter into body fluids thereby providing a toxicological mechanism of cell-cell communication. The present study aimed at assessing (a) the presence of EVs in mouse body fluids under physiological conditions, (b) the effect of exposure of mice to cigarette smoke for 8 weeks, and (c) modulation of smoke-related alterations by the nonsteroidal anti-inflammatory drug celecoxib, a selective cyclooxygenase-2 inhibitor. To this purpose, ICR (CD-1) mice were either unexposed or exposed to cigarette smoke, either treated or untreated with oral celecoxib. EVs, isolated from bronchoalveolar lavage fluid (BALF), blood serum, and urines, were analyzed by nanoparticle tracking analysis and flow cytometry. EVs baseline concentrations in BALF were remarkably high. Larger EVs were detected in urines. Smoking increased EVs concentrations but only in BALF. Celecoxib remarkably increased EVs concentrations in the blood serum of both male and female smoking mice. The concentration of EVs positive for EpCAM, a mediator of cell-cell adhesion in epithelia playing a role in tumorigenesis, was much higher in urines than in BALF, and celecoxib significantly decreased their concentration. Thus, the effects of smoke on EVs concentrations were well detectable in the extracellular environment of the respiratory tract, where they could behave as delivery carriers to target cells. Celecoxib exerted both protective mechanisms in the urinary tract and adverse systemic effects of likely hepatotoxic origin in smoke-exposed mice. Detection of EVs in body fluids may provide an early diagnostic tool and an end-point exploitable for preventive medicine strategies.




Bonzini, M., Pergoli, L., Cantone, L., Hoxha, M., Spinazzè, A., Del Buono, L., Favero, C., Carugno, M., Angelici, L., Broggi, L., Cattaneo, A., Pesatori, A.C., Bollati, V. 2017. Short-term particulate matter exposure induces extracellular vesicle release in overweight subjects. Environ. Res .155, 228-234.

Neven, K.Y., Nawrot, T.S., Bollati, V. 2017. Extracellular vesicles: how the external and internal environment can shape cell-to-cell communication. Curr. Environ. Health Rep. 4, 30-37.

Lötvall J, Hill AF, Hochberg F, Buzás EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW, Théry C. 2014.Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 3, 26913.

Larson, M.C., Hillery, C.A., Hogg, N. 2014. Circulating membrane-derived microvesicles in redox biology. Free Radic. Biol. Med. 7, 214-228.

Ragni, E., Banfi, F., Barilani, M., Cherubini, A., Parazzi, V., Larghi, P., Dolo, V., Bollati, V., Lazzari, L. 2017. Extracellular vesicle-shuttled mRNA in mesenchymal stem cell communication. Stem Cells 35, 1093-1105.

Robbins, P.D., Morelli, A.E. 2014. Regulation of immune responses by extracellular vesicles. Nat. Rev. Immunol. 14, 195-208.

Hunter, M.P., Ismail, N., Zhang, X., Aguda, B.D., Lee, E.J., Yu, L., Xiao, T., Schafer, J., Lee, M.L., Schmittgen, T.D, Nana Sinkam SP., Jarjoura D., Marsh, C.B. 2008. Detection of microRNA expression in human peripheral blood microvesicles. PLoS One 3, e3694

Raposo, G., Stoorvogel, W. 2013. Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol. 200, 373-383.

van Niel, G., D'Angelo, G., Raposo, G. 2018. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19, 213-228.

Prakash, P.S., Caldwell, C.C., Lentsch, A.B., Pritts, T.A., Robinson, B.R. 2012. Human microparticles generated during sepsis in patients with critical illness are neutrophil-derived and modulate the immune response. J. Trauma Acute Care Surg. 73, 401-406.

Jüngel, A., Distler, O., Schulze-Horsel, U., Hube,r L.C., Ha, H.R., Simmen, B., Kalden, J.R., Pisetsky, D.S., Gay, S., Distler, J.H. 2007. Microparticles stimulate the synthesis of prostaglandin E(2) via induction of cyclooxygenase 2 and microsomal prostaglandin E synthase 1. Arthritis Rheum. 56, 3564-3574.

Charoenviriyakul, C., Takahashi, Y., Morishita, M., Nishikawa, M., Takakura, Y. 2018. Role of extracellular vesicle surface proteins in the pharmacokinetics of extracellular vesicles. Mol. Pharm. 15, 1073-1080.

Nadaud, S., Poirier, O., Girerd, B., Blanc, C., Montani, D., Eyries, M., Imbert-Bismut, F., Pacheco, A., Vigne, J., Tregouet, D.A., Humbert, M., Soubrier, F. 2013. Small platelet microparticle levels are increased in pulmonary arterial hypertension. Eur. J. Clin. Invest. 43, 64-71.

Thomashow, M.A., Shimbo, D., Parikh, M.A., Hoffman, E.A., Vogel-Claussen, J., Hueper, K., Fu, J., Liu, C.Y., Bluemke, D.A., Ventetuolo, C.E., Doyle, M.F., Barr, R.G. 2013. Endothelial microparticles in mild chronic obstructive pulmonary disease and emphysema. The Multi-Ethnic Study of Atherosclerosis Chronic Obstructive Pulmonary Disease study. Am. J. Respir. Crit. Care Med. 188, 60-68.

Vykoukal, J., Sun, N., Aguilar-Bonavides, C., Katayama, H., Tanaka, I., Fahrmann, J.F., Capello, M., Fujimoto, J., Aguilar, M., Wistuba, I.I., Taguchi, A., Ostrin, E.J., Hanash, S.M. 2017. Plasma-derived extracellular vesicle proteins as a source of biomarkers for lung adenocarcinoma. Oncotarget 8, 95466-95480

International Agency for Research on Cancer 2012. A review of human carcinogens: personal habits and indoor combustions. IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans 100, part E. IARC, Lyon, France.

De Flora, S., Izzotti, A., D'Agostini, F., La Maestra, S., Micale, R.T., Ceccaroli, C., Steele, V.E., Balansky R 2014. Rationale and approaches to the prevention of smoking-related diseases: overview of recent studies on chemoprevention of smoking-induced tumors in rodent models. J.

Environ. Sci. Health C Environ. Carcinog. Ecotoxicol. Rev. 32, 105-120.

Li, C.J., Liu, Y., Chen, Y., Yu, D., Williams, K.J., Liu, M.L. 2013. Novel proteolytic microvesicles released from human macrophages after exposure to tobacco smoke. Am. J. Pathol. 182, 1552-1562.

Cordazzo, C., Petrini, S., Neri, T., Lombardi, S., Carmazzi, Y., Pedrinelli, R., Paggiaro, P., Celi, A. 2014. Rapid shedding of proinflammatory microparticles by human mononuclear cells exposed to cigarette smoke is dependent on Ca2+ mobilization. Inflamm. Res. 63, 539-547.

Benedikter, B.J., Volgers, C., van Eijck, P.H., Wouters, E.F.M., Savelkoul, P.H.M., Reynaert, N.L., Haenen, G.R.M.M., Rohde G.G.U., Weseler, A.R., Stassen, F.R.M. 2017. Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants. Free Radic. Biol. Med. 108, 334-344.

De Flora, S., Izzotti, A., D'Agostini, F., Balansky, R.M. 2001. Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points. Carcinogenesis 22, 999-1013. Armstrong, A., Eck, S.L. 2003. EpCAM: A new therapeutic target for an old cancer antigen. Cancer Biol. Ther. 2, 320-326.

Gordon, C., Gudi, K., Krause, A., Sackrowitz, R., Harvey, B.G., Strulovici-Barel, Y., Mezey, J.G., Crystal, R.G. 2011. Circulating endothelial microparticles as a measure of early lung destruction in cigarette smokers. Am. J. Respir. Crit. Care Med. 184, 224-232.

Mobarrez, F., Antoniewicz, L., Bosson, J.A., Kuhl, J., Pisetsky, D.S., Lundbäck, M. 2014. The effects of smoking on levels of endothelial progenitor cells and microparticles in the blood of healthy volunteers. PLoS One 9, e90314.

Héliot, A., Landkocz, Y., Roy Saint-Georges, F., Gosset, P., Billet, S., Shirali, P., Courcot, D., Martin, P.J. 2017. Smoker extracellular vesicles influence status of human bronchial epithelial cells. Int. J. Hyg. Environ. Health 220 (2 Pt B), 445-454.

Izzotti A, La Maestra S, Micale RT, Pulliero A, Geretto M, Balansky R, De Flora S. 2018a. Modulation of genomic and epigenetic end-points by celecoxib. Oncotarget.9, 33656-33681.

Izzotti, A., Longobardi, M., La Maestra, S., Micale, R.T., Pulliero, A., Camoirano, A, Geretto, M, D'Agostini, F, Balansky, R., Miller, M.S., Steele, V.E., De Flora, S. 2018b. Release of microRNAs into body fluids from ten organs of mice exposed to cigarette smoke. Theranostics 8, 2147-2160.

Grivennikov, S.I., Greten, F.R., Karin, M. 2010. Immunity, inflammation, and cancer. Cell 140, 883-899

Malkinson, A.M. 2004. Evidence that inflammation encourages pulmonary adenocarcinoma formation in mice: clinical implications. Chest 125, 154S-155S.

Takahashi, H., Ogata, H., Nishigaki,, R. .2010. Tobacco smoke promotes lung tumorigenesis by triggering IKKbeta- and JNK1-dependent inflammation. Cancer Cell 17, 89-97.

Mazhar, D., Gillmore, R., and Waxman, J. 2005. COX and cancer. QJM 98, 711-718.

Wolfe, F., Anderson, J., Burke, T.A., Arguelles, L.M., Pettitt, D. 2002. Gastroprotective therapy and risk of gastrointestinal ulcers: risk reduction by COX-2 therapy. J. Rheumatol. 29, 467-473.

Balansky, R., Ganchev, G., Iltcheva,, M., Nikolov, M., La Maestra, S., Micale, R.T., D'Agostini, F., Steele, V.E., De Flora, S. 2015. Modulation by licofelone and celecoxib of histopathological alterations in the lung and urinary tract of mice exposed to cigarette smoke. Curr. Cancer Drug Targets 15, 188-195.

Royo F, Zuñiga-Garcia P, Sanchez-Mosquera P, Egia A, Perez A, Loizaga A, Arceo R, Lacasa I, Rabade A, Arrieta E, Bilbao R, Unda M, Carracedo A, Falcon-Perez JM. 2016. Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples. J. Extracell. Vesicles. 15; 29497.

Murakami, T., Oakes, M., Ogura, M., Tovar, V., Yamamoto, C., and Mitsuhashi, M. 2014. Development of glomerulus, tubule and collecting duct-specific mRNA assay in human urinary exosomes and microvesicles. PLoS One 9, e109074.

Pollock, K., Albares, L., Wendt, C., Hubel, A. 2013. Isolation of fibroblasts and epithelial cells in bronchoalveolar lavage (BAL). Exp. Lung Res. 39, 146-154.

Litvinov, S.V., Balzar, M., Winter, M.J., Bakker, H.A., Briaire-de Bruijn, I.H., Prins, F., Fleuren, G.J., Warnaar, S.O. 1997. Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins. J. Cell Biol. 139,1337-1348.

Armstrong A., Eck, S:L. EpCAM: A new therapeutioc target for an old cancer antigen. Cancer Biol.ther.2,320-326.

Trzpis, M., McLaughlin, P.M., de Leij, L.M., Harmsen, M.C. 2007. Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule. Am. J. Pathol. 171, 386-395.

Slocombe, R. 1999. Surfactant and its role in pulmonary disease.


Effros, R.M. 1991. Permeability of the blood-gas barrier. In The Lung: Scientific Foundations (R.G. Crystal,RG, and J.B.West,JB, Eds.) pp. 1163–1175. Raven Press, New York.

De Flora, S., Balansky, R., D'Agostini, F., Cartiglia, C., Longobardi, M., Steele, V.E., Izzotti, A. 2012. Smoke-induced microRNA and related proteome alterations and their modulation by chemopreventive agents. Int. J. Cancer 131, 2763-2773.

De Flora, S., Ganchev, G., Iltcheva, M., La Maestra, S., Micale, R.T., Steele, V.E., Balansky, R. 2016. Pharmacological modulation of lung carcinogenesis in smokers: preclinical and clinical evidence. Trends Pharmacol. Sci. 37, 120-142.

Castleden, C.M., Cole, P.V. 1975. Carboxyhaemoglobin levels of smokers and non-smokers working in the City of London. Br. J. Ind. Med. 32, 115–118.

Camoirano, A., Bagnasco, M., Bennicelli, C., Cartiglia, C., Wang, J.B., Zhang, B.C., Zhu, Y.R., Qian, G.S., Egner, P.A., Jacobson, L.P., Kensler, T.W., De Flora, S. 2001. Oltipraz chemoprevention trial in Qidong, People's Republic of China: results of urine genotoxicity assays as related to smoking habits. Cancer Epidemiol. Biomarkers Prev. 10, 775-783.

Molina-Pinelo, S., Suárez, R., Pastor, M.D., Nogal, A., Márquez-Martín, E., Martín-Juan, J., Carnero, A., Paz-Ares, L. 2012. Association between the miRNA signatures in plasma and bronchoalveolar fluid in respiratory pathologies. Dis. Markers 32, 221-230.

Hunninghake, G.W., Gadek, J.E., Kawanami, O., Ferrans, V.J., Crystal, R.G. 1979. Inflammatory and immune processes in the human lung in health and disease: evaluation by bronchoalveolar lavage. Am. J. Pathol. 97, 149–206.

Ballinger, C.A., Brand, J.D., Postlethwait, E.M. 2010. In vitro systems for studying respiratory system toxicology. In Comprehensive Toxicology (C.A. McQueen, Ed.), pp. 243-259. Elsevier Ltd, Oxford, UK.

Sozer, S., Diniz, G., Lermioglu, F. 2011. Effects of celecoxib in young rats: histopathological changes in tissues and alterations of oxidative stress/antioxidant defense system. Arch. Pharm. Res. 34, 253-259.

Bryan, R.T., Shimwell, N.J., Wei, W., Devall, A.J., Pirrie, S.J., James, N.D., Zeegers, M.P., Cheng, K.K., Martin, A., Ward, D.G. 2014.Urinary EpCAM in urothelial bladder cancer patients: characterisation and evaluation of biomarker potential. Br. J. Cancer 110, 679-685.

Davies, N.M., McLachlan, A.J., Day, R.O., Williams, K.M. 2000. Clinical pharmacokinetics and pharmacodynamics of celecoxib: a selective cyclo-oxygenase-2 inhibitor. Clin. Pharmacokinet. 38, 225-242.

Liu, H., Wei, W., Li, X. 2009. Celecoxib exacerbates hepatic fibrosis and induces hepatocellular necrosis in rats treated with porcine serum. Prostaglandins Other Lipid Mediat. 88,63-67.

Koçkaya, E.A., Selmanoğlu, G., Kismet, K. 2010. Pathological and biochemical effects of therapeutic and supratherapeutic doses of celecoxib in Wistar albino male rats. Drug Chem. Toxicol. 33, 410-414.

Winterhalder, R.C., Hirsch, F.R., Kotantoulas, G.K., Kotantoulas, G.K., Franklin, W.A., Bunn, P.A. Jr. 2004. Chemoprevention of lung cancer. From biology to clinical reality. Ann. Oncol. 15, 185-196.

Tranah, G.J., Chan, A.T., Giovannucci, E., Ma, J., Fuchs, C., Hunter, D.J. 2005. Epoxide hydrolase and CYP2C9 polymorphisms, cigarette smoking, and risk of colorectal carcinoma in the Nurses' Health Study and the Physicians' Health Study. Mol. Carcinog. 44, 21-30.

Reilly, T.P., Brady, J.N., Marchick, M.R., Bourdi, M., George, J.W., Radonovich, M.F., Pise-Masison, C.A., Pohl, L.R. 2001. A protective role for cyclooxygenase-2 in drug-induced liver injury in mice. Chem. Res. Toxicol. 14, 1620-1628.

Royo F, Palomo L, Mleczko J, Gonzalez E, Alonso C, Martínez I, Pérez-Cormenzana M, Castro A, Falcon-Perez JM. 2017. Metabolically active extracellular vesicles released from hepatocytes under drug-induced liver-damaging conditions modify serum metabolome and might affect different pathophysiological processes. Eur. J. Pharm. Sci. 98, 51-57.Sereno, J., Parada, B., Reis, F., Cunha, F.X., Teixeira-Lemo,s E., Garrido, P., Pinto, R., Rocha-Pereira, P., Neto, P., Ruivo, J., Rodrigues-Santos, P., Nunes, S., Mota, A., Figueiredo, A., Teixeira, F. 2010. Preventive but not curative efficacy of celecoxib on bladder carcinogenesis in a rat model. Mediators Inflamm. 2010, 380937.

Boukouris, S., Mathivanan, S. 2015. Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteomics Clin. Appl. 9, 358-367.