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Ozone-primed neutrophils promote early steps of tumour cell metastasis to lungs by enhancing their NET production
  1. Natacha Rocks1,
  2. Céline Vanwinge1,
  3. Coraline Radermecker2,3,
  4. Silvia Blacher1,
  5. Christine Gilles1,
  6. Raphael Marée4,
  7. Alison Gillard1,
  8. Brigitte Evrard5,
  9. Christel Pequeux1,
  10. Thomas Marichal2,3,6,
  11. Agnes Noel1,
  12. Didier Cataldo1,7
  1. 1 Laboratory of Tumor and Development Biology, GIGA Research Center, Department of Biomedical and Preclinical Sciences, University of Liège, Liège, Belgium
  2. 2 Laboratory of Cellular and Molecular Immunology, GIGA Research Center, University of Liège, Liège, Belgium
  3. 3 Faculty of Veterinary Medicine, University of Liège, Liège, Belgium
  4. 4 Montefiore Institute, Department of Electrical Engineering and Computer Science, University of Liège, Liège, Belgium
  5. 5 Laboratory of Pharmaceutical Technology and Biopharmacy, Department of Pharmacy, Center for Interdisciplinary Research on Medicines (CIRM), University of Liège, Liège, Belgium
  6. 6 WELBIO, Walloon Excellence in Life Sciences and Biotechnology, Wallonia, Belgium
  7. 7 Respiratory Diseases, CHU Liège and University of Liège, Liège, Belgium
  1. Correspondence to Professor Didier Cataldo, Laboratory of Tumor and Development Biology, GIGA Research Center, University of Liège and Departement of Respiratory Diseases, CHU Liège 4000, Belgium; didier.cataldo{at}


Background Air pollution, including particulates and gazes such as ozone (O3), is detrimental for patient’s health and has repeatedly been correlated to increased morbidity and mortality in industrialised countries. Although studies have described a link between ambient particulate matter and increased lung cancer morbidity, no direct relation has yet been established between O3 exposure and metastatic dissemination to lungs.

Objectives To outline the mechanisms through which pulmonary O3 exposure modulates metastasis kinetics in an experimental mouse model of O3 exposure.

Methods Metastatic responses to pulmonary O3 exposure were assessed using a reliable experimental mouse model of concomitant pulmonary O3 exposure and tumour cell injection. Roles of neutrophils in O3-induced lung metastasis were highlighted using blocking anti-Ly6G antibodies; moreover, the implication of neutrophil extracellular traps (NETs) in metastatic processes was evaluated using MRP8cre-Pad4lox/lox mice or by treating mice with DNase I.

Results Pulmonary O3 exposure strongly facilitates the establishment of lung metastasis by (1) Inducing a pulmonary injury and neutrophilic inflammation, (2) Influencing very early steps of metastasis, (3) Priming neutrophils’ phenotype to release NETs that favour tumour cell colonisation in lungs. The ability of O3-primed neutrophils to enhance lung colonisation by tumour cells was proven after their adoptive transfer in Balb/c mice unexposed to O3.

Conclusions Pulmonary neutrophils induced by O3 promote metastatic dissemination to lungs by producing NETs. These findings open new perspectives to improve treatment and prevention strategies in patients affected by metastatic diseases.

  • ozone pollution
  • NET
  • metastasis
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  • Contributors NR contributed to conception and design of the work, development of methodology, data collection, analysis interpretation, preparation of figures, and manuscript preparation and critical revision. CV contributed to development of methodology, data collection, analysis interpretation, preparation of figures and critical revision of the manuscript. CR gave technical support and expertise in NET experiments. SB contributed to image analysis and interpretation of results. CG helped in interpreting and validating results, guided the research strategy and critically revised the manuscript. RM supported the project by affording his expertise in quantifications of tumour sizes in histology. AG and BE critically revised the manuscript. CP provided B16K1 cells and critically revised the manuscript. TM provided expertise with NET experiments, generated and provided Mrp8;Cre+;Pad4fl/fl mice and critically revised the manuscript. AN contributed to project supervision, manuscript preparation and critically revised the manuscript. DC conceived the research program, applied to grants for funding, supervised experiments and manuscript revision, and submitted the manuscript to the editor.

  • Funding This study was financially supported by grants from the WB health program of the Walloon Region (WB Health AEROGAL, Convention n°1318023), the Fonds National pour la Recherche Scientifique (FRS-FNRS Télévie, Grant n°7463012F), the Centre AntiCancéreux (University of Liège), the Foundation against Cancer (foundation of public interest, Belgium), Interuniversity Attraction Poles Program-Belgian State-Belgian Science Policy-project P7/30 and the Fonds Léon Fredericq (University of Liège). NR is a research fellow of the Walloon Region (DGO6, WB Health AEROGAL, Convention n°1318023); RM is a research fellow of the Walloon Region (DGO6, Histoweb, grant n°1318185; Cytomine, grant n°1017072); CR was a research fellow of the F.R.S-FNRS; TM is a Research Associate of the F.R.S.-FNRS and is supported by an ERC Starting Grant (801823), an 'Incentive Grant for Scientific Research' of the F.R.S.-FNRS (F.4508.18), by the FRFS-WELBIO under grant CR-2017s-04 and by the Acteria Foundation.

  • Competing interests DC is the founder of Aquilon Pharmaceuticals, received speaker fees from AstraZeneca, Boehringer-Ingelheim, Novartis, MundiPharma, Chiesi and GSK and received consultancy fees from AstraZeneca, Boehringer-Ingelheim, and Novartis for the participation to advisory boards. None of these activities have any connection with oncology or development of drugs in the field of oncology.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.

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