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C3a elicits unique migratory responses in immature low-density neutrophils

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Abstract

Neutrophils represent the immune system’s first line of defense and are rapidly recruited into inflamed tissue. In cancer associated inflammation, phenotypic heterogeneity has been ascribed to this cell type, whereby neutrophils can manifest anti- or pro-metastatic functions depending on the cellular/micro-environmental context. Here, we demonstrate that pro-metastatic immature low-density neutrophils (iLDNs) more efficiently accumulate in the livers of mice bearing metastatic lesions compared with anti-metastatic mature high-density neutrophils (HDNs). Transcriptomic analyses reveal enrichment of a migration signature in iLDNs relative to HDNs. We find that conditioned media derived from liver-metastatic breast cancer cells, but not lung-metastatic variants, specifically induces chemotaxis of iLDNs and not HDNs. Chemotactic responses are due to increased surface expression of C3aR in iLDNs relative to HDNs. In addition, we detect elevated secretion of cancer-cell derived C3a from liver-metastatic versus lung-metastatic breast cancer cells. Perturbation of C3a/C3aR signaling axis with either a small molecule inhibitor, SB290157, or reducing the levels of secreted C3a from liver-metastatic breast cancer cells by short hairpin RNAs, can abrogate the chemotactic response of iLDNs both in vitro and in vivo, respectively. Together, these data reveal novel mechanisms through which iLDNs prefentially accumulate in liver tissue harboring metastases in response to tumor-derived C3a secreted from the liver-aggressive 4T1 breast cancer cells.

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Fig. 1: iLDNs efficiently infiltrate metastasis bearing livers when compared with HDNs.
Fig. 2: iLDNs preferentially migrate toward conditioned media generated from liver-aggressive 4T1 breast cancer cells (2776).
Fig. 3: C3a–C3aR axis is engaged in iLDNs migrating toward conditioned media derived from liver-aggressive 4T1 breast cancer cells (2776).
Fig. 4: Inhibition of C3aR blocks the preferential migration of iLDNs toward conditioned media derived from liver-aggressive 4T1 breast cancer cells (2776).

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Data availability

The accession number for the RNA-sequencing data is GEO: GSE123669. All informatics analyses were performed with R, an open source software environment for statistical computer and graphics. The software is available for download at https://cran.r-project.org/. Chemotaxis analyses were performed in Matlab (Mathwork). Further details are presented in the supplementary data.

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Acknowledgements

We acknowledge the Goodman Cancer Research Centre histology core facility (McGill University) for routine histological services, the Cell Vision Core Facility (McGill University) for providing routine cell sorting service and the Genetics Perturbation Service for shRNAs (McGill University). We thank Anie Monast and Cynthia Lavoie for technical support and members of the Siegel laboratory for their thoughtful discussions and critical reading of the manuscript. This work was supported by an operating grant to P.M.S. from the Cancer Research Society and the Terry Fox Research Institute and Québec Breast Cancer Foundation (Grant #: 242122). S.C. acknowledges funding support from NSERC and Genome Canada and salary awarded from FRQS. J.D.S acknowledges funding support from AATS, MGH foundation and FRQS. BEH acknowledges support from the Charlotte and Leo Karassik Foundation PhD Fellowship and the Rolande and Marcel Gosselin Graduate Studentship. PMS is a McGill University William Dawson Scholar.

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Authors and Affiliations

  1. Goodman Cancer Research Centre, McGill University, Montréal, QC, H3A 1A3, Canada

    Brian E. Hsu, LeeAnn Ramsay, Sébastien Tabariès, Matthew G. Annis, Ian R. Watson & Peter M. Siegel

  2. Department of Medicine, McGill University, Montréal, QC, H3G 1Y6, Canada

    Brian E. Hsu & Peter M. Siegel

  3. Research Centre, Maisonneuve-Rosemont Hospital, Montréal, QC, H1T 2M4, Canada

    Joannie Roy & Santiago Costantino

  4. Biomedical Engineering Institute, University of Montréal, Montréal, QC, H3C 3T5, Canada

    Joannie Roy & Santiago Costantino

  5. Department of Surgery, Research Institute of the McGill University Health Center, Montréal, QC, H4A 3J1, Canada

    Jack Mouhanna, Roni F. Rayes & Jonathan D. Spicer

  6. Department of Biochemistry, McGill University, Montréal, QC, H3G 1Y6, Canada

    Ian R. Watson & Peter M. Siegel

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Contributions

Conceptualization, BEH, JR, JDS, SC, PMS; Methodology, BEH, JR, JM, ST, JDS, SC, PMS; Validation, BEH, JR, ST, JM, RFR; Formal analysis, BEH, JR, ST, JM, RFR, LR, JDS, SC, PMS; Investigation, BEH, JR, ST; Resources, BEH, JR, JM, RFR, ST, MGA, IRW, JDS, SC, PMS; Data Curation, BEH, JR, JM, RFR, ST; Writing-Original Draft, BEH, JR, PMS; Writing-Review & Editing, BEH, JR, SC, PMS; Visualization, BEH, JR, LR, PMS; Supervision, ST, SC, PMS; Project administration, PMS; Funding acquisition, PMS.

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Correspondence to Peter M. Siegel.

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Hsu, B.E., Roy, J., Mouhanna, J. et al. C3a elicits unique migratory responses in immature low-density neutrophils. Oncogene 39, 2612–2623 (2020). https://doi.org/10.1038/s41388-020-1169-8

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