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The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9

Nature Immunology volume 17, pages 1273–1281 (2016)Cite this article

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Abstract

Siglec-9 is a sialic-acid-binding lectin expressed predominantly on myeloid cells. Aberrant glycosylation occurs in essentially all types of cancers and results in increased sialylation. Thus, when the mucin MUC1 is expressed on cancer cells, it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). Here we found that this cancer-specific MUC1 glycoform, through engagement of Siglec-9, ‘educated’ myeloid cells to release factors associated with determination of the tumor microenvironment and disease progression. Moreover, MUC1-ST induced macrophages to display a tumor-associated macrophage (TAM)-like phenotype, with increased expression of the checkpoint ligand PD-L1. Binding of MUC1-ST to Siglec-9 did not activate the phosphatases SHP-1 or SHP-2 but, unexpectedly, induced calcium flux that led to activation of the kinases MEK-ERK. This work defines a critical role for aberrantly glycosylated MUC1 and identifies an activating pathway that follows engagement of Siglec-9.

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Figure 1: MUC1-ST binds to primary monocytes and macrophages through Siglec-9.
Figure 2: MUC1-ST induces monocytes to secrete factors associated with tumor progression in a Siglec-9-dependent manner.
Figure 3: Engagement of Siglec-9 by MUC1-ST during the differentiation of monocytes into inflammatory macrophages results in the generation of dysfunctional cells.
Figure 4: ‘MUC1-ST-educated’ macrophages differentiated via M-CSF secrete factors associated with tumor progression in a Siglec-9-dependent manner.
Figure 5: ‘MUC1-ST-educated’ macrophages differentiated via M-CSF differentiate into TAMs.
Figure 6: Binding of MUC1-ST to myeloid cells via Siglec-9 leads to calcium flux and activation of MEK-ERK.

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Acknowledgements

We thank V. Corrigall (King's College London) for tocilizumab; and N. O'Reilly for the lyophilization of samples. Supported by Breast Cancer Now (2011NovPR-43), the Medical Research Council (MR/J007196/1), the Department the Experimental Cancer Medicine Centre at King's College London, the National Institute for Health Research Biomedical Research Centre based at Guy's and St Thomas’ NHS Foundation Trust and King's College London. The views expressed are those of the authors and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.

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Authors and Affiliations

  1. Division of Cancer Studies, Breast Cancer Biology Group, King's College London, Guy's Hospital, London, UK

    Richard Beatson, Virginia Tajadura-Ortega, Gianfranco Picco, Theodora-Dorita Tsourouktsoglou, Matthew Hillier, Joyce Taylor-Papadimitriou & Joy M Burchell

  2. Division of Cancer Studies, CAR Mechanics Group, King's College London, Guy's Hospital, London, UK

    Daniela Achkova & John Maher

  3. Cell Culture Technology Group, University of Bielefeld, Bielefeld, Germany

    Sandra Klausing & Thomas Noll

  4. School of Life Sciences, University of Dundee, Dundee, UK

    Paul R Crocker

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Contributions

R.B. and J.M.B. designed the study and wrote the manuscript with comments from all authors; R.B. performed the experiments with the assistance of D.A., G.P., T.-D.T. and M.H.; V.T.-O. performed the quantitative RT-PCR; S.K. and T.N. cultured the Chinese hamster ovary cells in bulk; J.M. and P.R.C. supplied reagents; and J.T.-P. contributed to scientific discussions and approaches.

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Correspondence to Joy M Burchell.

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J.M.B. is a consultant to Palleon Pharma, and P.R.C. is a scientific co-founder of Palleon Pharma.

Integrated supplementary information

Supplementary Figure 1 MUC1-ST binds to Siglec-9.

(a) Representative flow cytometry histograms showing biotinylated MUC1-T or MUC1-ST binding to isolated or differentiated immune subsets from healthy donors. N=4 independent donors. (b,c) Monocytes were incubated with biotinylated MUC1-ST for (b) different time periods or (c) using different concentrations. N=2 independent donors. (d) Monocytes were treated ± neuraminidase before being incubated with 10µg/ml MUC1 glycoforms. N=3 independent donors. (e) Monocytes (N=3) and MCSF MΦ (N=2) were stained with antibodies to MUC1-ST binding Siglecs and analysed by flow cytometry. (f) Monocytes were treated with indicated concentrations of antibodies to Siglecs-3, 7 and 9 before being incubated with MUC1-ST. Graph illustrates % binding inhibition as calculated against MUC1-ST plus isotype MFI. N=3 independent donors. (g) Representative flow cytometry histograms showing the binding of MUC1-ST or PAA-ST in the presence of anti-Siglec-9 or isotype for both primary monocytes and U937 cells. Data shown are the mean and s.e.m. * p<0.05 ** p<0.01 *** p<0.001, paired or unpaired Student’s t-test where appropriate.

Supplementary Figure 2 MUC1-ST induces monocytes to secrete factors associated with tumor progression and modulates the differentiation of monocytes into dendritic cells (moDCs).

(a) The supernatant from MUC1-ST educated monocytes was analysed using a protein array. Highlighted factors: 1, CXCL5, 2, Chitinase 3-like 1, 3 IL-8, 4 CCL3, 5 IL17A, 6 MMP-9, 7 CCL2, 8 PAI-1, 9 IL6, 10 CXCL1. (b,c) Pooled MFI data showing CD40, CD83, HLA-DR and CD86 expression on day 7 immature (b) or mature (c) moDCs after treatment with MUC1-ST on day 0. N=6 independent donors. (d) Representative flow cytometry histograms showing CD86 expression on day 7 immature or mature moDCs after treatment with MUC1-ST on day 0 in the presence of isotype or anti Siglec-9 or anti IL-6Rα. (e,f,g) Pooled cytometric data showing CD86 (e,f) and CD83 (g) expression on day 7 immature (e) or mature (f,g) moDCs after treatment with MUC1-ST on day 0 in the presence of isotype or anti-Siglec-9 or anti-IL-6Rα. N=6 independent donors. (h) IL-12 p70 release from day 7 mature moDCs after treatment with MUC1-ST on day 0 in the presence of isotype or anti-Siglec-9 or anti-IL-6Rα. N= 6 independent donors. Data shown are the mean and s.e.m. * p<0.05 ** p<0.01 *** p<0.001, paired or unpaired Student’s t-test where appropriate.

Supplementary Figure 3 Proposed model of MUC1-ST-mediated modulation of the tumor microenvironment via engagement of Siglec-9.

Transformation associated inflammation induces the expression of MUC1 and COX-2 which in turn upregulates ST3Gal-I49. This increases the presence of MUC1-ST, which via Siglec-9, educates monocytes and macrophages to release factors involved in immune recruitment and tumor progression. Additionally, Siglec-9 engagement by MUC1-ST on monocytes results in altered differentiation and a dysfunctional phenotype, and on macrophages induces a TAM-like phenotype with increased expression of CD206, CD163, IDO and PD-L1, and poor CD8+ co-stimulatory ability. This cycle is maintained by MUC1-ST/Siglec-9 induced factors being able to upregulate ST3Gal-1 and MUC1, thus ensuring conservation of the microenvironment.

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Beatson, R., Tajadura-Ortega, V., Achkova, D. et al. The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9. Nat Immunol 17, 1273–1281 (2016). https://doi.org/10.1038/ni.3552

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