The mucin muc1 modulates the tumor immunological microenvironment through engagement of the lectin siglec-9

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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution

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about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS ENGAGEMENT OF SIALYLATED GLYCANS WITH SIGLEC RECEPTORS ON SUPPRESSIVE

MYELOID CELLS INHIBITS ANTICANCER IMMUNITY VIA CCL2 Article Open access 06 March 2024 CANCER-ASSOCIATED HYPERSIALYLATED MUC1 DRIVES THE DIFFERENTIATION OF HUMAN MONOCYTES INTO MACROPHAGES

WITH A PATHOGENIC PHENOTYPE Article Open access 04 November 2020 SIALIC ACIDS IN PANCREATIC CANCER CELLS DRIVE TUMOUR-ASSOCIATED MACROPHAGE DIFFERENTIATION VIA THE SIGLEC RECEPTORS SIGLEC-7

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references 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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * 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 * Division of Cancer Studies, CAR Mechanics Group, King's College London, Guy's Hospital, London, UK Daniela Achkova & John Maher *

Cell Culture Technology Group, University of Bielefeld, Bielefeld, Germany Sandra Klausing & Thomas Noll * School of Life Sciences, University of Dundee, Dundee, UK Paul R Crocker

Authors * Richard Beatson View author publications You can also search for this author inPubMed Google Scholar * Virginia Tajadura-Ortega View author publications You can also search for

this author inPubMed Google Scholar * Daniela Achkova View author publications You can also search for this author inPubMed Google Scholar * Gianfranco Picco View author publications You can

also search for this author inPubMed Google Scholar * Theodora-Dorita Tsourouktsoglou View author publications You can also search for this author inPubMed Google Scholar * Sandra Klausing

View author publications You can also search for this author inPubMed Google Scholar * Matthew Hillier View author publications You can also search for this author inPubMed Google Scholar *

John Maher View author publications You can also search for this author inPubMed Google Scholar * Thomas Noll View author publications You can also search for this author inPubMed Google

Scholar * Paul R Crocker View author publications You can also search for this author inPubMed Google Scholar * Joyce Taylor-Papadimitriou View author publications You can also search for

this author inPubMed Google Scholar * Joy M Burchell View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR

Correspondence to Joy M Burchell. ETHICS DECLARATIONS COMPETING INTERESTS 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. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–3 and Supplementary Tables 1 and 2 (PDF 976 kb) SOURCE DATA SOURCE DATA TO FIG. 1 SOURCE DATA TO FIG. 2 SOURCE DATA TO FIG. 3 SOURCE

DATA TO FIG. 4 SOURCE DATA TO FIG. 5 SOURCE DATA TO FIG. 6 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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

Download citation * Received: 28 April 2016 * Accepted: 03 August 2016 * Published: 05 September 2016 * Issue Date: November 2016 * DOI: https://doi.org/10.1038/ni.3552 SHARE THIS ARTICLE

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