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Anti-leukemia effect associated with down-regulated CD47 and up-regulated calreticulin by stimulated macrophages in co-culture

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Abstract

CD47 is over-expressed in Acute Myeloid Leukemia (AML) and functions as an inhibitory signal, suppressing phagocytosis by binding to signal regulatory protein α (SIRPα) on the surface of macrophages. Inhibition of CD47 restores the immune surveillance of AML cells. However, the inhibition of CD47 in AML by activated macrophages and the subsequent effects on different immune response parameters are not fully understood. Here, we demonstrate the use of a distinct co-culture method to inhibit CD47 and therefore eliminate AML cells by macrophages in vitro. Human chemically induced THP-1 macrophages were activated using different concentrations of lipopolysaccharide (LPS) and co-culturing with three AML cancer cell lines (HL-60, NB4, and THP-1), respectively, as well as normal human peripheral blood mononuclear cells (PBMC). CD47 inhibition was observed in and selective to AML but not observed in normal PBMC. Additionally, calreticulin (CRT) levels were elevated in the same cell lines simultaneously, after co-culturing with activated human macrophages, but not elevated in normal cells. We also show that the activated macrophages secreted high levels of cytokines, including IL-12p70, IL-6, and TNF-α, consistent with the elimination of AML by macrophages. Our study reveals the potential of this model for screening new drugs against AML and the possibility of using human macrophages in AML treatment in the future.

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Data availability

The datasets are available from the corresponding author on reasonable request.

Abbreviations

AML:

Acute myeloid leukemia

CRT:

Calreticulin

DMEM:

Dulbecco’s Modified Eagle’s Medium

FBS:

Fetal bovine serum

HBSS:

Hank’s balanced salt solution

LPS:

Lipopolysaccharide

MFI:

Mean of fluorescence intensity

PBMC:

Primary peripheral blood mononuclear cells

SIRPα:

Signal regulatory protein alpha

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Acknowledgements

We thank Mr. Henk van Faassen for his assistance on the flow cytometry measurements.

Funding

This work was financially supported by the National Research Council of Canada.

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Authors and Affiliations

  1. Metrology Research Centre, National Research Council Canada, 100 Sussex Drive, Ottawa, ON, K1A 0R6, Canada

    Eman M. Hassan & Shan Zou

  2. Department of Chemistry, University of Toronto, Toronto, ON, Canada

    Gilbert C. Walker

  3. Department of Pathology and Laboratory Medicine, Faculty of Medicine, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada

    Chen Wang

Authors
  1. Eman M. Hassan
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  2. Gilbert C. Walker
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  3. Chen Wang
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  4. Shan Zou
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Contributions

Conception and design: S.Z and G.W. Development of methodology: E.H and S.Z. Acquisition of data: E.H. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and computational analysis): E.H and S.Z. Writing, review, and/or revision of the manuscript: E.H, S.Z, G.W, and C.W. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): E.H and S.Z. Study supervision: S.Z.

Corresponding author

Correspondence to Shan Zou.

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No human or animal sample was involved in the study. The research is carried out in compliance with the biosafety and biosecurity protocols at the National Research Council Canada.

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A submission approval is granted by the National Research Council Canada, with consent emails from all authors.

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Hassan, E.M., Walker, G.C., Wang, C. et al. Anti-leukemia effect associated with down-regulated CD47 and up-regulated calreticulin by stimulated macrophages in co-culture. Cancer Immunol Immunother 70, 787–801 (2021). https://doi.org/10.1007/s00262-020-02728-z

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