Anti-leukemia effect associated with down-regulated CD47 and up-regulated calreticulin by stimulated macrophages in co-c
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ORIGINAL ARTICLE
Anti‑leukemia effect associated with down‑regulated CD47 and up‑regulated calreticulin by stimulated macrophages in co‑culture Eman M. Hassan1 · Gilbert C. Walker2 · Chen Wang3 · Shan Zou1 Received: 18 May 2020 / Accepted: 15 September 2020 © Crown 2020
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.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00262-020-02728-z) contains supplementary material, which is available to authorized users. * Shan Zou Shan.Zou@nrc‑cnrc.gc.ca 1
Metrology Research Centre, National Research Council Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada
2
Department of Chemistry, University of Toronto, Toronto, ON, Canada
3
Department of Pathology and Laboratory Medicine, Faculty of Medicine, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada
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Vol.:(0123456789)
Cancer Immunology, Immunotherapy
Graphic abstract
Keywords Acute myeloid leukemia (AML) · CD47 · Calreticulin · Lipopolysaccharide · Stimulated-macrophage phagocytosis · Co-culture 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
Introduction Acute myeloid leukemia (AML) is an aggressive malignancy with a generally poor prognosis. Chemotherapy may provide complete remission in most of AML patients (50–80%). Yet, relapse rate is high (60–90%) with approximate 5-year survival rate of 27% [1–3]. Therefore,
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