The Expression Level Of CD47 In Acute Myeloid Leukemia And Its Regulation On Macrophage-targeted Phagocytosis

Background and objective:Acute myeloid leukemia(AML)is a kind of life-threatening malignant disease originating from blood and bone marrow,with a high rate of incidence and mortality among Chinese adults.Anthracyclic drugs combined with standard dose cytarabine has been the first-line chemotherapy regimen for AML(except for acute promyelocytic leukemia)for many years,but 10-40% of young AML patients and 40-60% of elderly patients will relapse after induction treatment.Chemotherapy combined with immunotherapy is a promising combination therapy for AML patients.In recent years,treatments targeted at immune checkpoints has become the focus of attention in the field of tumor immunotherapy,such as the T cell immune checkpoint PD-1/PD-L1.In addition,many researchers began to pay attention to innate immunity in order to develop new anti-tumor drugs through the immune checkpoints of innate immunity.The macrophage immune checkpoint CD47 is a promising target for hematological malignancies.CD47 is a transmembrane protein that widely exists on the surface of normal cells,and it can regulate the signal of "don’t eat me" through combining with the signal regulatory protein α(SIRPα)on the phagocytes to limit the phagocytosis.Therefore,it is usually over-expressed on the surface of tumor cells and becomes the "accomplice" of tumor cell immune escape.The anti-CD47 monoclonal antibody can block the interaction of CD47-SIRPα to promote the elimination of tumor cells in vitro and in vivo.Since CD47 also broadly expresses on normal cells,CD47 blocking treatment may affect normal cells and cause a knockout effect.It has been pointed out that the targeting of CD47 blocking therapy depends on the relative expression of anti-phagocytic CD47 and prophagocytic signals.Surface calreticulin(CRT)is one of the key pro-phagocytic signals that have been found so far.It usually exists in the endoplasmic reticulum(ER)and can be transferred to the cell surface during cell damage or stress response used as a pro-phagocytic signal to mark cell clearance.Therefore,we hypothesized that up-regulating CRT exposure combined with anti-CD47 immunotherapy can promote macrophage phagocytosis targeted on AML cells,thus we can effectively "awaken" the innate tumor immunity.Some drugs such as anthracyclines,oxaliplatin,ultraviolet and γ radiation can induce the CRT exposure on the surface of cancer cells,but these induction methods can not only induce the CRT exposure on the surface of malignant cells,but also nonspecifically up-regulate this phagocytic signal on normal cells,thus leading to the potential toxicity of CD47 blocking immunotherapy.Metformin is an oral drug widely used for type 2 diabetes,and decades of clinical experience has proved that it is a safe and well tolerated drug.Many studies show that metformin has anti-cancer effects and immunomodulatory abilities in various types of cancer.In addition,some studies have pointed out that metformin targets the NNAT-mediated ER stress pathway by regulating the expression of mi R-708-5p,and the expression of the ER stress marker CRT is also up-regulated,finally inducing the apoptosis of prostate cancer cells.It is very attractive to explore metformin in the context of inducing CRT exposure and stimulating antitumor immune response.To sum up,we speculate that the addition of Metformin may up-regulate the expression of CRT exposure of AML cells,enhance the phagocytosis of macrophages targeting AML,and then enhance its immunotherapeutic effect in cooperation with CD47 blocker.The purpose of this study is to determine the expression level of CD47 in AML blasts and non-leukemia cells,verify the relationship between the phagocytosis of CD47 blocker mediated macrophage targeting AML cells and the CRT-CD47 phagocytosis signal axis,meanwhile analyze the expression level of CRT surface expression on AML blasts and non-leukemia cells and its feasibility of improving the targeting of CD47 blocking therapy.Then we aim at studying the effect of metformin on the CRT-CD47 phagocytosis signal axis,the effect of metformin combined with CD47 blocker on the phagocytosis of macrophage targeting AML cells and the related mechanisms.Research objectives:(1)The expression level of CD47 on AML blasts and non-leukemia cells and its feasibility as a new target for AML targeted immunotherapy.(2)Effects of metformin combined with CD47 blocker on macrophage phagocytosis targeting AML cells and its mechanism.Research methods:(1)The expression level of CD47 in AML blasts and non-leukemia cells and its feasibility as a new target for AML targeted immunotherapy.1.Using the peripheral blood samples of AML patients and healthy donors in our hospital to analyze the difference of CD47 expression level between AML blasts and non-leukemia cells and the correlation with clinical data of patients.2.Conducting phagocytosis test in vitro after using CD47 blocker to treat AML cells and mononuclear cells from healthy donors,then detecting the surface expression of CRT,and analyze the relationship between CRT exposure expression and CD47blocker-mediated macrophage targeted phagocytosis.3.Using the peripheral blood samples of AML patients and healthy donors in our hospital to analyze the difference in the expression level of surface CRT on AML blasts and non-leukemia cells,and determining whether this molecule has the potential to affect the targeting of CD47 blocking immunotherapy in AML.(2)Effects of metformin combined with CD47 blocker on macrophage phagocytosis targeting AML cells and its mechanism.1.Using flow cytometry,PCR and immunofluorescence to detect the surface CRT expression level,CD47 expression level,cell activity and apoptosis of AML cells treated with metformin.2.Using flow cytometry to select the AML blasts and lymphocytes from AML patients’ samples and then detecting the surface CRT expression level and CD47 expression level of these selected cell treated with metformin.3.Using flow cytometry to detect the effects of metformin combined with CD47 blocker on macrophage phagocytosis targeted AML cells.4.Using western blot to explore the effects of metformin combined with CD47 blocker on the expression of ER-related proteins.Results:(1)The expression level of CD47 in AML blasts and non-leukemia cells and its feasibility as a new target for AML targeted immunotherapy1.CD47 broadly expressed on different types of cells in peripheral blood of AML patients and healthy people.Compared with healthy donors,the expression level of CD47 on AML blasts of patients was significantly higher,but there was no significant difference of CD47 expression between AML blasts and non-leukemia cells of AML patients.There was no significant correlation between CD47 expression of AML blasts in AML patients and clinical data.2.The results of in vitro phagocytosis test showed that the macrophages in the untreated group could target and phagocytose AML cells,but almost did not phagocytose healthy human cells,and the function of macrophages phagocytosis targeted AML cells became stronger after CD47 blocking treatment.At the same time,CRT was highly expressed on the surface of AML cells,and almost no CRT was expressed on the surface of healthy human cells.CD47 blocking and CRT exposure expression were closely related to macrophage targeted phagocytosis.3.In patients with AML,compared with non-leukemia cells,the expression of CRT on the surface of AML blasts generally increased,while there was little or low expression of CRT on the surface of various cells in AML patients with remission or healthy donors,indicating that CRT exposure was closely related to the condition of AML patients.(2)Effects of metformin combined with CD47 blocker on macrophage phagocytosis of AML cells and its related mechanism.1.After metformin treatment,the CRT exposure level on the cell surface of AML cells significantly increased,which was related to the time and concentration of metformin.There was no significant difference in CD47 expression between AML cells treated with metformin and the untreated group.2.After metformin treatment,the exposure level of cell surface CRT of AML blasts selected from the samples of AML patients increased,and the expression level of CD47 showed an upward trend or no significant impact.After metformin treatment,the cell surface CRT level of lymphocytes selected from the samples of AML patients showed a downward trend,and the expression level of CD47 showed an upward or downward trend.3.Using metformin combined with CD47 blocker to treat AML cells can enhance the efficiency of macrophage phagocytosis targeted AML cells mediated by CD47 blocker.4.Using Metformin alone or Metformin combined with CD47 blocker to treat AML cells,the expression of endoplasmic reticulum stress pathway related proteins was upregulated.Conclusions:1.CD47 is widely expressed on almost all cells in AML patients and healthy people,and its expression difference between AML blasts and non-leukemia cells in AML patients is not obvious,indicating that there are other key factors that regulate the targeting of CD47 blocking therapy.2.The targeted phagocytosis of macrophages mediated by CD47 blocker therapy is closely related to the expression of CRT on the surface of AML cells.3.CRT exposure is highly expressed on AML blasts,and lowly or almost not expressed on non-leukemia cells.Regulating the expression of this molecule may be the key to affect the effect of CD47 blocking immunotherapy.4.Metformin can up-regulate the expression level of CRT exposure on the surface of AML cells.5.Metformin combined with CD47 blocker has synergistic effect on macrophage targeted phagocytosis of AML cells.6.Metformin combined with CD47 blocker may enhance the phagocytosis of macrophages targeting AML cells by up-regulating the expression of endoplasmic reticulum stress-related proteins.