Biological Characteristics Of Acute Myeloid Leukemia Stem Cell-derived Macrophage-like Cells

Background and objective:Acute myeloid leukemia(AML)is the most common type of leukemia,and it is the main malignant disease in middle-aged and elderly people.The incidence of AML increases with advancing age.Among them,40%to 50%of AML patients relapse in the course of treatment and eventually lead to death.Therefore,how to improve the efficacy of relapsed and refractory AML is an urgent clinical problem.Recurrence of AML is mainly studied in AML cells and AML leukemic stem cells(LSCs).In fact,AML LSCs do not exist in isolation,and their biological characteristics are closely related to the dysfunction of the hematopoietic microenvironment.At present,many studies at home and abroad show that abnormal hematopoietic microenvironment of leukemia can maintain the LSCs self-renew.Our previous study found that a large number of CD68-positive cells in bone marrow biopsy specimens of AML patients,and they were mixed with leukemia cells.Only a small number of CD68positive cells were found in normal human bone marrow biopsy specimens.CD68 is the surface marker of macrophages.Where did a large number of macrophages originate in bone marrow biopsy specimens of AML patients and what role they play in the development of leukemia deserve further exploration.Recent studies in solid tumors have found that there are a large number of macrophages in tumor tissues,which are called tumor-associated macrophages(TAMs).TAMs play an important role in promoting the proliferation and invasion of cancer cells,angiogenesis and immunosuppression.The research on TAMs has become a hot topic in cancer microenvironment research.Although the research on TAMs in the field of hematological tumors is still in its infancy,existing studies have shown that TAMs play an important role in the occurrence and development of hematological tumors.Macrophages originate from myeloid mononuclear cells,and we speculate that a large number of macrophages in AML microenvironment may originate from AML LSCs.In this study,AML LSCs were isolated from cell lines and patients bone marrow specimens,and cultured in macrophage conditioned medium to obtain AML LSCs-derived macrophage-like cells.The biological characteristics of AML LSCs-derived macrophage-like cells were further explored.These results will help us to fully understand the role of AML LSCs-derived macrophage-like cells on AML abnormal microenvironment,furthermore,and provides practical design ideals for exploring AML novel treatment strategy.Methods:1.After CD34~+CD38~-CD123~+AML LSCs were selected from AML cell line Kasumi-3and primary AML bone marrow samples by flow cytometry,the cells were cultured for 1week in the medium containing GM-CSF and IL-4.Then,these cells were transferred to a medium containing IL-4 and TGF-?to continue the culture,and finally AML LSCs-derived macrophage-like cells were obtained.We observed the ultrastructural changes of AML LSCs-derived macrophage-like cells by transmission electron microscopy,and observed surface marker expression of these cells by laser confocal microscopy.We selected AML1/ETO fusion gene AML patient bone marrow specimens for sorting and culture to obtain LSCs-derived macrophage-like cells,then detected AML LSCs and AML LSCs-derived macrophage-like cells AML1/ETO expression.2.The levels of IL-10 and IL-13 in AML LSCs-derived macrophage-like cell culture supernatants were detected by ELISA,and the biological function of LSCs-derived macrophage-like cells was verified by bacterial phagocytosis assay.3.Through the analysis of GEO(Gene Expression Omnibus),TCGA(The Cancer Genome Atlas),and other databases,we searched for leukemia-associated macrophages to play an important role in the development of tumorigenesis,and analyzed this group of genes.Correlation with the prognosis of AML,and then identify key genes involved in AML regulation.Finally,we analyzed the expression of these genes and analyzed their gene networks,signaling pathways,and functional regulation.Results:1.Most of AML LSCs-derived macrophage-like cells,in polygonal shape,were mainly polygonal and oval attached cells.Transmission electron microscopy displayed that the cells were oval,with plenty of cytoplasm and more vacuoles;the Golgi bodies were accumulated near the nucleus,and the capsular villi were rich and long.The cells specifically expressed CD11b,CD163,CD206.Using AML1/ETO as a marker,we demonstrated that AML LSCs-derived macrophage-like cells share homology with AML LSCs cells,and different levels of AML1/ETO fusion gene expression were detected in these cells.2.The cytokine levels of AML LSCs-derived macrophage-like cell culture supernatants were detected by ELISA.We found that the peak of IL-10 secretion was on the 8th day of culture(The secretion is 12.2pg/ml),and the peak of IL-13 secretion was on the 10th day(The secretion is 14.1pg/ml).Phagocytosis test showed that the cells could phagocytize a large amount of GFP~+E.coli in a time-dependent manner.3.After co-cultured with AML LSCs-derived macrophage-like cells,the Kasumi-3 cells expressed higher level of ABC protein,and presented lower DNR dosage(54.75±4.28 vs93.84±3.93,P<0.01 when compared with the Kasumi-3 cells in suspension cultivation)and certain drug resistances to adriamycin(ADM),cytarabine(Ara-C)and etoposide(VP-16).4.Through on-line analysis of GEO,GEO2R and TCGA database,34 genes significantly related to the prognosis of AML were obtained.Then,with the gene expression analysis and literature reading,GREM1 gene was finally identified as the key factor for LAMs to participate in leukemia regulation;Analysis of GO interaction gene database was further confirmed that GREM1 inhibits apoptosis of leukemia cells to promote the occurrence of AML by affecting mitochondrial energy metabolism.Conclusions:1.AML LSCs can be differentiated into AML LSCs-derived macrophage-like cells under appropriate conditions.2.AML LSCs-derived macrophage-like cells show M2 macrophage-like biological properties.3.AML LSCs-derived macrophage-like cells promote the expression of AML cell resistance protein and mediate leukemia cell resistance.4.GREM1 may be the key gene of AML LSCs-derived macrophage-like cells regulating the occurrence and development of AML.GREM1 inhibits apoptosis of leukemia cells to promote the occurrence of AML by affecting mitochondrial energy metabolism.