Detection, Sorting And Targeted Therapy Leukemia Stem Cells In Acute Monocytic Leukemia THP-1Cell Line

Leukemia is cancer of the blood cells. It starts in the bone marrow, and the bone marrow make a lot of abnormal white blood cells, called leukemia cells.Leukemia stem cells (LSCs) reside within a hierarchy of malignant hematopoiesis and possess the ability to instigate, maintain and serially propagate leukemia.In order to eradicate the leukemia and cure the patient, the LSCs must be eliminated. So the study to identify LSCs and then develop therapies that target it is critical in order to better understand both the genesis of leukemic disease and strategies by which such cells may be eradicated.Flow cytometry is an emerging technique which holds great promise for the separation, classification and quantitation of LSCs in leukemia cells.The THP-1cell line, which was originally established from an infant diagnosed with acute myeloid leukemia (AML), provides an experimental model for functional, preclinical therapeutics and target identification studies of AML. Cytarabine (Ara-C) is commonly used for the treatment of acute leukemia. Incorporation of Ara-C into DNA is a key event in the mechanism of killing of proliferating leukemic cells,but is relatively ineffective against LSCs which keep in quiescent status. So novel therapeutic strategies are urgently required to improve the unsatisfactory prognosis of this disease.This study was to investigate the percentage and biological characteristics of LSCs in THP-1cells. To find the target to eliminate LSCs by studying the expression of CD47in different cell subsets. To explore the tumorigenicity of LSCs in vivo by establishment NOD/SCID mice leukemia model using THP-1LSCs.In this study. Finally, to evaluate the anti-leukemia effect of anti-CD47monoclonal antibodies single, or combination Ara-C in vitro and in vivo in order to find out the best targeted treatment strategies for leukemia.Part One Sorting and Preliminary studying of leukemia stem cell in Acute Monocytic Leukemia THP-1Cell LineObjective:To isolate and preliminary identify whether the human acute monocytic leukemia THP-1cell line contains leukemia stem cell (LSCs) or not. To identify LSCs cells subsets and study the differences between THP-1LSCs and non-LSCs in the cell cycle, growth and proliferation resistance and the resistance protein and ends with death gene expression.Methods:Flow cytometry was used to detcet the percentage of LSCs in Logarithmic growth Period THP-1, which were then sorted by the fluorescence activating cell sorter, and then THP-1LSCs and non-LSCs subpopulations were collected. DNA content of two subpopulations were examined by flow cytometry. The proliferation and colony forming ability between THP-1LSCs and non-LSCs subpopulations were compared in terms of colony assay and growth curve.Two groups of cells dealed with different concentrations of cytarabine (Ara-C), respectively, and calculated the IC5o value in each group at each time point, then drawed growth curve according to the standard curve. The mRNA expression of multidrug resistance proteins (ABCG2、ABCB1) and apoptosis gene (BCL-2、BAX) were examined by real time quantitative PCR(RQ-PCR) in two subpopulations.Results:1.LSCs almost accounted for0.12±0.06%of viable cells in THP-1. The LSCs tube cells were concentrated in the LSCs regional, LSCs cell ratio reached (97.0±1.7%)and non-LSCs tube cells is still concentrated in sub-region of the main group of cells before the election.2. TheG0/G1Phase cells in the THP-1LSCs subpopulation accounted for about94.3%of the total cells and more highly than non-LSCs(P<0.05)3. The proliferation and colony forming ability of THP-1LSCs subpopulation wre significantly higher than those of non-LSCs (P<0.05)4. At different time point, the IC50value of THP-1LSCs were higher than the non-LSCs (P<0.05).5. The mRNA expression of multidrug resistance proteins (ABCG2、ABCB1) of THP-1LSCs were higher than the non-LSCs (P<0.05).Although there were no differences between these two subpopulations in and Bax expression (P>0.05), Bcl-2/Bax value were significantly higher than those of in the non-LSCs (P<0.05) examined by RQ-PCR.Conclusions:The THP-1cell line contains LSCs, and the proportion of LSCs is much lower. and the LSCs is different with the main group of cells in the colony forming ability, growth and proliferation ability and expression of drug resistance protein gene, apoptosis gene and most LSCs stay quiescent, it may be associated with tumor resistance and recurrence. Part Two Establishment of an Animal Model of Human Acute monocytic leukemia in NOD/SCID Mice Using THP-1stem cellsObjective:To find the target of the treatment of childhood acute myelocytic leukemia by studying the expression of CD47in different cell subsets. To explore the feasibility of establishment of NOD/SCID mice model using microdose acute monocytic leukemia THP-1stem cells and to evaluate the tumorigenic ability of THP-1stem cells.Methods:Flow cytometry was used to detcet the percentage of LSCs in Logarithmic growth Period THP-1, which were then sorted by the fluorescence activating cell sorter, and LSCs in THP-1were enriched using cytarabine, and then the expression of CD47in THP-1LSCs and non-LSCs and other subpopulations were detected. NOD/SCID mice divided into eight groups, inoculated with different subpopulations and different number of THP-1cells by vein,or abdominal. And human AML engraftment (hCD45+CD33+cells) was assessed in the peripheral blood and bone marrow by tail bleed and aspiration of the femur using flow cytometry, respectively, and compare the survivalrate,histopathology changes in different groups.Results:l.Using flow cytometry, CD47was more highly expressed on multiple pecimens of THP-1LSCs than bulk THP-1and the LSC-enriched fraction(P<0.05). This increased expression extended to the bulk leukemia cells, which expressed CD47similarly to the LSC-enriched fraction (P>0.05)2. The results indicated that the systemic disseminated leukemia model was established successfully by injecting1×103THP-1LSCs. Unsorted THP-1cells requires at least1X106to established tumor model. Statistically,there were significant differences in the incidence of leukemia among different groups (P<0.05)3.The local tumors appeared in the abdominal cavity or on the greater omentumof NOD/SCID mice. On the third week after inoculation, the leukemia cells were found on peripheral blood smear, and the leukemia cell infiltration was observed in liver, spleen and bone marrow. On the brink of death, the count of peripheral blood WBC was much more than that before inoculation. Human AML engraftment (hCD45+CD33+cells) was detected in the peripheral blood and bone marrow,but not in control group mice (P<0.05)Conclusions:1. Microdose THP-1LSCs inoculation can be used to establish an animal model of human leukemia in NOD/SCID mice successfully,and this reveals leukemic stem cells have a strong tumorigenic in the body. The established THP-1NOD/SCID mice model with leukemia well imit-ates the process of leukemia in human body, so it is a good model for the research on the effects of new drugs and target or gene therapy.2. The expression of CD47is higher in THP LSCs,and CD47may be used as an important target for clearing the leukemic stem cells. Part Three Studying of Effectiveness of Ara-C and Anti-CD47Antibody Combination Targeting Therapy Acute Myeloid LeukemiaObjeetive:To explore the prognostic significance of CD47in acute myeloid leukemia by studying the expression of CD47in human acute monocytic leukemia THP-1cells transplanted leukemia mice. To study the effectiveness of Ara-C and anti-CD47antibody combination therapy eliminates THP-1cells in vivo and in vitro and to find best strategies targeted treatment acute myeloid leukemia.Methods:CD47expression on peripheral blood and bone marrow of NOD/SCID normal and leukemia-bearing mice were examined. Then, according to different levels of CD47expression, leukemia-bearing mice be divided into CD47low and CD47gh two groups and survival time be surveyed. Moribund mice were sacrificed and the peripheral blood, bone marrow and organ such as liver,spleen were analyzed for AML. THP-1LSCs incubated with either mouse or human macrophages in the presence of7μg/ml IgGl isotype control, anti-CD45IgG1, anti-CD47for2hours. Cells were then analyzed by microscopy to determine the phagocytic index (number of cells ingested per100macrophages). Engrafted mice were given daily intraperitoneal injections of single anti-CD47antibody,single Ara-C or Ara-C and anti-CD47antibody combination therapy. Human AML engraftment (hCD45+CD33+cells) was assessed in the peripheral blood and bone marrow by tail bleed and aspiration of the femur, respectively.Treatment was then stopped and mice were followed for survival analysis.Results:1.Compared to both normal mononuclear cells of peripheral blood (14.1 ±2.14%) and bone marrow (30.7±3.09%),CD47was more highly expressed on THP-1LSCs (73.8±5.34%)(P<0.05)2. Higher CD47expression was also associated with a poor clinical outcome. The peripheral blood, bone marrow and organ such as liver, spleen of CD47hlgh mice were packed with monomorphic leukemic blasts, while organ such as liver, spleen of CD47low mice were hypocellular and AML cells infiltrate can not be found in lung,liver, spleen,didymus.3. In vitro, incubation of THP-1LSCs in the presence of IgG1isotype control or anti-CD45IgGl antibody did not result in significant phagocytosis; however, anti-CD47antibodies enabled phagocytosis of THP-1LSCs. For THP-1LSC, the differences between isotype or anti-CD45antibody with blocking anti-CD47antibody treatment was statistically significant (p=0.000)4. Anti-CD47antibody treatment decreased the leukemia burden in these mice and signficantly prolonged survival compared to control IgG, although all mice eventually died. Similar results were seen with Ara-C and were not statistically different compared to anti-CD47antibody. In contrast, combination therapy with anti-CD47antibody and Ara-C eliminated leukemia in70%of mice as indicated by long-term survivalConclusions:1.Increased CD47expression correlates with a worse clinical prognosis and adverse pathology features in human THP-1LSCs engrafted mice.2. Combination therapy with Anti-CD47antibody and Ara-C eliminates leukemia in human THP-1xenotransplant models.It has an important methodological significance to clear leukemia stem cells, completely cure acute myeloid leukemia.