Normal human bone marrow mononuclear cells were purchased from AllCells Inc. (Emeryville, CA). Human AML samples () were obtained from patients at the Stanford Medical Center with informed consent, according to an IRB-approved protocol (Stanford IRB# 76935 and 6453). Human CD34-positive cells were enriched with magnetic beads (Miltenyi Biotech).
Flow Cytometry Analysis and Cell Sorting
A panel of antibodies was used for analysis and sorting of AML LSC (Lin−CD34+CD38−CD90−, where lineage included CD3, CD19, and CD20), HSC (Lin−CD34+CD38−CD90+), and MPP (Lin−CD34+CD38−CD90−CD45RA−) as previously described (Majeti et al., 2007
). Analysis of CD47 expression was performed with an anti-human CD47 PE antibody (clone B6H12, BD Biosciences, San Jose CA). For analysis of mouse bone marrow, the following antibodies were used: Sca1 PB, cKit Alexa 750, Flk2 PE, CD34 FITC, Lineage (CD3, CD4, CD5, CD8, B220, Mac1) PeCy5 (Ebiosciences. San Diego, CA).
Anti-Human and Mouse CD47 and Anti-Mouse SIRPα Antibodies
Monoclonal mouse anti-human CD47 antibodies included: BRIC126, IgG2b (Abcam, Cambridge, MA), 2D3, IgG1 (Ebiosciences), and B6H12.2, IgG1. Monoclonal rat anti-mouse CD47 antibody used was MIAP301, IgG2a. The B6H12.2 and MIAP301 hybridomas were obtained from the American Type Culture Collection (Rockville, MD). Antibody was either purified from hybridoma supernatant using protein G affinity chromatography according to standard procedures or obtained from BioXCell (Lebanon, NH). Monoclonal rat anti-mouse SIRPα, P84, IgG1 was purchased from BD Pharmingen. Isotype controls included mouse IgG1 and rat IgG2a antibodies (Ebiosciences).
In Vitro Phagocytosis Assays
Human AML LSC or normal bone marrow CD34+ cells were CFSE-labeled and incubated with either mouse or human macrophages in the presence of 7μg/ml IgG1 isotype control, anti-CD45 IgG1, anti-CD47 (clones B6H12.2, BRIC126, or 2D3), or anti-mouse SIRPα antibody for 2 hours. Mouse GFP-positive leukemia cells were incubated with mouse macrophages in the presence of 10 μg/ml of rat IgG2a isotype control or anti-mouse CD47 (MIAP301) for 2 hours. Cells were then analyzed by fluorescence microscopy to determine the phagocytic index (number of cells ingested per 100 macrophages). In some experiments, cells were then harvested and stained with either a mouse or human macrophage marker and phagocytosed cells were identified by flow cytometry as macrophage+CFSE+. Statistical analysis using Student’s t-test was performed with GraphPad Prism. See Supplemental Methods
for detailed procedures.
In Vivo Antibody Treatment of Human AML LSC Engrafted Mice
1–2.5×105 FACS-purified LSC were transplanted into NOG pups. Eight to twelve weeks later, human AML engraftment (hCD45+CD33+ cells) was assessed in the peripheral blood and bone marrow by tail bleed and aspiration of the femur, respectively. Engrafted mice were then treated with daily intraperitoneal injections of 100 micrograms of anti-CD47 antibody or IgG control for 14 days. On day 15 mice were sacrificed and the peripheral blood and bone marrow were analyzed for AML.
In Vivo Human AML Phagocytosis Assay
A GFP encoding lentivirus was prepared from the pCDH-CMV construct (System Biosciences, Mountain View, CA) using standard techniques. AML LSC from sample SU028 were transduced overnight and transplanted into newborn NOG pups as described. Twelve weeks later human CD45+CD33+GFP+ leukemia engraftment was assessed in the peripheral blood, and GFP+ human leukemia-engrafted mice were injected intraperitoneally with a single 100 microgram dose of either anti-CD47 antibody (clone B6H12.2) or IgG control. Four hours later, mice were sacrificed and bone marrow, spleen, and liver were analyzed for the presence of GFP+ leukemia cells within F4/80 positive mouse phagocytes by flow cytometry. The presence of human CD45−GFP+mouse F4/80+ events identified mouse phagocytes with ingested human leukemia cells.
In Vivo Macrophage Depletion
Liposomal clodronate and control liposomes were prepared as described (Jaiswal et al., companion manuscript). Macrophages were depleted in AML LSC-engrafted NOG mice with the following treatment schedule: 200 microliters of either clodronate or liposomal control was injected intravenously via the retro-orbital sinus two days prior to treatment of these mice with anti-CD47 antibody for IgG control. 100μl of either clodronate or liposomal control was then injected in the same manner on days 2, 6, and 10 after initiation of daily antibody treatment. Mice were then sacrificed on day 14 to assess human leukemic engraftment as described.
AML LSC-engrafted mice treated with daily injections of either IgG control or anti-CD47 antibody were sacrificed at the end of 14 days of treatment. 5×105 whole bone marrow cells were transplanted into newborn NOG mice. Twelve weeks later peripheral blood and bone marrow was harvested and analyzed for human CD45+CD33+ leukemia engraftment as described.
AML Patients, Microarray Gene Expression Data, and Statistical Analysis
Gene expression and clinical data were analyzed for three previously described cohorts of adult AML patients: (1) a training dataset of 285 patients with diverse cytogenetic and molecular abnormalities described by Valk et al.
(Valk et al., 2004
), (2) a test dataset of 242 patients with normal karyotypes described by Metzeler et al.
(Metzeler et al., 2008
), and (3) a validation dataset of 137 patients with normal karyotypes described by Bullinger et al.
(Bullinger et al., 2008
) See Supplemental Methods
for details of therapy. The clinical end points analyzed included overall and event-free survival, with events defined as the interval between study enrollment and removal from the study owing to a lack of complete remission, relapse, or death from any cause, with data censored for patients who did not have an event at the last follow-up visit. See Supplemental Methods
for detailed procedures.