In vitro evaluation of the therapeutic efficacy of CD47 mAb against osteosarcoma cells

In vitro studies were performed in three human osteosarcoma cell lines (Saos-2, U-2 OS, MNNG/HOS, ATCC, Manassas, VA, USA), and one murine osteosarcoma cell line (K7M2, ATCC, Manassas, VA, USA). Saos-2, U-2 OS, and K7M2 cells were cultured in Dulbecco’s modified Eagle's medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 mg/mL of streptomycin. MNNG/HOS cells were grown in Eagle's minimum essential medium (ATCC) supplemented with 10% FBS, 100 units/mL of penicillin, and 100 mg/mL of streptomycin. A normal bone cell line hFOB 1.19, ATCC was used as a negative control. All cell lines used were authentic and confirmed to be mycoplasma negative using the MycoAlert Mycoplasma Activity kit (Lonza).

The following antibodies were used for treatment purpose: anti-human CD47 (BioXcell, Clone B6.H12), anti-mouse CD47 (BioXcell, clone MIAP301), human IgG control, and mouse IgG1 isotype control (BioXcell).

To evaluate macrophage-mediated tumor phagocytosis⁴² in the presence of CD47 mAb, Saos-2 osteosarcoma cells were labeled with 1,1′-Dioctadecyl-3,3,3,’,3′-tetramethylindodicarbocyanine (CellBrite™ Green, Biotium) according to the manufacturer’s protocol, and incubated at a 1:1 ratio with bone marrow-derived mouse macrophages in serum-free (Iscove's modified Dulbecco's medium (IMDM), with or without 10 μg/mL CD47 mAb at 37 °C for 4 h.

To evaluate uptake of iron nanoparticles by macrophages in the presence of CD47 mAbs, we co-cultured murine bone marrow derived macrophages with unlabeled MNNG/HOS cells and FITC-labeled ferumoxytol (0.01 mM) with or without 10 μg/mL CD47 mAb at 37 °C for 4 h.

Macrophages were then stained with F4/80 antibody and subsequently imaged using Leica SP8 confocal microscopy. Tumor phagocytosis was calculated as the percentage of macrophages positive for phagocytized CellBrite™ Green⁺ cells. FITC-ferumoxytol signal in F4/80⁺macrophage was quantified to demonstrate nanoparticle uptake by macrophages.

Flow cytometry assay was used to confirm macrophage-mediated phagocytosis of osteosarcoma cells and macrophage-mediated tumoricidal effects. For this CellBrite™ Green-labeled MNNG/HOS cells were co-cultured with murine M1 and M2 macrophages as described above. Fluorescently labeled antibodies targeting macrophage markers (CD11b,F4/80, CD80, and C206) were used to identify the M1 and M2 macrophage population. Phagocytosis was quantified by the percentage of CellBrite™ Green events among CD11b+F4/80+ macrophage events. Tumor cell death in macrophage co-cultures was assessed with Aqua amine cell viability staining in the CellBrite™ Green+ population.

Time-lapse tumor phagocytosis and cell death of MNNG/HOS osteosarcoma cells caused by M1 macrophages in the presence of CD47 mAb was performed to identify whether tumor cell death preceded phagocytosis or vice versa. Osteosarcoma cells were labeled with JC-1 mitochondrial membrane dye and M1 macrophages were stained with Hoechst33342, and co-incubated at a 1:1 ratio with CD47 mAb or control mAb and time-lapse imaged every 180 s for 4 h using LSM 880 Airyscan confocal microscope using Zen Black software at 63× objectives. In viable cells with normal, polarized mitochondria, JC-1 shows red J-aggregate fluorescence (590 nm), but upon losing viability as mitochondrial potential reduces, the red J-aggregate fluorescence changes to cytoplasmic diffusion of green monomer fluorescence (530 nm). Intensity of JC-I fluorescence at 590 nm and 530 nm for 100 tumor cells was calculated using velocity software. Ratio of JC-1 fluorescence at 590 and 530 was used to represent mitochondrial membrane potential at different time points during phagocytosis.

For preparation of mouse macrophages, 7–11-week-old NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) mice were euthanized and the femora and tibiae were isolated. The bones were kept in ice-cold phosphate buffered saline (PBS) and sterilized in 70% ethanol. By flushing them with mouse macrophage medium (IMDM with 10% FBS, 1× penicillin/streptomycin, 200 mM glutamine, and 25 mM HEPES, all from Corning Inc.), bone marrow cells were gathered and plated at 1 × 10⁶/ml in Petri dishes in mouse macrophage medium. To generate macrophages, bone marrow cells were treated for 7 days with recombinant mouse macrophage colony-stimulating factor (25 ng/mL, Shenandoah Biotech). For polarization of M1 and M2 macrophages⁴², murine bone marrow cells were treated for 7 days with either recombinant human or mouse macrophage colony-stimulating factor (M-CSF; 25 ng/mL). M2 polarization was achieved by further treatment on days 5 and 6 with IL-4 (20 ng/mL) and IL-13 (20 ng/mL). To generate M1 macrophages, bone marrow cells were treated for 7 days with either recombinant human or mouse granulocyte macrophage colony-stimulating factor (GM-CSF; 25 ng/mL). M1 polarization was achieved with further treatment on day 5 by interferon-γ (20 ng/mL) stimulation for 1 h, followed by lipopolysaccharide for 48 h (100 ng/mL; Sigma-Aldrich). Unless otherwise stated, all cytokines were purchased from Shenandoah Biotech.