Membrane Bound CRT Fragment Accelerates Tumor Growth of Melanoma B16 Cell In Vivo through Promoting M2 Polarization via TLR4

Figure 2

tmCRT/39-272 enhanced macrophage phagocytosis and M2 macrophage polarization. (a) Female C57BL/6 mice () were s.c. injected with B16-EGFP or B16-tmCRT/39-272 cells ( μL/mouse) and sacrificed 18 days postinoculation to obtain the solid tumors. B16-EGFP and B16-tmCRT/39-272 tumor tissues were digested with collagenase Ⅳ (1 mg/mL) and DNase Ⅰ (0.05 mg/mL). Single cells were filtered through a 70-μm cell strainer and stained with fluorescence-labeled anti-F4/80 and anti-CD206 antibodies. The percentage of F4/80⁺CD206⁺ M2 macrophages among the tumor cells was analyzed by FACS. (b) Immunofluorescence staining of tumor tissues with FITC-labeled anti-CD206 antibody (green) and DAPI (blue). Images were acquired by laser-scanning confocal microscopy. (c and d) FACS dot plots and the statistical results of percentage of F4/80⁺CD206⁺ cells and CD11b⁺TNF-α⁺ cells from C57BL/6 mouse bone marrow cells analyzed by flow cytometry. (e) B16-EGFP or B16-tmCRT/39-272 cells were labeled with CFSE and cocultured with macrophages at a ratio of 3:1 for 2 h, 3 h, and 4 h. The cells were stained with fluorescence-labeled antibody against anti-CD11b and the phagocytic capability of the macrophages was analyzed by flow cytometry. (f) 293T-EGFP cells (labeled with dye) were added to macrophage cocultured with B16-EGFP or B16-tmCRT/39-272 cells. The cell ratio was 1:1:3 (macrophage:293T-EGFP:B16-EGFP or B16-tmCRT/39-272). At 2 h, 3 h, 4 h, and 5 h, the cells were collected for staining with fluorescence-labeled anti-CD11b antibody and the phagocytic capability of the macrophages on 293T-EGFP cells was assessed by flow cytometry. The experiments were repeated three times., , .

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