Figure 4
Figure 4. Impairment of macrophage function reduces tumor cell survival despite clot formation. (A) Experimental design for the ablation of macrophages in CD11b-DTR mice. DT was administered intraperitoneally (15 ng/g) on 2 consecutive days. Control mice were injected with saline at the same time points. Four hours after the second administration of DT or saline, 5 × 105 B16F10-wt cells were intravenously injected. The population of myeloid cells expressing F4/80, CD11b, and Gr-1 obtained at the time of the tumor cell injection or 8 hours after (as indicated), from either the peripheral blood or the lung, was analyzed by flow cytometry. (B-C) Immunohistochemistry against CD11b (B, AlexaFluor-488, green) and the platelet-specific integrin αIIb (C, AlexaFluor-488, green) in lung sections from C57BL/6-wt or CD11b-DTR mice treated with DT as in panel A or Mac1 KO mice, 8 hours after intravenous injection of 5 × 105 CMRA-stained B16F10-wt cells (red, imaged with a confocal microscope). The percentage of tumor cells associated with platelets was scored; n = 3 mice, approximately 40 cells per mouse analyzed (1-way ANOVA). (D) Left panel: C57BL/6-wt or CD11b-DTR mice, treated as in panel A, were intravenously injected with 5 × 105 CMFDA-stained B16F10-wt cells. Lungs were isolated 24 hours after and imaged as intact organ with an epifluorescence microscope. The total number of tumor cells observed from images of consecutive fields of the entire left lobe of the lungs was scored; n = 3 mice (1-way ANOVA and Tukey test). Middle and right panels: C57BL/6-wt or Mac1 KO mice were intravenously injected with 5 × 105 CMFDA-stained (middle panel) or 2.5 × 105 unstained (right panel) B16F10-wt cells. Middle panel: Lungs were isolated 8 or 24 hours after tumor cell injection, imaged as intact organ with an epifluorescence microscope, and the number of tumor cells observed from images of consecutive fields of the entire left lobe of the lungs were scored; n ≥ 3 mice (Mann-Whitney). Right panel: Metastatic lung nodules were scored 3 weeks after intravenous injection of tumor cells; n ≥ 4 mice (Mann-Whitney). (E) NK-cell depletion assay. C57BL/6-wt or Mac1 KO mice were treated with anti–asialo-GM1 antibody (20 μL, intraperitoneally) on days 1, 8, and 15 and intravenously injected with 2.5 × 105 B16F10-wt cells on day 4. Lungs were isolated on day 25, and metastatic lung nodules were scored; n ≥ 4 mice (Mann-Whitney). (C-E) Data are mean + SD. *P < .05. **P < .01. (B-C) Scale bars represent 50 μm.

Impairment of macrophage function reduces tumor cell survival despite clot formation. (A) Experimental design for the ablation of macrophages in CD11b-DTR mice. DT was administered intraperitoneally (15 ng/g) on 2 consecutive days. Control mice were injected with saline at the same time points. Four hours after the second administration of DT or saline, 5 × 105 B16F10-wt cells were intravenously injected. The population of myeloid cells expressing F4/80, CD11b, and Gr-1 obtained at the time of the tumor cell injection or 8 hours after (as indicated), from either the peripheral blood or the lung, was analyzed by flow cytometry. (B-C) Immunohistochemistry against CD11b (B, AlexaFluor-488, green) and the platelet-specific integrin αIIb (C, AlexaFluor-488, green) in lung sections from C57BL/6-wt or CD11b-DTR mice treated with DT as in panel A or Mac1 KO mice, 8 hours after intravenous injection of 5 × 105 CMRA-stained B16F10-wt cells (red, imaged with a confocal microscope). The percentage of tumor cells associated with platelets was scored; n = 3 mice, approximately 40 cells per mouse analyzed (1-way ANOVA). (D) Left panel: C57BL/6-wt or CD11b-DTR mice, treated as in panel A, were intravenously injected with 5 × 105 CMFDA-stained B16F10-wt cells. Lungs were isolated 24 hours after and imaged as intact organ with an epifluorescence microscope. The total number of tumor cells observed from images of consecutive fields of the entire left lobe of the lungs was scored; n = 3 mice (1-way ANOVA and Tukey test). Middle and right panels: C57BL/6-wt or Mac1 KO mice were intravenously injected with 5 × 105 CMFDA-stained (middle panel) or 2.5 × 105 unstained (right panel) B16F10-wt cells. Middle panel: Lungs were isolated 8 or 24 hours after tumor cell injection, imaged as intact organ with an epifluorescence microscope, and the number of tumor cells observed from images of consecutive fields of the entire left lobe of the lungs were scored; n ≥ 3 mice (Mann-Whitney). Right panel: Metastatic lung nodules were scored 3 weeks after intravenous injection of tumor cells; n ≥ 4 mice (Mann-Whitney). (E) NK-cell depletion assay. C57BL/6-wt or Mac1 KO mice were treated with anti–asialo-GM1 antibody (20 μL, intraperitoneally) on days 1, 8, and 15 and intravenously injected with 2.5 × 105 B16F10-wt cells on day 4. Lungs were isolated on day 25, and metastatic lung nodules were scored; n ≥ 4 mice (Mann-Whitney). (C-E) Data are mean + SD. *P < .05. **P < .01. (B-C) Scale bars represent 50 μm.

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