Figure 3.
Skin CD169+ macrophages surround the tumor. (A) Immunostaining of IBA1 (red), CD169 (green), and DAPI (blue) in mouse B16-F10–bearing skin of day 11–12 with isotype mAb or anti-CSF1R mAb treatment (representative of four mice for each group). Scale bar = 500 and 100 μm. (B) The ratio of CD169+ macrophages inside or outside of the tumor (left) in isotype mAb mice and the number of CD169+ macrophages in the tumor-bearing skin with treatment with isotype mAb or anti-CSF1R mAb (right) were analyzed using Imaris (n = 4 for each group, combined data from two experiments). P value was calculated using Student’s t test. (C) The number of intratumoral macrophages (CD169− IBA1+ cells inside the tumor) with treatment with isotype mAb or anti-CSF1R mAb was analyzed using Imaris (n = 4 for each group, combined data from two experiments). P value was calculated using Student’s t test. (D) The numbers of LN SSM and CD169+ Mϕ after the two treatments of 200 μg isotype mAb or anti-CSF1R antibodies were subcutaneously injected into the mouse back skin were analyzed using flow cytometry (n = 3–5 for each group, data are representative of three independent experiments). P value was calculated using one-way ANOVA. (E) The tumor volumes of B16-F10 melanoma with isotype mAb in WT and B cell KO mice versus anti-CSF1R mAb in B cell KO mice (n = 15 for each group, data are combined from three independent experiments with four to five mice per group). Each circle represents one mouse. P value was calculated using one-way ANOVA. Refer to the image caption for details. Panel A shows two sets of confocal microscopy images. The left set is stained with isotype monoclonal antibody (mAb) and the right set with alpha-CSF1R mAb. The images are stained for IBA1 (red), CD169 (green), and DAPI (blue). The images show the dermis, DWAT, tumor, and adventitia regions. The scale bars are 500 micrometers for the larger images and 100 micrometers for the zoomed-in images. Panel B shows bar graphs quantifying percentages and counts of CD169 positive macrophages located inside or outside tumor regions under treatments. Panel C shows bar graphs comparing intratumoral macrophage counts per tumor area between isotype-treated and anti-CSF1R-treated experimental mouse groups. Panel D shows bar graphs comparing lymph node SSM and skin CD169 positive macrophage counts in B cell knockout experimental groups. Panel E shows a bar graph comparing tumor volumes on day 11 among WT-isotype, B cell KO-isotype, and anti-CSF1R-treated groups.

Skin CD169+ macrophages surround the tumor. (A) Immunostaining of IBA1 (red), CD169 (green), and DAPI (blue) in mouse B16-F10–bearing skin of day 11–12 with isotype mAb or anti-CSF1R mAb treatment (representative of four mice for each group). Scale bar = 500 and 100 μm. (B) The ratio of CD169+ macrophages inside or outside of the tumor (left) in isotype mAb mice and the number of CD169+ macrophages in the tumor-bearing skin with treatment with isotype mAb or anti-CSF1R mAb (right) were analyzed using Imaris (n = 4 for each group, combined data from two experiments). P value was calculated using Student’s t test. (C) The number of intratumoral macrophages (CD169− IBA1+ cells inside the tumor) with treatment with isotype mAb or anti-CSF1R mAb was analyzed using Imaris (n = 4 for each group, combined data from two experiments). P value was calculated using Student’s t test. (D) The numbers of LN SSM and CD169+ Mϕ after the two treatments of 200 μg isotype mAb or anti-CSF1R antibodies were subcutaneously injected into the mouse back skin were analyzed using flow cytometry (n = 3–5 for each group, data are representative of three independent experiments). P value was calculated using one-way ANOVA. (E) The tumor volumes of B16-F10 melanoma with isotype mAb in WT and B cell KO mice versus anti-CSF1R mAb in B cell KO mice (n = 15 for each group, data are combined from three independent experiments with four to five mice per group). Each circle represents one mouse. P value was calculated using one-way ANOVA.

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