Figure 4. Panel A: The image shows raw... | Rockefeller University Press

Figure 4.

$Skin CD169+ macrophages ingest tumor cells. (A) Direct interaction of LYZM+ cells and B16-F10 cells using intravital imaging on day 1. LysmCre/+.KikumeLSL/LSL mice were subcutaneously treated with isotype mAb or anti-CSF1R mAb on days −3 and −1, and then mCherry-expressing B16-F10 cells were subcutaneously injected on day 0. Magnified images of the areas outlined in yellow rectangles (a and b) in isotype mAb-treated skin. Representative of four mice for each group. Scale bar = 50 and 20 μm. (B) Violin plots of total contact time (minutes) and contact enrichment (tumor cell–macrophage’s overlap fraction/the tumor cell occupancy) in the tumor-bearing skin with treatment of isotype mAb or anti-CSF1R mAb (day 1). P value was calculated using the Wilcoxon rank-sum test (representative of three mice for each group). (C) Engulfment of B16-F10 cells (mCherry+ cells) by LYZM+ cells using intravital imaging on day 7 (n = 4). Scale bar = 50 μm. (D) The number and percentages of mCherry-containing LYZM+ cells in the tumor-bearing skin with treatment of isotype mAb or anti-CSF1R mAb were quantified using Imaris (representative of four mice for each group, combined data of two experiments). P value was calculated using Student’s t test. (E) Immunostaining of IBA1 (red), CD169 (green), and DAPI (blue) in mouse mCherry-expressing (magenta) B16-F10–bearing skin on day 8 with treatment of isotype mAb or anti-CSF1R mAb (representative of four mice for each group). Scale bar = 50 μm. (F) Histograms of mCherry in CD169+ Mϕ, CD169− MHCII+ Mϕ, and CD169– MHCII− Mϕ on day 8 of control (Ctrl) or mCherry-expressing B16-F10–bearing skin (B16-F10). MFI of mCherry in each macrophage subset in B16-F10 skin was subtracted by that in Ctrl skin (n = 4 for each group, data are representative of two independent experiments). P value was calculated using one-way ANOVA. (G) Subtracted MFI of mCherry in CD169+ Mϕ, CD169– MHCII+ Mϕ, and CD169– MHCII− Mϕ on day 8 of mCherry-expressing B16-F10–bearing skin in WT or MERTK KO mice (n = 3–4 for each group, data are representative of two independent experiments). P value was calculated using one-way ANOVA. (H) The tumor volumes of B16-F10 melanoma in WT or MERTK KO mice (n = 8–9, data are combined from two independent experiments with four to five mice per group). Each circle represents one mouse. P value was calculated using Student’s t test. Refer to the image caption for details.$ Panel A: The image shows raw and magnified images of tumor-bearing skin on Day 1. The raw images display the interaction of LYZM positive cells and B16-F10 cells using intravital imaging. The magnified images focus on areas outlined in yellow rectangles in isotype mAb-treated skin. Panel B: Violin plots illustrate the total contact time and contact enrichment in tumor-bearing skin treated with isotype mAb or anti-CSF1R mAb on Day 1. Panel C: Surface-rendered images of tumor-bearing skin on Day 7 show the engulfment of B16-F10 cells by LYZM positive cells. Panel D: Bar graphs quantify the number and percentages of mCherry-containing LYZM positive cells in tumor-bearing skin treated with isotype mAb or anti-CSF1R mAb. Panel E: Immunostaining images display IBA1, CD169, and DAPI in mouse mCherry-expressing B16-F10-bearing skin on Day 8. Panel F: Histograms show mCherry levels in different macrophage subsets on Day 8 of control or mCherry-expressing B16-F10-bearing skin. Panel G: Bar graphs present the subtracted mean fluorescence intensity (MFI) of mCherry in different macrophage subsets on Day 8 of mCherry-expressing B16-F10-bearing skin in WT or MERTK KO mice. Panel H: A bar graph shows the tumor volumes of B16-F10 melanoma in WT or MERTK KO mice.

Skin CD169⁺ macrophages ingest tumor cells. (A) Direct interaction of LYZM⁺ cells and B16-F10 cells using intravital imaging on day 1. Lysm^Cre/+.Kikume^LSL/LSL mice were subcutaneously treated with isotype mAb or anti-CSF1R mAb on days −3 and −1, and then mCherry-expressing B16-F10 cells were subcutaneously injected on day 0. Magnified images of the areas outlined in yellow rectangles (a and b) in isotype mAb-treated skin. Representative of four mice for each group. Scale bar = 50 and 20 μm. (B) Violin plots of total contact time (minutes) and contact enrichment (tumor cell–macrophage’s overlap fraction/the tumor cell occupancy) in the tumor-bearing skin with treatment of isotype mAb or anti-CSF1R mAb (day 1). P value was calculated using the Wilcoxon rank-sum test (representative of three mice for each group). (C) Engulfment of B16-F10 cells (mCherry⁺ cells) by LYZM⁺ cells using intravital imaging on day 7 (n = 4). Scale bar = 50 μm. (D) The number and percentages of mCherry-containing LYZM⁺ cells in the tumor-bearing skin with treatment of isotype mAb or anti-CSF1R mAb were quantified using Imaris (representative of four mice for each group, combined data of two experiments). P value was calculated using Student’s t test. (E) Immunostaining of IBA1 (red), CD169 (green), and DAPI (blue) in mouse mCherry-expressing (magenta) B16-F10–bearing skin on day 8 with treatment of isotype mAb or anti-CSF1R mAb (representative of four mice for each group). Scale bar = 50 μm. (F) Histograms of mCherry in CD169⁺ Mϕ, CD169⁻ MHCII⁺ Mϕ, and CD169^– MHCII⁻ Mϕ on day 8 of control (Ctrl) or mCherry-expressing B16-F10–bearing skin (B16-F10). MFI of mCherry in each macrophage subset in B16-F10 skin was subtracted by that in Ctrl skin (n = 4 for each group, data are representative of two independent experiments). P value was calculated using one-way ANOVA. (G) Subtracted MFI of mCherry in CD169⁺ Mϕ, CD169^– MHCII⁺ Mϕ, and CD169^– MHCII⁻ Mϕ on day 8 of mCherry-expressing B16-F10–bearing skin in WT or MERTK KO mice (n = 3–4 for each group, data are representative of two independent experiments). P value was calculated using one-way ANOVA. (H) The tumor volumes of B16-F10 melanoma in WT or MERTK KO mice (n = 8–9, data are combined from two independent experiments with four to five mice per group). Each circle represents one mouse. P value was calculated using Student’s t test.