Characterization of the myeloid cells recruited to the premetastatic niche. (A) Cx₃cr1^gfp/+ mice were subcutaneously injected with 5 × 10³ B16F10 cells. After 21 days, lungs were isolated and sections imaged with a confocal microscope (top panels) and scored by software for the number (bottom left) and area (bottom middle) of CX₃CR1-GFP cells per field; n = 3 mice, ≥ 35 single fields analyzed per mouse (Mann-Whitney). The number of CX₃CR1-GFP cell clusters (as seen in top right panel) per field was manually scored (bottom right); n = 3 mice, ≥ 35 single fields analyzed per mouse (Mann-Whitney). (B) Cx₃cr1^gfp/+ mice were intravenously injected with 5 × 10⁵ CMRA-stained B16F10-wt cells after the establishment of the premetastatic niche, as described in panel A. Lungs, isolated either 8 or 24 hours after intravenous injection of tumor cells, were sectioned and directly imaged with a confocal microscope (top panels, tumor cell in red) or after immunohistochemistry (bottom panels) against the platelet-specific integrin α_IIb (AlexaFluor-633, red; and tumor cells, blue; 8 hours only). Representative images from 3 independent experiments performed per time point are shown. (C) Immunohistochemistry of sections from panel B, imaged with a confocal microscope (8 hours after the intravenous injection of tumor cells) against CD11b, CD11c, CD68, Gr-1, CD3ϵ, and CD45 (AlexaFluor-633, red; and tumor cells, blue). The percentage of CX₃CR1-GFP cells within the clusters that coexpressed the indicated markers was scored; n = 3 mice, ≥ 10 clusters analyzed per mouse (1-way ANOVA and Tukey test). Scale bars represent 50 μm. (A,C) Data are mean + SD. *P < .05. ***P < .001.