CCL5 recruits monocytes to the metastatic microenvironment. (A) Representative immunofluorescence images of CCL5 detection (red) in lung sections from mice injected with MC-38GFP cells (green). Endothelial cells were stained with CD31 antibody (gray) and nuclei by DAPI (blue). CCL5 expression colocalized with endothelial cells (left side of the 12-hour image). Bar represents 20 μm. (B) Quantification of CCL5 signal in a defined area around MC-38GFP tumor cells (15 000 μm3). CCL5 detection was significantly higher at 12 hours compared with 6 hours and 48 hours after tumor cell injection (**P < .01; *P < .05), tested by ANOVA and Bonferroni posthoc test. (C) Representative images of lung sections from mice treated with Met-RANTES, injected with MC-38GFP cells (green), and killed 48 hours later. Monocytes were detected with F4/80 antibody (red) and nuclei by DAPI (blue). Magnification 40×/0.75 NA oil objective. Bar represents 100 μm. Insets show tumor cell microenvironment at higher magnification (63×/1.30 NA oil objective). (D) Quantification of tumor-associated F4/80-positive cells. Statistical analysis of Met-RANTES– and PBS-treated mice (n = 3); ***P < .001.
