Fig. 3.

Metastatic effects of 27HC require polymorphonuclear neutrophils. a Myeloid-derived immune cells are required for the colonizing effects of 27HC, as clodronate-liposome depletion of phagocytic cells ablates its effects. Mice were pretreated prior to cellular graft (i.v.) with placebo or 27HC and/or liposomes containing either PBS or clodronate as outlined in Fig. 2a. Representative images of iRFP-expressing Met1 colonies (depicted as black) within the lungs are presented above quantified data for both Met1 and E0771 mammary cancer models (Met1 in order from left to right N = 5, 3, 3, 4, E0771 in order from left to right N = 3, 3, 4, 4, scale bars indicate 5 mm). b 27HC treatment of metastasis bearing mice results in the recruitment of inflammatory monocytes (IMs) and polymorphonuclear neutrophils (PMNs) to the lung, as assessed by FACS analysis (in order from left to right N = 3, 3, 6). For comparison, one group of mice received only an i.v. PBS injection (no cells). c 27HC increases in vitro migration of bone marrow-derived neutrophils in a CXCR2-dependent manner, as the small-molecule inhibitor of CXCR2, SB225002 attenuates its effects (N = 3/group). d Colonizing effect of 27HC requires activation of CXCR2. Mice were pretreated with indicated ligands for 5 days prior to engraftment with Met1 cells expressing iRFP (as in Fig. 2a). Representative images of iRFP-expressing Met1 colonies (depicted as black) within the lungs are presented above quantified data (N = 6/group, scale bar indicates 5 mm). e PMNs are required for the colonizing effects of 27HC, as immune-depletion of PMNs with an antibody against Ly6G (α-Ly6G) ablates its effects. Mice were pretreated with indicated ligands for 5 days prior to engraftment with Met1 cells expressing iRFP (as in Fig. 2a). Representative images of iRFP-expressing Met1 colonies (depicted as black) within the lungs are presented above quantified data (placebo groups N = 4, isotype control 27HC group N = 6, α-Ly6G 27HC group N = 7, scale bar indicates 5 mm). Results are depicted as mean +/− SEM. Lines and asterisks denote statistical differences between groups (p < 0.05). [One-way ANOVA followed by a Student Newman-Keuls multiple comparison test]