Expression of CCL3 in MAMs promotes metastatic seeding of cancer cells. (A) Number and size of lung foci were assessed in WT irradiated mice transplanted with BM cells from C57BL/6 (WT, n = 13), Ccl3−/− (n = 14), or Ccr2−/− (n = 15) mice (three independent experiments). After reconstitution of their BM, mice were injected with E0771-LG cells and euthanized after 11 d. Data are means ± SEM. *, P < 0.01 versus WT. (B) Number and size of lung foci were assessed in FVB mice injected with Met-1 cells (three independent experiments). Mice were treated with IgG (n = 9) or anti-CCL3 antibodies (n = 6) 24 and 2 h before tumor injection. Data are means ± SEM. *, P < 0.01. (C) Number and size of lung foci were assessed at day 11 after E0771-LG tumor cell injection in mice transplanted with WT (n = 8) or Ccl3−/− (n = 7) BM. The Ccl3−/− BM transplanted mice were also injected with WT (Ccl3+/+, n = 7) or Ccl3−/− (n = 6) inflammatory monocytes (IMs). Data are means ± SEM from four independent experiments. *, P < 0.01 versus WT; †, P < 0.05 versus Ccl3−/−. (D) Number of transmigrated E0771-LG cells was measured in the transendothelial migration assay in the absence (–) or presence of BMDMs from C57BL/6 or Ccl3−/− mice (n = 3, 2 independent experiments). Mean cell number in each insert was determined by the counts from five fields. Data are means ± SEM. *, P < 0.05 versus BMM (–); †, P < 0.01 versus WT. (E) mRNA expression of CC-chemokine ligands and their receptors in cultured mouse macrophages (BMDM), cancer cells (Met-1 and E0771), and endothelial cells (3B-11), as indicated (two independent experiments). PCR product size (bp) is shown on the right. (F) Secretion of CCL3 protein was assessed by ELISA in cultured mouse macrophages (BMDM), cancer cells (Met-1 and E0771), and endothelial cells (3B-11), as indicated (n = 3, three independent experiments). ND, not detected. (G) mRNA expression of CCL3 and its receptors in cultured human macrophages (MDM) and breast cancer cell lines (two independent experiments). PCR product size (bp) is shown on the right.