Inactivation of CCL9-CCR1 signaling blocks accumulation of the iMCs in the liver. (A) A metastatic focus in the liver immunostained for CCR1 and CD34. (Inset) A higher magnification of the boxed area. T, tumor; L, liver. (Scale bar, 100 μm.) (B) Expression of mRNAs for CCR1 ligands determined by RT-PCR. Total RNA was isolated from cultured CMT93 cells. Total RNA from intestinal polyps of Apc/Smad4 mice was used as a control (p.c.). Negative controls (n.c.) had distilled water instead of RNA. (C) A metastatic focus in the liver immunostained for CCL9 (brown DAB staining with hematoxylin counterstaining). (Inset) A higher magnification of the boxed area. (Scale bar, 100 μm.) (D) Liver metastasis foci in mice injected with CMT93 cells expressing scramble RNA (Scramble) and shRNA against Ccl9 (shCcl9#1). Serial sections were immunostained for CK, CD31 and the iMC markers (CD34 and CD11b). T, tumor; L, liver. (Scale bars, 100 μm.) (E) Liver metastasis foci in wild-type and Ccr1−/− mice injected with CMT93 cells. (Scale bars, 100 μm.)