Skip to main content
. 2022 Jun 9;13(6):540. doi: 10.1038/s41419-022-04986-4

Fig. 4. CCL8 derived from TAMs is associated with collective migration of tumor cells.

Fig. 4

A The mRNA levels of a panel of cytokines in TAMs versus PBMCs isolated from five primary breast tumors (MMTV-PyMT) were detected by qRT-PCR. B The intracellular CCL8 and IL-10 in TAMs versus PBMCs isolated from normal mouse or tumor-bearing mouse (MMTV-PyMT tumor) were determined by ELISA. n = 5/group. Bars correspond to mean ± SD. *p < 0.05, **p < 0.01, ns, nonsignificant. C The secreted CCL8 and IL-10 in TAMs versus primary BrCa cells were determined by ELISA. Cells were isolated from five primary breast tumors (MMTV-PyMT) and cultured separately. Bars correspond to mean ± SD. **p < 0.01, ns, nonsignificant. D Fluorescence in situ hybridization analysis of human BrCa tissue sections revealed that CCL8 mRNA is found in CD206+ TAMs but not in cancer cells. The expression of CD206 was detected using immunochemical staining. Inset representing a CD206+ macrophage-expressing CCL8 mRNA. Scale bars, 100 μm (n = 3). E Blocking of CCL8 inhibited the collective migration induced by THP-1-derived M2-like macrophages. In vitro scratch assay of untreated MCF7 or treated with THP-1-derived M2-like macrophages, THP-1-derived M2-like macrophages plus normal IgG, or THP-1-derived M2-like macrophages plus CCL8 antibody for the indicated period of time. Red line, cell culture margins (n = 4). F Blocking of CCL8 inhibited the re-distribution of invadopodia molecules in collective migration induced by THP-1-derived M2-like macrophages. Representative confocal images showed the re-distribution of Cortactin (red) and TKS5 (green) at the migrating front of clusters after CCL8 blocking for 3 days. MCF7 cells co-cultured with THP-1-derived M2-like macrophages in a non-contact transwell system, followed by treatment with normal IgG or CCL8 antibody. Scale bars, 20 μm. G Knocking down of CCL8 in THP-1-derived M2-like macrophages inhibited the increase of Vinculin at the migrating front of clusters. Wound healing assay was used to observe the changes of Vinculin expression at the migrating front. M2-like macrophages derived from THP-1 were transfected with siRNAs (si-Control or si-CCL8) and then co-cultured with MCF7 cells in a non-contact transwell system for 3 days. The expression of Vinculin was determined by immunofluorescence assay. Scale bars, 10 μm. H Blocking of CCL8 inhibited the onset of xenografts induced by THP-1-derived M2-like macrophages in vivo. MCF7 cells were orthotopically implanted into mammary pad of nu/nu mice, followed by intraperitoneal administration of a neutralizing antibody for CCL8. I HE staining of MCF7 xenografts in normal IgG or CCL8 antibody-treated mice. J CD44 (brown) expression of MCF7 xenografts in normal IgG- or CCL8 antibody-treated mice. K CD31 (red) expression of MCF7 xenografts in normal IgG- or CCL8 antibody-treated mice.