Fig. 6. Knockdown of CSF1 blocks NCOA1-promoted macrophage recruitment in culture and in mice and inhibits metastasis.

A. The numbers of macrophages recruited by non-targeting siRNA-transfected Tg(Neu) (Neu-siCtrl) and Tg(NCOA1)×Tg(Neu) (NCOA1×Neu-siCtrl) tumor cells, hNCOA1 and Csf1 siRNA-transfected Tg(NCOA1)×Tg(Neu) (NCOA1×Neu-siNCOA1 and NCOA1×Neu-siCsf1) tumor cells, and untransfected Tg(Neu) (Neu) and Tg(NCOA1)×Tg(Neu) (NCOA1×Neu) tumor cells. Cells were treated with or without recombinant Csf1 (rCsf1) protein or CSF1 antibody (a-CSF1) as indicated. The assay was performed using a transwell co-culture system with the indicated tumor cells in the lower chambers and macrophages above a matrigel layer in the upper chambers. The knockdown efficiency of hNCOA1 and Csf1 mRNAs were evaluated by qPCR (Supplementary Fig. S4). B. qPCR measurement of relative NCOA1 and CSF1 mRNA levels in MDA-MB-231^shCtrl, MDA-231-LM3.3^shCtrl, MDA-231-LM3.3^shCSF1-1 and/or MDA-231-LM3.3^shCSF1-2 cells. These cells were generated by lentiviral expression of control shRNA (shCtrl) and CSF1 shRNAs (shCSF1-1 and shCSF1-2). The expression levels of these mRNAs were normalized to 18S rRNA. C and D. MDA-MB-231^shCtrl, MDA-231-LM3.3^shCtrl, MDA-231-LM3.3^shCSF1-1 and MDA-231-LM3.3^shCSF1-2 cells (2×10⁶) were injected into the 4^th pair of mammary fat pads of SCID mice (n=5). Mice were sacrificed 30 days after injection. F4/80 immunostaining was performed to identify macrophages (brown) on the xenograft tumor (T) sections. F4/80-positive cells were counted as described in Fig. 2B. Data are presented as mean ± SD (Panel C). H&E staining of non-adjacent lung sections was performed to determine metastasis index, which was presented as the average ratio of lung metastasis (M) area to the total metastasis and lung (L) area (Panel D). * in all panels, p < 0.05 by Student’s t-test.