Tumor cell chemotaxis to LECs is mediated by CCL1. (A) Migration of breast cancer and melanoma cells to LEC-CM in a Boyden chamber assay. MCF-10F and 184-B5, nontumorigenic human breast epithelial cell lines; MCF-7, MDA-MB-231, and MDA-MB-435, breast cancer cell lines; SK-MEL-28, SK-MEL-25, and MEL-501, melanomas. (B) Migration of MDA-MB-435 cells to LEC-CM in presence of pertussis toxin (PTx) or cholera toxin (CTx). (C) Effects of a monoclonal anti-CCL1 neutralizing antibody on MDA-MB-435 and SK-MEL-25 chemotaxis to LEC-CM. (D) Tumor cell migration to LEC-CM depleted of CCL1 with a polyclonal anti-CCL1 antibody. (E) Migration of tumor cells to 50 ng/ml rhCCL1, in comparison to LEC-CM. (F) Cell shape change of MDA-MB-435 cells stimulated with 50 ng/ml human CCL1. Cells are stained with rhodamine-labeled phalloidin (red) and Hoechst (nuclei; blue). Data are representative of three experiments. Migration (fold increase) is calculated as cell migration (total cell area) to LEC-CM/cell migration to basal medium. Error bars indicate SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bar, 10 µm.