Figure 4. CCR4 ligands secreted by human stromal brain cells induce melanoma cell migration.

(A–B) FACS analysis for CCR4 expression by local melanoma cells infected with empty plasmid (CON- red) (A), or with CCR4 cDNA (CCR4^hi-green) (B). (C) Viability (XTT-based assay) of control (CON) cells and of CCR4 over-expressing melanoma cells (CCR4^hi). (D–I) Migration assays.(D) Representative images of migrated CCR4^hi and CON cells following 24 h incubation with astrocyte-derived soluble factors (conditioned medium - HA CM) as compared to cells that were allowed to migrate towards serum free medium (SFM). (E) Quantification of (D). (F) Representative images of migrated local and brain metastasizing melanoma cells (HBMMC) 24 h following incubation with 10 ng/ml recombinant CCL17 (rCCL17). (G) Quantification of (F). (H–I) Neutralizing anti –CCL17 Ab reduce melanoma cell migration and transendothelial migration (TEM). (I) Melanoma cells were incubated with HA CM (control) or with HA CM supplemented with 1 μg/ml neutralizing Ab to CCL17. Representative images of migration assays are shown. I Quantification of (H). Data shown are mean pixel density normalized to control (SFM). Experiments were performed in triplicate and four independent fields/well were quantified. *P < 0.05 **P < 0.005, n = 3.