Does Macrophage Stimulation by CCL5 Alter Melanoma Migratory Behavior?

Abstract

BackgroundCCL5 (RANTES, regulated upon activation, normal T‐cell expressed and secreted) is secreted not only by T‐cells, but also by melanoma cells into the tumor microenvironment. The current project is part of a bigger effort by our laboratory to determine how the stimuli present in the tumor microenvironment may contribute to the progression of melanoma. Here, we explore CCL5 receptor expression on monocytes, macrophages, and melanoma cells, and whether endogenous or exogenous CCL5 stimulation of THP‐1 macrophages produce phenotypical changes that affect melanoma migratory behavior.MethodsWe used WM278 and LM‐Mel‐45 melanoma cells and THP‐1 monocytes before and after conversion to macrophages by 2 days exposure to 50 ng/ml PMA. To examine expression of the 3 key CCL5 receptors, we compared median fluorescence intensity (MFI) of antibody‐exposed cells to MFI of unlabeled cells using antibodies for CCR1, CCR3, and CCR5. We gated out debris and multiples, corrected for spillover, and analyzed the samples on a Bio‐Rad S3e cell sorter. We used recombinant CCL5 (100 ng/ml), the CCR1 & CCR3 receptor antagonist, UCB35625 (100 nM) and the CCR5 antagonist, maraviroc (100 ng/ml). Melanoma migration rate was measured by a 16 h wound‐closure assay. Treatments included: Control, CCL5 only, maraviroc only, UCB35625 only, maraviroc and UCB35625 together, and CCL5 plus both blockers. Wound closure of LM‐Mel‐45 melanoma alone, a 1:4 mixture of macrophages to melanoma, or LM‐Mel‐45 melanoma treated with macrophage‐conditioned medium were compared in each treatment condition.ResultsCCL5 receptor expression was not found on WM278 melanoma cells. LM‐Mel‐45 melanoma express CCR1 but not CCR3 or CCR5. CCR1 and CCR5, but not CCR3, are expressed by both THP‐1 monocytes and THP‐1 macrophages. Melanoma cells alone or when co‐cultured with previously treated macrophages migrated at 14.3 ± 0.4 μm/h, but neither CCL5 stimulation nor receptor blockade changed migration significantly. When LM‐Mel‐45 melanoma cells were treated with conditioned macrophage medium, velocity increased to 16.8 ± 0.7 μm/h.ConclusionsThis work suggests that CCL5 does not directly impact melanoma migration either by endogenous expression or by exogenous addition. Even in the presence of CCL5 or antagonist‐exposed macrophages, we found no phenotypical changes that altered the physical interactions of macrophages on melanoma migratory rate. There is a small but significant increase in velocity of melanoma cells when treated with conditioned macrophage medium.