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There is evidence to support the view that inflammatory processes are important in the development of local progression and metastases in patients with breast cancer. The sphingosine kinase-1/sphingosine-1-phosphate (SK1/S1P) pathway, which is a known mediator of inflammation, is critically implicated in breast cancer progression and chemotherapy resistance and is linked with poor prognosis. In this study we have investigated the implication of the SK1/S1P pathway in the interaction between tumour-associated macrophages and breast cancer cells.
We have used modified Boyden chambers to perform macrophage-tumour cell co-culturing. Cytokine production and alterations in gene expression were measured by quantitative RT-PCR. Proteome profiler assays were used to identify secreted cytokines. Cell motility and chemotaxis were assayed in 96-well plates of Dunn chambers respectively using high-throughput video time-lapse scanning microscopy.
MDA-MB-231 breast cancer cells were pretreated with docetaxel and subsequently co-cultured with THP-1 macrophages. Macrophages exhibited increased chemotaxis towards apoptotic tumour cells (aTCs) or aTC conditioned media. Co-culturing with aTCs has transiently increased macrophage SK1 activity. Proteome profiling of media from macrophages revealed that aTCs induced an SK1-mediated secretion of IL-6 and siCAM-1. Interestingly, co-culturing with macrophages increased aTC chemoresistance. Incubation of untreated cancer cells with macrophages pretreated with conditioned media from aTCs induced an IL-6-mediated upregulation of cancer cell SK1 expression in cancer cells, which has lead to an increase in cancer cell motility and chemotaxis in gradients of macrophage conditioned media. These enhanced migratory phenotypes were reversed following treatment of cancer cells with SK1 siRNA.
Our results suggest a novel IL-6/SK1-dependent mechanism of macrophage-induced breast cancer chemoresistance and metastasis.