TITLE PAGE Tumor cell apoptosis polarizes macrophages – role of sphingosine-1-phosphate

Macrophage polarization contributes to a number of human pathologies. This is exemplified for tumor-associated macrophages (TAM), which display a polarized M2 phenotype, closely associated with promotion of angiogenesis and suppression of innate immune responses. We present evidence that induction of apoptosis in tumor cells and subsequent recognition of apoptotic debris by macrophages participates in the macrophage phenotype shift. During coculture of human primary macrophages with human breast cancer carcinoma cells (MCF-7) the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-α and interleukin 12-p70 production, but increased formation of interleukins 8 and 10. Alternative macrophage activation required tumor cell death because a coculture with apoptosisresistant colon carcinoma cells (RKO) or Bcl-2 overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-κB signaling. These findings suggest that tumor cell apoptosisderived S1P contributes to macrophage polarization.