In this study, tissue specimens of normal, non-inflamed human testis with intact spermatogenesis and testicular germ cell neoplasia (GCNIS, seminoma) containing immune cell infiltrations were analysed by immunohistochemistry, immunfluores-cence and qRT-PCR to reveal phenotypic and functional differences among the immune cells that are contributing to the respective environment, reflected by cytokine expression profiles. The focus is set on macrophages and dendritic cells. Using specific macrophage and dendritic cell (DC) markers, different subsets of these immune cell types were identfied in testicular germ cell neoplasia suggesting a functional polarization. Under physiological conditions in the testis, anti-inflammatory M2 macrophages as supported by the presence of TGF-beta and IL-10 have been detectable, whereas DCs that express CD11c were rare. In contrast, GCNIS (known as precursor of seminoma) and seminoma was associated with infiltration of different DC subsets, i.e. mDC and pDC. A detailed analysis of macrophages revealed that, pro-inflammatory M1 and anti-inflammatory M2 macrophages are involved in immune cell infiltrates associated with testicular germ cell neoplasia. In line with the detection of M1 macrophages, increased levels of trancripts encoding IL-12 and TNF-alpha were found in neoplasia. To delinate why both, anti- and pro-inflammatory macrophages could be associated with testicular cancer, chemokines as well as potential macrophage polarization factors were analysed. A detailed chemokine expression profile revealed CCL2, CCL5, CCL18 and CCL22 as well as TGF-beta1, TNF-alpha and IL-6 as potential macrophage polarization factor to be associated with GCNIS and seminoma. To understand how these factors influence macrophages regarding their migration and differentiation/ polarization, an in vitro cell culture model using human monocyte derived THP-1 cells was estabilished. The results obtained indicate that CCL2 and CCL5 recruit macrophages and also influence macrophage polarization. To test whether tumour cells could drive macrophage differentiation, a co-culture model was estabilished using a human seminoma cell line (TCam-2) and THP-1 cells that were differentiated into M0, M1 and M2 macrophages. The co-culture of M0 and M1 macrophages with TCam-2 revealed a functional polarization of the THP-1-derived cells into an immunosuppressive M2 phenotype that may be driven by TGF-beta1, IL-6 and CCL2. TGF-beta1 signalling pathway inhibitor blocked the polarization towards an immunosuppressive M2 phenotype. Furthermore, a functional assessment of THP-1-derived M0 and M1 macrophages was conducted to reveal potential tumoricidial activities. In a phagocytosis assay, M1 macrophages showed high phagocytic activity enabling these cells to reduce TCam-2 cell numbers in the co-culture model. However, TCam-2 cells are likely to escape the tumoricidal activity by driving these macrophages into an immunosuppressive phenotype. In conclusion, M2 macrophages are the dominating immune cell population that support tumour grow. Therefore, as clinical outcome an inhibition of typical M2 polarization factors should be considered rather than an inhibition of recruitment factors (chemokines) since this would also influence the recruitment of phagocytic, tumour suppressive M1 macrophages. Future work should address the role of specific immune modulators, such as TGF-beta and activin A, and the impact and regulation of macrophage phagocytic activity on tumour cell survival and proliferation. Thus, potential macrophage differentiation inhibitors should be considered, as therapeutics to reinforce strong immune reactions against tumour cells.
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