Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

Background
Tumor-associated inflammatory cells (TAIC) are a major component of the tumor microenvironment and can contribute to both tumor progression and metastasis for instance by direct cell-cell interaction via membrane-bound proteins. Tumor cells show varying expression of Eph receptors and their ephrin ligands, which both are receptor tyrosine kinases. Eph/ephrins are hypothesized to be possible mediators of tumor-associated inflammation. The aim of our study was to analyze the distribution of ephrinB2 and its receptors EphB4 and EphB6 in inflammatory and melanoma cells and to clarify proinflammatory effects due to Eph/ephrin-mediated cell-cell contact.

Material and Methods
HL-60 promyelocytes and THP-1 monocytes, differentiated into granulocytes and macrophages, were used as a model for TAIC. Undifferentiated and differentiated cells were co-cultivated with Mel-Juso and A2058 melanoma cells. EphrinB2, EphB4 and EphB6 mRNA expression and protein synthesis was investigated using qRT-PCR and flow cytometry. Secretion of the proinflammatory cytokines IL-6 and TNF-α was analyzed using ELISA.

Results
No alteration in gene expression of ephrinB2, EphB4 and EphB6 could be observed during differentiation of HL-60 and THP-1 cells. In contrast, protein synthesis of ephrinB2, EphB4 and EphB6 was two- to threefold higher in HL-60 granulocytes compared to HL-60 promyelocytes and HL-60 macrophages. THP-1 macrophages showed a slightly increased protein synthesis of EphB4 and EphB6 compared to THP-1 monocytes whereas ephrinB2 protein content remained constant. Co-culture of both THP-1 monocytes and macrophages with Mel-Juso cells caused a substantial increment in secretion of proinflammatory cytokines. Co-culture of both HL-60 granulocytes and THP-1 monocytes with A2058 cells did not affect cytokine secretion. By contrast, co-culture of HL-60 macrophages with A2058 cells resulted in increased IL-6 secretion whereas co-culture of THP-1 macrophages with A2058 cells resulted in increased IL-6 secretion but decreased TNF-α release.

Conclusions
To our knowledge, mRNA expression and protein synthesis of ephrinB2, EphB4 and EphB6 was investigated for the first time in undifferentiated and differentiated HL-60 and THP-1 cells and, moreover, in Mel-Juso and A2058 melanoma cells. Co-culture of TAIC with melanoma cells resulted in proinflammatory effects. To differentiate the role of various Eph receptors and ephrin ligands in mediation of these effects after direct cell-cell contact of TAIC and melanoma cells selective inhibitors for Eph are applied in ongoing studies.