Effect of hypoxic stress on induction and plasticity of tumor-induced immune cells in uveal melanoma

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Abstract

Purpose

Highly malignant uveal melanoma contain increased numbers of lymphocytes and macrophages. We wondered whether hypoxia plays a role in the development of this inflammation. We analysed whether hypoxia induces uveal melanoma cells to release pro-inflammatory cytokines, and whether tumor supernatant (TSN) affects monocyte migration and differentiation.

Methods

The expression of pro-inflammatory genes in freshly cultured uveal melanoma was studied in an in vitro 24 hour hypoxic culture system using qPCR. Cell lines were cultured under normoxic and hypoxic conditions. Chemotaxis was tested using a transwell system with purified monocytes and TSN. Differentiation was tested by adding TSN to a monocyte-DC culture. CCL2, IL-6 and PGE2 levels in TSN were determined by ELISA.

Results

Exposure of freshly-cultured uveal melanoma cells to hypoxia led to an increased expression of the pro-inflammatory cytokines PLGF, TGFß, END1, ICAM1 and a lower expression of AIMP1 (EMAP2), CCL2 (MCP-1), and IL1b. TSN from melanoma cells lines, cultured in normoxic as well as hypoxic conditions, was able to attract monocytes. Migration was independent of tumor-produced CCL2. Uveal melanoma supernatant inhibits monocyte differentiation.

Conclusion

Under hypoxic conditions, immune response genes are differentially expressed in cultured primary uveal melanoma cells. TSN from uveal melanoma cell lines is capable of affecting the chemotactic response of monocytes in vitro, irrespectively of the hypoxic or normoxic conditions. Our data suggest that the induction of immune cells is not dependent on intratumoral oxygen level, and the uveal melanoma TSN does not skew macrophage polarization phenotype.

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