Effect of Hypoxic Stress on Migration and Characteristics of Monocytes in Uveal Melanoma

Abstract

IMPORTANCE Among the characteristics of uveal melanoma that are associated with a poor prognosis are a large tumor size and the presence of increased numbers of lymphocytes and macrophages. In rapidly growing tumors, reduction in oxygen tension may occur with increased distance from blood vessels, which we hypothesize may lead to an inflammatory microenvironment, further stimulating tumor growth.OBJECTIVES To analyze whether hypoxia induces uveal melanoma cells to express proinflammatory cytokines and whether tumor supernatant (TSN) affects monocyte migration and differentiation. DESIGN AND SETTINGThe expression of proinflammatory genes in freshly cultured uveal melanoma samples was studied in an in vitro 24-hour hypoxic culture system using quantitative polymerase chain reaction. In addition, cell lines cultured under normoxic and hypoxic conditions were used. The effect of TSN on monocyte chemotaxis was tested using a transwell migration system and by analyzing monocyte differentiation. The levels of the cytokines CCL2, IL6, and PGE2 in TSN were determined by enzyme-linked immunosorbent assay.PARTICIPANTS Five cell lines (OCM8, 92.1, Mel270,Mel290 and OMM2.5) and 11 primary short-term cultures.RESULTS Exposure of freshly cultured uveal melanoma cells to hypoxia led to an increased expression of the proinflammatory cytokines PLGF (OMIM 601121), TGF␤ (OMIM 190180), END1 (OMIM +131240), and ICAM1 (OMIM 147840) and a lower expression of AIMP1 (OMIM 603605) (EMAP2), CCL2 (MCP-1) (OMIM +158105), and IL1b (OMIM *147720). The TSN from cultured melanoma cell lines induced chemotaxis of monocytes, but this was independent of the normoxic or hypoxic state. The TSN of 1 cell line and 2 primary uveal melanoma cultures inhibited the dendritic cell maturation and did not induce M2 macrophage polarization in vitro. CONCLUSIONS AND RELEVANCEOur results indicate that under hypoxic conditions, immune response genes are differentially expressed in cultured primary uveal melanoma cells. The TSN from uveal melanoma cell lines is capable of affecting the chemotactic response and maturation of monocytes in vitro, but this is irrespective of hypoxia. 31. Cools-Lartigue J, Marshall JC, Caissie AL, Saraiva VS, Burnier MN Jr. Secretion of interleukin-6 and prostaglandin E2 during uveal melanoma-monocyte in vitro interactions. Exp Eye Res. 2004;79(4):451-454. 32. Müller-Quernheim UC, Potthast L, Müller-Quernheim J, Zissel G. Tumor-cell co-culture induced alternative activation of macrophages is modulated by interferons in vitro.

Paracrine Anti-inflammatory Effects of Adipose Tissue-Derived Mesenchymal Stem Cells in Human Monocytes

“…Cell migration was assayed with 8-μm-pore size Transwell migration chambers (Thermo Fisher Scientific) ( Bronkhorst et al, 2014 ). Monocytes (10 6 cells) in 1 ml of DMEM/Ham’s F-12 with antibiotics and 10% human serum or in 1 ml of CM were added to the upper chamber.…”

Hypoxia Alters the Expression of CC Chemokines and CC Chemokine Receptors in a Tumor–A Literature Review

“…Many studies show that CCL2/MCP-1 expression is reduced or increased in response to reduced oxygen levels. Chronic hypoxia has been reported to decrease the expression of this chemokine in glioma cells [154,155], HUVEC [156], monocytes and macrophages [144,157,158], and in uveal melanoma [159]. In contrast, it has also been reported to increase in breast cancer cells [142], cervical squamous carcinoma cells [46], hepatoma cells [160], in another study on HUVEC [161], dermal fibroblasts [162], Lewis lung carcinoma cells [163,164], multiple myeloma [165], primary mouse astrocytes [166], and vascular endothelial cells [167,168].…”

HLA Class I Antigen Expression in Conjunctival Melanoma Is Not Associated With PD-L1/PD-1 Status

“…The isolation of total cellular RNA, complementary DNA conversion, and quantitative PCR (qPCR) was performed based on a standard protocol of our laboratory, as described previously. 31 Primer sequences are in Supplementary Table S2. CFX Manager 3.1 (Bio-Rad, Hercules, CA, USA) software was used to analyze the data: the Ct values of each sample were normalized against the geometric mean Ct values of housekeeping genes (RPS11 and RPL13).…”