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Original Investigation | Laboratory Sciences

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

Inge H. G. Bronkhorst, MD, PhD1; Tina M. L. Jehs, MSc2; Eveline M. Dijkgraaf, MD3; Gregorius P. M. Luyten, MD, PhD1; Pieter A. van der Velden, PhD1; Sjoerd H. van der Burg, PhD3; Martine J. Jager, MD, PhD1
[+] Author Affiliations
1Department of Ophthalmology, Leiden University Medical Center, Leiden, the Netherlands
2Department of International Health, Microbiology and Immunology, Copenhagen University, Copenhagen, Denmark
3Department of Clinical Oncology, Leiden University Medical Center, Leiden, the Netherlands
JAMA Ophthalmol. 2014;132(5):614-621. doi:10.1001/jamaophthalmol.2014.43.
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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 Setting  The 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), TGFβ (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 Relevance  Our 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.

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Figure 1.
Uveal Melanoma (UM) Cell Transcriptional Responses to Hypoxic Culture

RNA was prepared from 8 primary UM cultures grown under hypoxic or normoxic conditions for 24 hours and subjected to quantitative polymerase chain reaction analysis. The ordinate shows fold difference in transcript level in hypoxic culture relative to the normoxic culture reference (reference = 1). Asterisks indicate P < .05 derived from paired t tests of ΔCT values comparing hypoxic and normoxic cultures. Error bars indicate SEM.

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Figure 2.
Effect of Chromosome 3 Status on Immune Response Gene Expression in Primary Uveal Melanoma Cultures

Hypoxia stimulates vascular endothelial growth factor (VEGF) production (A) but not CCL2 (B) and IL6 (C). Under normoxic conditions, CCL2 and IL6 (P = .11) expression seems higher in M3 tumors, whereas VEGF (P = .88) showed no difference (Mann-Whitney test). IL6 was significantly increased in M3 tumors under hypoxic conditions (P = .03). Asterisk indicates P < .05.

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Figure 3.
Effect of Tumor Supernatant (TSN) From Normoxic and Hypoxic Uveal Melanoma Cell Lines on In Vitro Migration of Monocytes

Monocytes were added in an insert, and TSN was added underneath the insert. After 16 hours, the number of migrated cells was counted with flow cytometry. A mixture of CCL2 and lipopolysaccharide (LPS) served as a positive control. The TSN from all 5 cell lines had a migration-inducing capacity, which was independent of normoxic or hypoxic conditions.

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Figure 4.
Migration Assay

The presence of CCL2 was determined in tumor supernatants (TSNs) from the uveal melanoma cell lines OCM8 (A) and OMM2.5 (B) grown under hypoxic and normoxic conditions for 24, 48, 72, and 96 hours. The CCL2 concentrations in the TSN of cell line 92.1 and Mel270 were less than 5 pg/mL. The TSN of cell line Mel290 only showed a low level of CCL2 after 96 hours (not shown). C, Primary uveal melanoma cell cultures 10-019 and 12-009 and the lung adenocarcinoma cell culture 10-015 express CCL2.

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Figure 5.
Effect of Tumor Supernatant on Monocyte Differentiation

Peripheral blood monocytes were cultured for 6 days with granulocyte-macrophage colony-stimulating factor and interleukin 4 in 20% control medium or in culture medium with 20% tumor supernatant (TSN) of the indicated cultures or cell lines. Cells were analyzed for the expression of the monocyte-derived dendritic cell (mo-DC) marker CD1, macrophages and DC marker CD206, the macrophage marker CD14, and M2 marker CD163. The TSN of CC8 and of the lung adenocarcinoma cell line 10.015 were able to skew mo-DCs toward CD14+ macrophages with a high CD163 expression, whereas the TSN from the primary UM cell cultures (10-019 and 12-009) or the UM cell line OCM8 (under either normoxic or hypoxic conditions) inhibited DC maturation (low CD1a) but did not affect macrophage differentiation.

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