Inhibition of chemokine receptor expression on uveal melanomas by CXCR4 siRNA and its effect on uveal melanoma liver metastases

Abstract

Purpose: To determine whether blocking the expression of the chemokine receptor CXCR4 using siRNA inhibits chemotactic responses of human uveal melanoma cells to liver-derived factors and prevents liver metastases.

Methods: Human uveal melanoma cells were transfected with CXCR4 siRNA or control siRNA and tested in vitro for chemotactic and invasive behavior in response to soluble factors produced by human liver cells. The effect of CXCR4 siRNA transfection on the formation of liver metastases was tested by injecting transfected melanoma cells into the spleen capsules of NOD-SCID mice, and metastases were quantified by measuring the human housekeeping gene hHPRT in livers.

Results: Blocking CXCR4 interaction with its ligand using anti-CXCL12 antibody resulted in a significant reduction in the chemotactic responses of uveal melanoma cells to soluble factors produced by human liver cells. Similarly, blocking CXCR4 gene expression by transfection with CXCR4 siRNA inhibited both the chemotactic and the invasive properties of uveal melanoma cells exposed to factors produced by human livers. Uveal melanoma cells transfected with CXCR4 siRNA produced fewer liver metastases than untreated uveal melanoma cells or uveal melanoma cells transfected with control siRNA.

Conclusions: CXCR4 is a key chemokine receptor that may account for the organ-specific homing of human uveal melanomas to the liver, which contains significant quantities of CXCL2, the only known ligand for CXCR4. CXCR4 is a potential therapeutic target for preventing the initial establishment of liver metastases but has limited application for use in advanced liver tumors.

Figure 1

Blockade of CXCR4/CXCL12 interactions with anti-CXCL12 antibody inhibits uveal melanoma chemotactic responses to liver-derived chemoattractants. Uveal melanoma cells were placed in the top chambers of 24-well transwell culture plates, and protein extracts of either human liver or human smooth muscle (40 μg/mL) were added to the bottom chambers. Top and bottom chambers were separated by a membrane with 8-μm pore size. In some experiments, anti-CXCL12 or isotype control antibody was added to the lower chamber (20 μg/mL). Twenty-four hours later, the number of melanoma cells that migrated to the bottom chamber was determined by counting the melanoma cells in 10 random HPFs using a compound microscope. Mean ± SEM. *P < 0.05; **P < 0.01.

Figure 2

CXCR4 siRNA inhibits CXCR4 message and protein expression in human uveal melanoma cells. RT-PCR results of OCM3 melanoma cells transfected with CXCR4 siRNA compared with control siRNA and nontransfected OCM3 melanoma cells. CXCR4 gene expression was downregulated in the CXCR4 siRNA group (A). FACS analysis of CXCR4 protein expression (B). Compared with the control siRNA group, the CXCR4 protein expression was 50% lower in the CXCR4 siRNA group. *P < 0.05.

Figure 3

CXCR4 siRNA inhibits chemotactic and chemoinvasive responses of human uveal melanoma cells but does not affect melanoma cell proliferation. Untreated and siRNA-transfected OCM3 uveal melanoma cells were placed in the top chambers of transwell culture plates. Protein extracts of either human liver or human smooth muscle (40 μg/mL) were added to the bottom chambers and served as chemoattractants to stimulate uveal melanoma cell migration to the bottom chambers. For chemotaxis assays, the top and bottom chambers were separated by a membrane with 8-μm pore size (A). For invasion assays, chambers were separated by a synthetic basement membrane created by coating an 8-μm pore membrane with basement membrane matrix (B). Twenty-four hours later, the number of melanoma cells that migrated to the bottom chamber was determined by counting the melanoma cells in 10 random HPFs using a compound microscope. This experiment was performed three times with similar results. Bars represent mean ± SEM. ***P < 0.001; **P < 0.01. OCM3 melanoma cells were transfected with CXCR4 siRNA or control siRNA. Melanoma cell proliferation was assessed by uptake of [³H]thymidine after 48 hours in culture (C). P > 0.05 in all groups.

Figure 4

CXCR4 siRNA treatment inhibits liver metastasis of human uveal melanomas in NOD-SCID mice. OCM3 uveal melanoma cells were transfected with either CXCR4 siRNA or control siRNA. OCM3 uveal melanoma cells (2 × 10⁵) were injected into the spleen capsules of NOD-SCID mice. Mice underwent necropsy 18 days later, and the metastatic tumor burden was quantified by gel electrophoresis (A) and real time RT-PCR (B) of the human housekeeping gene hHPRT. There were five mice in each group. Mean ± SEM. *P = 0.027

Figure 5

Histopathology of liver metastases arising from CXCR4 siRNA-transfected OCM3 uveal melanoma cells. OCM3 uveal melanoma cells were transfected with either CXCR4 siRNA or control siRNA. OCM3 uveal melanoma cells (2 × 10⁵) were injected into the spleen capsules of NOD-SCID mice. Mice underwent necropsy 35 days later, and livers were processed for conventional hematoxylin and eosin staining. (A, D) Untreated. (B, E) siRNA control-treated OCM3 uveal melanoma cells. (C, F) CXCR4 siRNA-treated OCM3 melanoma cells. Arrow: metastatic tumor nodules. (A–C) Low power and (D–F) high-power photographs of A–C, respectively.