Bevacizumab suppression of establishment of micrometastases in experimental ocular melanoma

Abstract

Purpose: This study was undertaken to determine whether anti-vascular endothelial growth factor (VEGF) therapy inhibits growth of primary uveal melanoma and spread of its hepatic micrometastases.

Methods: The human uveal melanoma cell lines Mel290 and Mel 270, HUVECs, mouse B16LS9 melanoma cells, and mouse vascular endothelial cells were separately cultured or co-cultured and incubated with bevacizumab or IgG1. The level of VEGF protein in the culture medium was measured by ELISA. In vitro angiogenesis and invasion assays were performed under bevacizumab or IgG1 treatment. Mel290 or B16LS9 cells were inoculated into NU/NU or C57Bl/6 mouse eyes which were enucleated after 7 days. The sizes of the intraocular tumors were determined. Time and dosage experiments were performed by using 50 or 250 microg bevacizumab starting at day 1 or 4 after inoculation. Hepatic micrometastases were enumerated. Proliferation, apoptosis, and angiogenesis markers were detected in the ocular tumor by immunofluorescence staining.

Results: Bevacizumab significantly reduced the level of VEGF in the culture media from human uveal melanoma cells, mouse melanoma cells, and co-cultured cells. It also inhibited cell tube formation and decreased in vitro invasion of tumor cells. In the mouse model, bevacizumab suppressed primary ocular melanoma growth and the formation of hepatic micrometastases in a dose-dependent manner. Furthermore, immunohistochemical staining showed decreased Ki67 and unchanged caspase 3 expression after treatment with bevacizumab.

Conclusions: Treatment with bevacizumab suppressed in vitro growth and in vivo hepatic micrometastasis of ocular melanoma cells. Bevacizumab is a potential therapeutic agent for the treatment of uveal melanoma micrometastases.

Figure 1.

VEGF expression after in vitro treatment with bevacizumab. (A) Human uveal melanoma cell lines and HUVECs expressed from 150 to 500 pg/mL VEGF, with a significant decrease after treatment with 10 or 100 μg/mL bevacizumab. (B) Human uveal melanoma cells co-cultured with HUVECs expressed higher levels of VEGF (1750–3000 pg/mL) than did individually cultured cells, which significantly decreased after treatment with 10 or 100 μg/mL bevacizumab. (C) Mouse B16LS9 melanoma cells expressed VEGF, which was significantly decreased after treatment with 10 or 100 μg/mL bevacizumab. Mouse B16LS9 melanoma cells co-cultured with mouse vascular endothelium expressed higher levels of VEGF did individually cultured cells, with a significant decrease in VEGF expression after treatment with 10 or 100 μg/mL bevacizumab.

Figure 2.

Bevacizumab decreased angiogenesis in vitro and in vivo. Bevacizumab decreased tube formation: IgG1-treated HUVECs (A, left) and Mel290-GFP co-cultured with HUVECs (B, left) exhibited meshlike patterns of tube formation (grade 5). Corresponding cells treated with bevacizumab showed a migration/alignment pattern (grade 1) in HUVECs (A, right) and sprouting new capillary tubes (grade 3) in the melanoma-GFP co-cultured cells (B, right). Bevacizumab decreased the vascular density in our mouse model of ocular melanoma (C). CD31+ vascular channels constitute 1% of the area of the tumor in mice treated with bevacizumab 250 μg/mL (C, left, arrow), compared with 26% of the area of the tumor in control mice treated with PBS (C, right, arrows).

Figure 3.

Bevacizumab inhibited intraocular melanoma growth. Groups with IP injections of (A) 250 or (B) 50 μg bevacizumab starting at day 4 after inoculation had smaller intraocular melanomas than did the PBS-treated control group (C). The 50- and 250-μg IP bevacizumab groups, both starting on day 4 after inoculation, exhibited decreased intraocular tumor size compared with that in the PBS-treated control (D). The size of the intraocular melanoma was significantly smaller in the day-1 and -4 groups compared with the control, and the tumors in the day-1 groups were significantly smaller than those in the day-4 groups (E). Each group contained 10 mice.

Figure 4.

Bevacizumab decreased Ki67 in human uveal melanoma and mouse melanoma cells. Melanoma cells grown in vitro and treated with 100 μg/mL bevacizumab for 48 hours showed inhibition of nuclear Ki67. (A) Top: IgG1-treated Mel290 cells showed 53% of nuclei stained. Bottom: bevacizumab-treated Mel 290 cells showed no stained nuclei. (B) Top: PBS-treated B16LS9 tumor showed 98% of nuclei stained. Bottom: bevacizumab-treated B16LS9 tumor showed 11% of nuclei stained.

Figure 5.

Bevacizumab did not cause apoptosis in melanoma cells. (A) Treatment of Mel 290 cells with IgG1 (top, 6% nuclear staining) or bevacizumab (middle, 4% nuclear staining) did not result in increased expression of caspase 3, compared with staurosporine-treated positive control cells (bottom, 74% nuclear staining). (B) Similarly, intraocular melanoma in the PBS control (top) and the bevacizumab-treated mice (bottom) both showed a lack of expression of caspase 3, with no nuclear staining detected in either group.