Interferon Alpha 2b Decreases Hepatic Micrometastasis in a Murine Model of Ocular Melanoma by Activation of Intrinsic Hepatic Natural Killer Cells

Purpose: To investigate in a murine model the mechanism by which micrometastatic melanoma, which spreads from the eye to the liver, is controlled by interferon (IFN)-alpha 2b.

Methods: Major histocompatibility (MHC) class I antigen (H-2, all haplotypes) expression in three murine melanoma cell lines (Queens, B16LS9, B16F10) was determined by flow cytometric immunophenotyping. The cell lines were heterotopically inoculated into the posterior compartments (PCs) of C57Bl/6 mice, and the mice were given intraperitoneal (IP) injections of IFN-alpha 2b or PBS for 1 or 4 days before enucleation at 7 days after inoculation. Groups of mice were made NK deficient or depleted with subcutaneous (s.c.) injection of anti-asialo GM1. The mice were killed at 28 days or 56 days (survival experiment) after inoculation, and the number of hepatic micrometastases was histologically determined. NK cells were isolated from the spleen and liver at necropsy, and propidium iodide labeled target-specific cytolysis was determined by flow cytometry. The micrometastases were evaluated for apoptosis and proliferation with TUNEL and MIB1 immunostaining, respectively, and TUNEL-to-MIB1 ratios were determined. Hepatic NK cells were immunostained with CD49b.

Results: MHC class I antigen was expressed in the three cell lines in the order of Queens < B16LS9 < B16F10. All cell lines grew, were confined to the PC, and formed hepatic micrometastases. A decrease in micrometastases, an increase in target-specific cytolysis, and an increase in survival correlated with decreased HLA class I expression by the melanoma cells. The IFN-alpha 2b treatment resulted in a boost of intrinsic hepatic NK cells, demonstrated in NK-deficient but not NK-depleted mice. The treatment effect corresponded to increased apoptosis (TUNEL)-proliferation (MIB1) ratios in the micrometastases. Immunostaining demonstrated an increased number of intrahepatic NK cells associated with the micrometastases in treated groups.

Conclusions: Neoadjuvant IFN-alpha 2b results in decreased hepatic micrometastasis and increased survival time through increased intrinsic hepatic NK cell-mediated tumor apoptosis in a murine model of metastatic ocular melanoma.