Inhibition of B16 melanoma experimental metastasis by interferon-gamma through direct inhibition of cell proliferation and activation of antitumour host mechanisms

Abstract

Interferon-gamma (IFN-gamma) has pleiotropic activities other than its antivirus action, including cell growth inhibition, natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activation, and angiogenesis inhibitory activity, and these activities are supposed to be involved in its antitumour activity. However, it has not been completely elucidated which activity is mainly involved in the tumour suppression in vivo. In this study, we analysed inhibitory mechanisms of endogenous IFN-gamma against B16 melanoma experimental metastasis. After intravenous injection of tumour cells, tumour deposits in the lungs and liver were increased and life span was shorter in IFN-gamma(-/-) mice, indicating important roles for IFN-gamma in antitumour mechanisms. Interestingly, tumour deposits were not increased in IFN-gamma receptor (R)(-/-) mice. Furthermore, only low levels of cell-mediated immunity against the tumour and activation of NK cells were observed, indicating that antimetastatic effects of IFN-gamma is not mediated by host cells. The survival period of B16 melanoma-bearing IFN-gamma R(-/-) mice was, however, shorter than wild-type mice. These observations suggest that IFN-gamma prevents B16 melanoma experimental metastasis by directly inhibiting the cell growth, although antitumour host functions may also be involved in a later phase.

Figure 1

Experimental metastasis of B16 melanoma cells in IFN-γ^−/−, IFN-γR^−/−, and wild-type mice. B16 melanoma cells (1 × 10⁵ cells) were injected through the tail vein, and after 14 days, the lungs (a–c) and the liver (d–f) were removed, and the photographs were taken. Wild-type: a, d; IFN-γ^−/−: b, e; IFN-γR^−/−: c, f. Arrowheads, B16 metastatic colonies.

Figure 2

Number of the B16 metastatic colonies on the liver and the lung in IFN-γ^−/−, IFN-γR^−/−, and wild-type mice. (a) Number of the colonies on the liver and the lungs shown in Fig. 1 was counted, and the average ±SD is shown. Results are reproducible in three independent experiments. (b) The diameter of each colony was measured on the histological sections, and the average size and SD are shown. *P < 0·05, **P < 0·01 (IFN-γ^−/− versus wild-type and IFN-γR^−/− mice).

Figure 3

IFN-γ sensitivity of B16 melanoma cells. Anti-cell growth activity was accessed by counting the number of B16 melanoma cells in two wells in a six-well dish periodically in the presence of the indicated concentrations of IFN-γ.

Figure 4

IFN-γ production by splenocytes prepared from IFN-γ^−/−, IFN-γR^−/− and wild-type mice. (a) Splenocytes were cultured in anti-TCR-β antibody-coated culture plate for 4 hr and the concentration of IFN-γ in the supernatants was measured by ELISA. (b) Splenocytes were prepared from the mice 14 days after injection of B16 cells, and cultured with irradiated B16 cells for 96 hr in the presence or absence of IL-2. The concentration of IFN-γ in the supernatants was measured by ELISA. *, not detected.

Figure 5

Cytotoxic activities of splenocytes from IFN-γ^−/−, IFN-γR^−/− and wild-type mice against B16 melanoma cells (a) or YAC-1 cells (b). Splenocytes were prepared 14 days after the injection of B16 cells, and cultured with irradiated B16 cells for 96 hr in the presence or absence of IL-2. Cytotoxic activity of the splenocytes was determined by a standard 4-hr ⁵¹Cr-release assay using B16 and YAC-1 cells as the target cells at various effector : target cell ratios. Similar results were obtained in two independent experiments. Wild-type: circle; IFN-γ^−/−: square; IFN-γ R^−/−: triangle. Open symbol: +IL-2, closed symbol: −IL-2.

Figure 6

Experimental metastasis of B16 melanoma cells in anti-asialo GM1 antibody-treated mice. Photographs of the metastatic colonies in the lungs (a–c, g–i) and the liver (d–f, j–l) were taken 14 days after the intravenous injection of 1 × 10⁵ B16 melanoma cells. Wild-type: a, d, g, j; IFN-γ^−/−: b, e, h, k; IFN-γR^−/−: c, f, i, l; treated with anti-asialo antibody: g–l; without treatment: a–f.

Figure 7

Number of the B16 melanoma metastatic colonies on the liver and the lungs in antiasialo GM1 antibody-treated mice. Colonies on the lung (a) and liver (b) of IFN-γ^−/−, IFN-γR^−/− and wild-type mice were counted, and the average ±SD is shown. *P < 0·05, **P < 0·01 IFN-γ^−/− versus IFN-γR^−/− and wild-type mice. Similar results were obtained in another experiment.

Figure 8

Survival of IFN-γ^−/− and IFN-γR^−/− mice inoculated with B16 melanoma cells. Mice were intravenously inoculated with 1 × 10⁵ B16 melanoma cells. Closed circle: wild-type mice, n = 15; open circle: IFN-γ^−/− mice, n = 21; open square: IFN-γR^−/−, n = 8. *P < 0·05: IFN-γR^−/− versus wild-type, **P < 0·01: IFN-γ^−/− versus wild-type mice.

Figure 9

B16 melanoma cell nodule formation in IFN-γ^−/− and IFN-γR^−/− mice. Mice were subcutaneously inoculated with 2 × 10⁵ B16 melanoma cells. Sizes of the tumour were measured from 8 days after the inoculation. Open circle: IFN-γ^−/− mice, n = 5; square: IFN-γR^−/−, n = 5; closed circle: wild-type mice, n = 5. *P < 0·05: IFN-γ^−/− versus wild-type mice. Similar results were obtained in three independent experiments.