Effect of interleukin-18 on metastasis of mouse osteosarcoma cells

Abstract

The effect of interleukin-18 (IL-18) on metastasis of highly metastatic LM8 mouse osteosarcoma cells was investigated using nude mice treated with anti-asialo GM1 serum to exclude anti-tumor actions of IL-18 through activation of T and natural killer cells. Injection of LM8 cells which do not express IL-18 receptor beta into a tail vain resulted in the formation of pulmonary and hepatic metastatic foci. Daily injection of mice with IL-18 starting the fifth day from the cell injection had no significant effect on the number of metastatic foci, while five daily injections of IL-18 before and after the cell injection resulted in marked decreases. Culture of LM8 cells with IL-18 for 5 days before the injection into mice produced no significant effect on the number of pulmonary and hepatic metastatic foci. In contrast, pretreatment of mice with IL-18 for 5 days before the cell injection markedly decreased metastatic foci. The retention of LM8 cells in the lung 24 h after their injection was also reduced by the pretreatment of mice with IL-18. Serum obtained from mice pretreated with IL-18 for 5 days suppressed mobility of LM8 cells but IL-18 itself did not. These results suggest that IL-18 inhibits metastasis of LM8 cells partly by inducing a factor(s) in the host which suppresses cell mobility.

Fig. 1

Reverse-transcriptase polymerase chain reaction (RT-PCR) analaysis of mRNAs of interleukin-18 (IL-18) receptor subunits (IL-18 R α and β) in LM8 cells. 5E3 cells were used as a positive control for the expression of mRNAs of IL-18 R α and β

Fig. 2

Effect of IL-18 on the growth of LM8 cells in vitro. LM8 cells (1×10⁴ cells/35 mm-dish) were cultured in Eagle’s minimum essential medium (EMEM) containing 10% fetal calf serum (FCS) for 4 or 7 days in the presence or absence of IL-18 (10 or 100 ng/ml). Each bar represents a mean + SE of four dishes

Fig. 3

Effect of IL-18 on the growth of LM8 cells in nude mice. Mice were injected with anti-asialo GM1 serum (20 μl/mouse) and, 5 days later, inoculated with LM8 cells (1×10⁷ cells) subcutaneously. Injection of anti-asialo GM1 serum continued every 5 days. Starting the fifth day from the cell inoculation, mice received daily injection of IL-18 (2 μg/mouse) or vehicle. The upper figure shows the experimental design and the lower one the results. Each point represents a mean ± SE of eight mice

Fig. 4

Effect of IL-18 on the growth of LM8 cells metastasized to the lung and liver. LM8 cells (2×10⁵ cells) were injected into a tail vein of nude mice. Anti-asialo GM1 serum and IL-18 or vehicles were injected as described in the legend of Fig. 3. On the 19th day from the injection of LM8 cells, metastatic foci on the lung and liver were counted. The upper figure shows the experimental design and the lower one the results. Each bar represents a mean + SE of ten mice

Fig. 5

Effect of IL-18 on pulmonary and hepatic metastases of LM8 cells. LM8 cells (2×10⁵ cells) were injected into a tail vein of nude mice. These mice were daily injected with IL-18 (2 μg/mouse) or vehicle from 2 days before the cell injection to 2 days after (total 5 days). Anti-asialo GM1 serum (20 μl/mouse) was injected every 5 days starting 5 days before the injection of LM8 cells. On the 21st day from the injection of LM8 cells, metastatic foci on the lung and liver were counted. The upper figure shows the experimental design and the lower one the results. Each bar represents a mean + SE of ten mice. **P<0.01, significant difference from the values of mice injected with vehicle only

Fig. 6

Effect of pretreatment of mice with IL-18 on the pulmonary and hepatic metastases of LM8 cells. LM8 cells (2×10⁵ cells) were injected into a tail vein of nude mice. These mice were daily injected with IL-18 (2 μg/mouse) or vehicle from 5 to 1 day before the injection of LM8 cells (total 5 days). Anti-asialo GM1 serum (20 μl/mouse) was injected every 5 days starting 5 days before the injection of LM8 cells. On the 21st day from the injection of LM8 cells, metastatic foci on the lung and liver were counted. The upper figure shows the experimental design and the lower one the results. Each bar represents a mean + SE of ten mice. **P<0.01, significant difference from the values of mice injected with vehicle only

Fig. 7

Effect of treatment of LM8 cells with IL-18 in vitro on their pulmonary and hepatic metastasis. LM8 cells (2×10⁵ cells) which had been cultured in medium with or without IL-18 (100 ng/ml) for 5 days, were injected into a tail vein of nude mice. Anti-asialo GM1 serum (20 μl/mouse) was injected every 5 days starting 5 days before the injection of LM8 cells. On the 21st day from the injection of LM8 cells, metastatic foci on the lung and liver were counted. The upper figure shows the experimental design and the lower one the result. Each bar represents a mean + SE of ten mice

Fig. 8

Effect of pretreatment of mice with IL-18 on the retention of LM8 cells in the lung. LM8 cells (2×10⁵ cells) labeled with ¹²⁵I by culturing them with [¹²⁵I]iodo-2′-deoxyuridine, were injected into a tail vein of nude mice. Anti-asialo GM1 serum and IL-18 or vehicles were injected as described in the legend of Fig. 6. All mice were killed 24 h after the injection of LM8 cells, and the radioactivity retained in the whole lung was counted. The upper figure shows the experimental design and the lower one the results. Each bar represents a mean + SE of ten mice. **P<0.01, significant difference from the values of mice injected with vehicle only

Fig. 9

Distribution of LM8 cells in the lung. LM8 cells (2×10⁵ cells) labeled with a fluorescent dye were injected into a tail vein of nude mice and, 24 h later, the lungs were removed, cut into sections and examined under a fluorescence microscope . Fluorescent LM8 cells were located in an alveolar cavity, not in the lumen of capillaries. The asterisk shows an alveolar cavity and the arrows show LM8 cells in it. Bar: 50 μm

Fig. 10

Effect of serum obtained from mice treated with IL-18 on the motility of LM8, B16 melanoma and Lewis lung carcinoma cells, and the proliferation of LM8 cells. Nude mice were treated with anti-asialo GM1 serum and IL-18 or vehicle as described in Fig. 6, and sera of these mice were obtained 24 h after the last injection of IL-18 or vehicle. The effect of serum (50% in medium) of mice treated with IL-18 on the motility of LM8, Lewis lung carcinoma and B16 melanoma cells (a), and on the proliferation of LM8 cells (b) were estimated by the wound assay and ³H-thymidine uptake, respectively. Each bar represents a mean + SE of ten wells. *P<0.05, **P<0.01, significant difference from the values of mice treated with vehicle only

Fig. 11

Effect of serum obtained from mice treated with IL-18 on the attachment of LM8 cells to MS1 endothelial cells and effect of treatment of mouse MS1 endothelial cells with IL-18 on the attachment of LM8 cells. a Effect of serum on LM8–MS1 interaction. LM8 cells were incubated with MS1 endothelial cells in the presence of 50% serum from mice which were treated with IL-18 or vehicle as described in Fig. 10. b Effect of treatment of MS1 endothelial cells with IL-18 on the attachment to LM8 cells. MS1 cells were cultured in the presence or absence of IL-18 (100 ng/ml) for 24 h, and incubated with LM8 cells for 3 h in the presence of 50% serum from mice treated with vehicle. Each bar represents a mean + SE of ten wells