Interleukin-15 suppresses gastric cancer liver metastases by enhancing natural killer cell activity in a murine model

Abstract

Interleukin (IL)-15 is a promising cytokine for cancer immunotherapy as it is a critical factor for the proliferation and activation of natural killer (NK) cells. Previous studies have suggested critical roles of IL-15 in tumor invasion and metastasis. However, the association between IL-15 and liver metastasis of gastric cancer (LMGC) remains unknown. The present study investigated the therapeutic efficacy of recombinant mouse IL-15 (rmIL-15) in murine LMGC models, in which stable green fluorescent protein (GFP)-expressing MKN45 cells (MKN45-GFP cells) were injected into the spleen parenchyma of mice for liver metastasis. At different treatments (high dose group: 2.5 µg of rmIL-15; low dose group: 0.2 µg of rmIL-15; control group: PBS), it was found that rmIL-15 decreased the formation of liver metastasis sites. Additionally, this treatment lead to improved survival of mice following tumor cell transplantation. Treatment with a high dose of rmIL-15 provided greater therapeutic efficacy by prolonged survival of the mice compared with low dose group and control group. It was found that NK cells isolated from the liver that received the high dose of rmIL-15 showed stronger cytotoxic activity compared with the other two groups on the target cells. These findings hold significant importance for the use of IL-15 as a potential adjuvant/therapeutic for liver metastasis from gastric cancer.

Keywords: cancer immunotherapy; gastric cancer; interleukin-15; liver metastasis; natural killer cell.

Figure 1.

Establishing a mouse model of liver metastasis from gastric cancer. Representative fluorescence image of MKN45-GFP and representative image of spleen during injection of 1×10⁶/0.2 ml of MKN45-GFP cells into the spleen parenchyma of nude mice. MKN45-GFP cells, stable green fluorescent protein (GFP)-expressing MKN45 cells (scale bar, 50 µm).

Figure 2.

Representative fluorescence images of low-dose, high-dose and control groups (scale bar, 20 µm). MKN45-green fluorescent protein cells were injected into the spleen parenchyma and the livers were cut into 5-µm thick serial sections for fluorescence imaging. Female BALB/c nu/nu mice aged 4–6 weeks old were assigned randomly into a low-dose group (0.2 µg rmIL-15), high-dose group (2.5 µg rmIL-15) and control group (PBS), with 6 mice/group. All mice were treated five times/week for 3 weeks. On day 28, 6 mice/group were sacrificed. (A-C) Representative fluorescence images of the high-dose group, (D-F) Representative fluorescence images of the low dose group. (G-I) Representative fluorescence images of the control group. rmIL-15, recombinant mouse interleukin-15.

Figure 3.

Liver metastasis and survival of nude mice. Mice were inoculated with 1×10⁶ MKN45 cells into the spleen parenchyma. After 24 h, they were divided into three groups: Control (PBS treatment), 0.2 µg rmIL-15 and 2.5 µg rmIL-15. At day 28, metastasis nodules on the livers were counted. The rest of the mice were monitored daily until mortality. (A) 66.7% (4/6) and 83.3% (5/6) of mice given high or low dose rmIL-15 developed liver metastases, respectively, in contrast to 100% (6/6) among PBS-treated mice. (B) Mean liver weight of the three experimental groups; no significant difference among three groups (P>0.05). (C) rmIL-15 treatment caused a decrease in the number of liver metastasis nodules compared with PBS treatment, (P<0.05), and the number of liver metastasis nodules of the high dose rmIL-15 group was significantly less than the low dose group (P<0.05). (D) Kaplan-Meier survival curves, (E) average survival and (F) increased life-span rate in the liver metastasis model confirmed an association between 2.5 µg rmIL-15 therapy and longer survival time. Data are presented as the mean ± standard deviation. All statistics were calculated by the long-rank test (*P>0.05 vs. control, **P<0.05 vs. 0.2 µg, ***P<0.01 vs. control). rmIL-15, recombinant mouse interleukin-15.

Figure 4.

The serum concentrations of IL-15 and IFN-γ with different treatments indicated that IL-15 induced IFN-γ secretion both in nude mice and Balb/c mice. (A) Serum concentrations of IL-15 in nude mice, *P<0.05 vs. control. (B) Serum concentrations of IL-15 in nude mice, *P<0.05 vs. control. (C) Serum concentrations of IL-15 in Balb/c mice, *P<0.05 vs. control. (D) Serum concentrations of IFN-γ in Balb/c mice, *P<0.05 vs. control. Data are presented as the mean ± standard deviation. IL-15, interleukin-15; IFN-γ, interferon-γ.

Figure 5.

NK-mediated cytotoxicity of liver isolated from the tumor-bearing mice. Livers were isolated and incubated with MKN45 cells, or NK cell-sensitive YAC-1 in nude mice or Balb/c mice. (A) Percentage of CD49b⁺CD3⁻NK cells in nude mice, *P<0.05 vs. control. (B) Number of CD49b⁺CD3⁻NK cells in nude mice, *P<0.05 vs. control. (C) Percentage of CD49b⁺CD3⁻NK cells in Balb/c mice, *P<0.05 vs. control. (D) Number of CD49b⁺CD3⁻NK cells in Balb/c mice, *P<0.05 vs. control. NK, natural killer; CD3, cluster of differentiation 3; CD49b, cluster of differentiation 49b; rmIL-15, recombinant mouse interleukin-15.

Figure 6.

NK-mediated cytotoxicity of liver isolated from the tumor-bearing mice. Livers were isolated and incubated with NK cell-sensitive YAC-1 in (A) nude mice or (B) Balb/c mice, respectively. Values are presented as the mean ± standard deviation. Difference between the control and low dose rmIL-15 group was significant, also between the control and high dose rmIL-15 group for NK activities (*P<0.05). NK, natural killer; rmIL-15, recombinant mouse interleukin-15.