Inhibition of iNOS activity enhances the anti-tumor effects of alpha-galactosylceramide in established murine cancer model

Abstract

Alpha-garactosylceramide (GalCer) has been shown to have anti-tumor effect in the basic research and clinical studies. However, anti-tumor effect of GalCer is limited. The administration of GalCer increases the production of IFN-γ which is involved in the suppression of tumor growth. On the other hand, the enhancement of IFN-γ production increases immunosuppressive factors such as nitric oxide. This suppressive action might impair the anti-tumor effect of GalCer. In the present study, we evaluated the anti-tumor effect of GalCer in the absence of inducible nitric oxide synthase (iNOS). In lung metastatic model, the number of tumor nodules in the lung of iNOS-KO mice treated with GalCer was significantly reduced compared with that of WT mice treated with GalCer. Moreover, L-NAME, which is the inhibitor for iNOS, enhanced the anti-tumor effect of GalCer in lung metastatic model. The frequency of CD8+ cells in bronchoalveolar lavage fluid increased in iNOS-KO mice treated with GalCer. The administration of GalCer increased the frequency of myeloid-derived suppressor cells (MDSCs) in the lung from tumor-bearing WT mice, but the increase of MDSCs in the lung was not induced in iNOS-KO mice. The subcutaneous tumor experiments revealed that the administration of GalCer in the absence of iNOS expression significantly enhanced the induction of tumor antigen-specific response. Finally, our results indicated that the inhibition of iNOS expression could enhance the therapeutic efficacy of GalCer via the increase of tumor antigen-specific immune response and the suppression of MDSCs.

Keywords: MDSC; alpha-garactosylceramide; cancer immunotherapy; induced nitric oxide synthase; tumor antigen-specific immune response.

Figure 1. Up-regulation of iNOS expression after GalCer administration in tumor-bearing mice

B16F10 cells (3 × 10⁵/mouse) were intravenously administered to WT mice. WT mice were intraperitoneally injected with GalCer (2 μg/mouse) at 7 days after the inoculation of tumor cells. A. The relative expression levels of iNOS mRNA in the lung of tumor-bearing mice treated with GalCer were measured by real-time RT-PCR. B. CD11b+ cells were magnetically isolated from BALF, and iNOS mRNA expression of CD11b+ cells were measured by real-time RT-PCR. The results were normalized to the expression of 18S rRNA. Each value is shown as mean and SEM for three mice. * indicates statistically significant differences.

Figure 2. Inhibition of iNOS expression enhanced the anti-tumor effect of GalCer on lung metastatic cancer model

A. B16F10 cells (3 × 10⁵/mouse) were intravenously administered to WT and iNOS-KO mice on day 0. Tumor-bearing mice were intraperitoneally injected with GalCer (2 μg/mouse) at 7 days after the inoculation of tumor cells. Mice were killed at 7 days after the injection of GalCer, and their lungs were removed to count superficial metastatic nodules. The number of superficial metastatic lung tumor was significantly decreased in iNOS-KO mice treated with GalCer (P < 0.05). B. B16F10 cells (3 × 10⁵/mouse) were intravenously administered to WT mice on day 0. WT mice were administered with L-NAME orally at 2 mg/ml in drinking water for 1 days before and 7 days after GalCer injection. Mice were killed at 7 days after the injection of GalCer. The number of superficial metastatic lung tumor was significantly reduced in mice with L-NAME and GalCer (P < 0.05). C. CT26 cells (2 × 10⁵/mouse) were intravenously administered to WT mice on day 0. The mice were orally administered L-NAME in drinking water at 6 days after tumor injection, and intraperitoneally injected with GalCer 1 day later. Mice were killed at 7 days after the injection of GalCer. The number of superficial metastatic lung tumor was significantly reduced in mice with L-NAME and GalCer (P < 0.05). Each value is shown as mean and SEM for 4-6 mice.

Figure 3. Changes of the proportion of CD8+ and CD11b+/Ly6G+ cells in BALF cells from tumor-bearing WT and iNOS-KO mice treated with GalCer

The lymphocytes in BALF from tumor-bearing WT and iNOS-KO mice were isolated 3 days after GalCer administration. A. Data show the percentage of CD4+, CD8+, and CD11b+/Ly6G+ cells. Each data point and error bar represent the mean and SEM, respectively, of data from triplicate samples. * indicates statistically significant differences. Closed bar; WT mice, open bar; iNOS-KO mice. B. BALF cells were isolated from WT and iNOS-KO mice administered with GalCer. Naïve splenocytes labeled with CFSE were co-cultured with BALF cells in 96-well plates at a cell-count ration of 1/1 for 3 days in the presence of PMA. Data were representative of at least three independent experiments with similar result.

Figure 4. The mRNA expression of IFN-γ, FasL, and CXCL9 in the lung from tumor-bearing iNOS-KO mice was up-regulated after GalCer administration

B16F10 cells (3 × 10⁵/mouse) were intravenously administered to WT mice on day 0. WT mice were intraperitoneally injected with GalCer at day 7 after the inoculation of tumor cells. The relative expression levels of IFN-γ, FasL, CCL2, and CXCL9 mRNA in the lung of WT and iNOS-KO mice treated with GalCer were measured using real-time RT-PCR. The results were normalized to the expression of 18S rRNA. Each data point and error bar represent the mean and SEM, respectively, of data from triplicate samples. * indicates statistically significant differences.

Figure 5. Inhibition of iNOS expression enhanced the anti-tumor effect of GalCer in a subcutaneous tumor model

EG7 cells (1 × 10⁶/mouse) were subcutaneously inoculated into the flank of WT and iNOS-KO mice on day 0. After the tumors became palpable, the mice were intraperitoneally injected with GalCer (2 μg/mouse). A. Data show the mean ± SEM increase in tumor size 6-8 mice per group, from two experiments. B. DLN in tumor-bearing WT and iNOS-KO mice were isolated 7 days after GalCer intraperitoneal injection. These DLN cells were stimulated with OVA peptide in vitro and monitored for IFN-γ production using ELISPOT assay. Results represent the mean ± SEM of 4 mice per group. The number of spot was significantly increased in DLN cells from iNOS-KO mice treated with GalCer (P < 0.05). C. Isolated effector cells (CD8+ T cells) from DLN of WT and iNOS-KO mice treated with GalCer were incubated for 6 h with CFSE-labeled EG7 at an effector to target cell ratio of 20 to 1. Each data point and error bar represent the mean and SE, respectively, of results for triplicate samples. D. The relative expression of IFN-γ mRNA in DLN of WT and iNOS-KO mice treated with GalCer was measured using real-time RT-PCR. The results were normalized to the expression of 18S rRNA. The expression of IFN-γ mRNA was significantly increased in iNOS-KO mice treated with GalCer (P < 0.05).

Figure 6. Changes of the proportion of CD8+ and CD11b+/Ly6G+ cells in the tumor infiltrating lymphocytes from tumor-bearing WT and iNOS-KO mice treated with GalCer

The tumor infiltrating lymphocytes from tumor-bearing WT and iNOS-KO mice were isolated 1 and 3 days after GalCer administration. A. Data show the percentage of CD4+, CD8+, and CD11b+/Ly6G+ cells. Each data point and error bar represents the mean and SEM, respectively, of data from triplicate samples. * indicates statistically significant differences. Closed bar; WT mice, open bar; iNOS-KO mice. B. Splenic CD11b+ cells were isolated from WT and iNOS-KO mice administered with GalCer. Naïve splenocytes labeled with CFSE were co-cultured with CD11b+ cells in 96-well plates at a cell-count ration of 1/1 for 3 days in the presence of PMA. Data were representative of at least three independent experiments with similar result.