Inhibition of IL-17A in tumor microenvironment augments cytotoxicity of tumor-infiltrating lymphocytes in tumor-bearing mice

Abstract

It remains controversial whether IL-17A promotes or inhibits cancer progression. We hypothesized that IL-17A that is locally produced in the tumor microenvironment has an important role in angiogenesis and tumor immunity. We investigated the effect of inhibiting IL-17A at tumor sites on tumor growth and on local and systemic anti-tumor immunity. MC38 or B16 cells were inoculated subcutaneously into mice, and intratumoral injection of an adenovirus vector expressing siRNA against the mouse IL-17A gene (Ad-si-IL-17) significantly inhibited tumor growth in both tumor models compared with control mice. Inhibition of IL-17A at tumor sites significantly suppressed CD31, MMP9, and VEGF expression in tumor tissue. The cytotoxic activity of CD8(+) T cells from tumor-infiltrating lymphocytes in mice treated with Ad-si-IL-17 was significantly higher than in control mice; however, CD8(+) T cells from splenocytes had similar activity levels. Suppression of IL-17A at tumor sites led to a Th1-dominant environment, and moreover, eliminated myeloid-derived suppressor cells and regulatory T cells at tumor sites but not in splenocytes. In conclusion, blockade of IL-17A at tumor sites helped suppress tumor growth by inhibiting angiogenesis as well as cytotoxic T lymphocytes activation at tumor sites.

Figure 1. Inhibition of IL-17A at tumor sites suppresses tumor growth.

(A, left panel) 1×10⁶ B16 tumor cells were injected subcutaneously into C57BL/6 mice, and PBS, Ad-SNC, or Ad-si-IL-17 with 1×10⁹ PFU was injected intratumorally at 7, 10, and 13 d. Data represent means ± SE (n = 7 mice per group of two independent experiments). *P<0.05, one-way ANOVA. (A, right panel) 5×10⁵ MC38 tumor cells were injected subcutaneously into mice, in the same way adenovirus vectors were injected at 5, 8, and 11 d. Data represent means ± SE (n = 7 mice of two independent experiments). *P<0.05, one-way ANOVA. (B).Levels of IL-17A protein were measured by ELISA in tumor lysates from mice after having been treated with PBS, Ad-SNC, or Ad-si-IL-17. In B16 and MC38 tumor tissues, Ad-si-IL-17 treatment showed lower levels of IL-17A compared with PBS or Ad-SNC treatment. Data are representative of two independent experiments. Data are presented as means ± SE (n = 4). *P<0.05, Student's t test.

Figure 2. Inhibition of IL-17A at tumor sites decreases the intratumoral microvessel density.

(A) The endothelial maker CD31-stained sections of tumor tissue are shown in Ad-SNC and Ad-si-IL-17 treatment models, Ad-si-IL-17 treatment decreased the intratumoral microvessel density compared with Ad-SNC. Vessels in five high-power fields (×200 magnification) were counted. Positive cells were quantified by an image-processing application. Data are representative of two independent experiments. Data are presented as means ± SE (n = 5). *P<0.05, Student's t test. (B) Intratumoral MMP-9 expression was decreased by the inhibition of IL-17. MMP-9 stained sections of tumor tissue were shown in Ad-SNC and Ad-si-IL-17 treatment models. Ad-si-IL-17 treatment decreased the intratumoral MMP-9 density compared with Ad-SNC. MMP-9 in five high-power fields (×200 magnification) was counted. Positive cells were quantified by an image-processing application. Data are representative of two independent experiments. Data are presented as means ± SE (n = 5). *P<0.05, Student's t test. (C) VEGF and IL-17A levels produced by MC38 cells were measured by ELISA. MC38 cells did not secretion IL-17A protein and produce VEGF protein. Levels of VEGF in vivo were measured by ELISA in tumor lysates from mice at 14 d after having been treated with Ad-SNC, or Ad-si-IL-17. In tumor tissues, Ad-si-IL-17 treatment showed lower levels of VEGF compared with Ad-SNC treatment. Data are representative of two independent experiments. Data are presented as means ± SE (n = 4). *P<0.05, Student's t test. ND: not detected.

Figure 3. Inhibition of IL-17A in tumor sites promotes CTLs activation, especially in the tumor microenvironment.

The cytotoxicity assay used CD8⁺ T cells from splenocytes or TILs in MC38 subcutaneous tumor treated Ad-si-IL-17 and Ad-SNC. (A) The cytotoxic activities against MC38 cells of CD8⁺ T cells from splenocytes in mice treated with intratumoral injection of Ad-si-IL-17 were almost the same as those in control mice (n = 5). The cytotoxic activities against B16 cells were less than 10% in both groups (n = 5). (B) The cytotoxic activities against MC38 cells in CD8⁺ T cells from TILs in mice treated with intratumoral injection of Ad-si-IL-17 were significantly higher than those with Ad-SNC (n = 3). Data are representative of two independent experiments. Data are presented as means ± SE. *P<0.05, Mann-Whitney test.

Figure 4. Inhibition of IL-17A in tumor sites increases Th1 cells in tumor microenvironment.

Splenocytes and TILs were collected from spleens or tumor tissues of MC38 subcutaneous tumor model. Th1/Th2 cells were detected by intracellular staining assay of anti-IFN-γ mAb and anti-IL-4 mAb. Representative flow cytometry analysis profiles gated on anti-CD4 mAb. Ad-si-IL-17 treatment increased Th1 phenotype of TILs compared to control, but that of splenocytes was similar level in both treatments. Th2 phenotype was similar ratio of TILs and splenocytes in both treatment. Data are representative of two independent experiments (n = 5).

Figure 5. Inhibition of IL-17A at tumor sites reduces MDSCs and Treg cells in the tumor microenvironment.

(A) Splenocytes and TILs were collected from spleens or tumor tissues of MC38 subcutaneous tumor model. Treg cells were detected by intracellular staining assay of anti-Foxp3 mAb. Representive flow cytometry analysis profiles gated on anti-CD3 and anti-CD4 mAb. Ad-si-IL-17 treatment decreased the ratio of Treg cells in TILs, but those in splenocytes were similar in both treatments. Data are representative of two independent experiments (n = 5). (B) Splenocytes and TILs were collected from spleens or tumor tissues of the MC38 subcutaneous tumor model. MDSCs cells were detected by double-staining of anti-CD11b mAb and anti-Gr1 mAb. Representive flow cytometry analysis profiles gated on anti-CD45 mAb. Ad-si-IL-17 treatment decreased the ratio of MDSCs in TILs, but those in splenocytes were at similar levels in both treatments. Data are representative of two independent experiments (n = 5). (C) Levels of IL-6 and CCL2 protein were measured in tumor lysates from mice at 14 d after having been treated with Ad-SNC, or Ad-si-IL-17. IL-6 and CCL2 were significantly decreased in tumors from Ad-si-IL-17–injected mice compared with control mice. Data are presented as means ± SE (n = 5). *P<0.05, Student's t test.