ST2/IL-33 signaling promotes malignant development of experimental squamous cell carcinoma by decreasing NK cells cytotoxicity and modulating the intratumoral cell infiltrate

Abstract

Squamous cell carcinoma (SCC) is the second most common form of skin cancer and the mechanism(s) involved in the progression of this tumor are unknown. Increases in the expression of IL-33/ST2 axis components have been demonstrated to contribute to neoplastic transformation in several tumor models and interleukin-33 is correlated with poor prognosis of patients with squamous cell carcinoma of the tongue. Based on these observations, we sought to determine the role of the IL-33/ST2 pathway during the development of SCC. Our findings show that ST2-deficiency led to a marked decrease in the severity of skin lesions, suggesting that ST2 signaling contributed to tumor development. An analysis of tumor lesions in wild-type and ST2KO mice revealed that a lack of ST2 was associated with specific and significant reductions in the numbers of CD4⁺ T cells, CD8⁺ T cells, dendritic cells, and macrophages. In addition, NK cells that were isolated from ST2KO mice exhibited higher cytotoxic activity than cells isolated from wild-type mice. Notably, ST2 deficiency resulted in lower IFN-γ, TNF-α, IL-10, and IL-17 production in tumor samples. Our findings indicate that the IL-33/ST2 pathway contributes to the development of SCC by affecting leukocyte migration to tumor microenvironment and impairing NK cytotoxic activity.

Keywords: IL-33; ST2; chemical carcinogenesis; immune modulation; squamous cell carcinoma.

Figure 1. ST2/IL-33 signaling favors SCC development

(A) Graphic showing the frequencies of tumor-free wild-type (WT, empty squares) and ST2KO mice (full squares). The results are expressed as the percentage of mice that were free of skin tumors (n = 15 per group). (B) Representative pictures of lesion growth patterns (40x). (C) Graphic showing the frequencies of exophytic and endophytic tumors. (D) Representative pictures of tumor inflammatory infiltration (100x). Scale bars, 50μM (E) Graphic showing the inflammation score in wild type and ST2KO mice. (F) Representative pictures are shown of H&E-stained skin sections that were obtained from wild type and ST2KO mice at 18 weeks after SCC induction (100x (upper) and 400x (lower) magnification). Scale bars, 50μM (lower), 100μM (upper). (G) H&E stained sections were scored to grade their level of dysplasia. Scale bars, 50μM. The data represent the mean ± SEM from 3 independent experiments. ^**P < 0.01.

Figure 2. ST2 deficiency is associated with reduced immune cell infiltration in tumors

The frequencies of macrophages (F4/80⁺), B (CD19⁺) and T cells (CD4⁺ and CD8⁺), and dendritic cells (CD11c⁺) were determined using immunostaining and FACS analysis in tumor lesions that were harvested from mice on day 126. The bars show (A) the absolute number of leukocytes and the number of (B) CD4⁺ T lymphocytes, (C) CD8⁺ T lymphocytes, (D) B lymphocytes, (E) dendritic cells, (F) and macrophages present in the tumor lesions. (G) Frequencies of CD11b⁺ (myeloid cells), CD119⁺ (M1) and CD124⁺ (M2) cells in the tumor microenvironment. The data are representative of three experiments (n = 5 per group). ^*P < 0.05.

Figure 3. A lack of ST2 is associated with increased NK cytotoxicity

(A) Flow cytometry was used to quantify the proportions of NK cells (CD11b⁺CD27⁺) in tumor samples obtained from wild type and ST2KO mice. (B) Flow cytometry analysis of splenic NK cell activity against YAC-1 cells and (C) KLRG1 and NKp46 expression: WT, grey histogram; and ST2KO, empty histogram. The data shown are representative of the mean ± SEM from at least three independent experiments (n = 5 per group). ^*P < 0.05, ^**P < 0.01; ^***P < 0.001.

Figure 4. A lack of ST2 is associated with a decrease in the expression of pro-inflammatory cytokines

IFN-γ, TNF-α, IL-12p70, IL-4, IL-10, IL-13, TGF-β, IL-17, and IL-33 levels were measured in supernatants recovered during tumor processing using ELISA. The data are shown as the mean ± SEM and are representative of at least three independent experiments (n = 5 per group). ^*P < 0.05.