Deletion of IL-33R attenuates VEGF expression and enhances necrosis in mammary carcinoma

Abstract

Interleukin-33 (IL-33)/IL-33 receptor (IL-33R, ST2) signaling pathway promotes mammary cancer growth and metastasis by inhibiting anti-tumor immunity. However, the role of IL-33/IL-33R axis in neoangiogenesis and tumor necrosis is not elucidated. Therefore, the aim of this study was to investigate the role of IL-33/IL-33R axis in mammary tumor necrosis. Deletion of IL-33R (ST2) gene in BALB/c mice enhanced tumor necrosis and attenuated tumor growth in 4T1 breast cancer model, which was associated with markedly decreased expression of vascular endothelial growth factor (VEGF) and IL-33 in mammary tumor cells. We next analyzed IL-33, IL-33R and VEGF expression and microvascular density (MVD) in breast tumors from 40 female patients with absent or present tumor necrosis. We found significantly higher expression of IL-33, IL-33R and VEGF in breast cancer tissues with absent tumor necrosis. Both, IL-33 and IL-33R expression correlated with VEGF expression in tumor cells. Further, VEGF expression positively correlated with MVD in perinecrotic zone. Taking together, our data indicate that IL-33/IL-33R pathway is critically involved in mammary tumor growth by facilitating expression of pro-angiogenic VEGF in tumor cells and attenuating tumor necrosis. These data add an unidentified mechanism by which IL-33/IL-33R axis facilitates tumor growth.

Keywords: IL-33; IL-33R; breast neoplasm; necrosis; neoangiogenesis.

Figure 1. Genetic deletion of IL-33R favors tumor necrosis and attenuates tumor growth in 4T1 breast carcinoma in mice

A. Mammary tumor necrosis was analyzed using Image Pro Plus software (v. 6.0.0.206) and presented as a percentage of necrotic area (red pixels) compared to the area of tumor tissue (ROI). B. The presence of IL-33R reduced tumor necrosis C. Tumor diameter D. Tumor volume E. Tumor weight in WT and IL-33R^−/− mice. Statistical significance was tested by Mann-Whitney Rank Sum test.

Figure 2. IL-33 and VEGF expression in mammary carcinoma is lower in IL-33R deficient mice

A. IL-33 expression in mammary tumor cells significantly increased over time in WT mice (*p = 0.019, **p = 0.003). B. H&E staining (original magnifications: x100, scale bar: 200μm) and immunohistochemical expression of IL-33 (Upper panel) and VEGF (Lower panel) (original magnifications: x200, scale bar: 100μm) in tumor tissue in IL-33R^−/− and WT mice, representative tissue sections. C. Percentage of cytoplasmic expression of IL-33 and difference in percentage of cytoplasmic expression of IL-33 in tumor cells between IL-33R^−/− and WT mice. D. Correlation between tumor weight and IL-33 expression in tumor cells in WT and IL-33R^−/− mice. E., F. Expression of VEGF in tumor tissue at 29th and 36th day after inoculation of 4T1 cells in IL-33R^−/− and WT mice. G. Correlation between the extent of tumor necrosis and IL-33 expression in tumor cells in WT mice. H. Correlation between the extent of tumor necrosis and VEGF expression in tumor cells in WT mice. I. Correlation between expression of VEGF and IL-33 in tumors in WT mice. Statistical significance was tested by Mann-Whitney Rank Sum test and by Spearman correlation coefficient.

Figure 3. IL-33, IL-33R and VEGF expression in human breast carcinoma with present or absent tumor necrosis

A. Representative images of human mammary carcinoma with and without necrosis (H&E, original magnification: x100, scale bar: 200μm; x200, scale bar: 100μm). IL-33, IL-33R and VEGF expression was evaluated by immunohistochemistry for each patient (original magnification: x100, scale bar: 200μm; x200, scale bar: 100μm). The difference in the expression of immunohistochemical markers was examined between tumors with and without necrosis. B., C. Correlation between IL-33 and IL-33R with VEGF expression in tumor cells in human mammary carcinoma tissues. Statistical significance was tested by Mann-Whitney Rank Sum test, independent samples t-test or Spearman correlation coefficient.

Figure 4. VEGF expression in tumor cells correlate with the expression of IL-33 and IL-33R and MVD in human breast cancer

A. The difference in the MVD was examined between tumors with present or absent necrosis. B. Representative images of CD105 immunostaining in human breast carcinoma with and without necrosis (original magnification: x100, scale bar: 200μm; x200, scale bar: 100μm). C., D. Correlation between the expression of IL-33 and IL-33R in perinecrotic tumor cells with MVD in human breast carcinoma. E. Correlation between the expression of VEGF in perinecrotic tumor cells with MVD in human breast carcinoma. Statistical significance was tested by Mann-Whitney Rank Sum test or Spearman correlation coefficient.