Marsdenia tenacissima enhances immune response of tumor infiltrating T lymphocytes to colorectal cancer

Abstract

Introduction: Tumor-infiltrating T lymphocytes in the tumor microenvironment are critical factors influencing the prognosis and chemotherapy outcomes. As a Chinese herbal medicine, Marsdenia tenacissima extract (MTE) has been widely used to treat cancer in China. Its immunoregulatory effects on tumor-associated macrophages is well known, but whether it regulates tumor-infiltrating T-cell functions remains unclear.

Method: We collected 17 tumor samples from MTE-administered colorectal cancer patients, 13 of which showed upregulation of CD3+/CD8+ tumor-infiltrating T cells. Further in vitro and in vivo experiments were performed to investigate the regulatory effects of MTE on tumor-infiltrating T cells and immune escape of tumors.

Results: Under single and co-culture conditions, MTE inhibited TGF-β1 and PD-L1 expression in the colorectal cancer (CRC) cell lines HCT116 and LoVo. In Jurkat cells, MTE inhibited FOXP3 and IL-10 expression, increased IL-2 expression, but had no effect on PD-1 expression. These findings were confirmed in vitro using subcutaneous and colitis-associated CRC mouse models. MTE also increased the density of CD3+/CD8+ tumor-infiltrating T cells and exhibited considerable tumor-suppressive effects in these two tumor mouse models.

Conclusions: Our findings suggested that MTE inhibits the immune escape of cancer cells, a precipitating factor increasing the immune response of T lymphocytes.

Keywords: CD8; colorectal cancer; marsdenia tenacissima extraction; tumor infiltrating T cells; tumor microenvironment.

Figure 1

Immunohistochemistry findings from tissue samples of CRC patients before and after MTE treatment. (A) Shown are the findings from one patient. Results of other 16 patients are shown in Figure S1 . (B) The bar graph compares the expression of PD-1, PD-L1, TGF-β1(tumor), and FOXP3 before and after MTE treatment in all 17 patients. (C) Serum levels of IL-2, IL-10 and TGF-β1 were compared before and after treatment in all 17 patients. Cytokine concentrations were normalized against the untreated group. *P < 0.05.

Figure 2

Human XL Cytokine Array of 105 proteins in MTE treated Jurkat T cells. (A) The array included multiple cytokines, chemokines, growth factors and other soluble proteins in the culture supernatants. Each protein was spotted in duplicate, and three pairs of positive controls were added in the three corners (top and bottom left and top right) and a pair of negative control in the bottom right corner. (B) Images of the exposed membranes. ‘Control’ represents untreated Jurkat cells. (C) Statistical results of some proteins which relate to function and differentiation of T cells are shown.

Figure 3

Western blotting and ELISA of HCT116, LoVo and Jurkat T cells treated with MTE treated in single culture or co-culture condition. (A) Western blot analysis of PD-L1, TGF-β1 expression in HCT116 or LoVo cells, single cultured or co-cultured with Jurkat T cells and treated with MTE, which were labeled in top row of blots or bottom row of column chart. (B) Western blot analysis of PD-1 and FOXP3 expression in Jurkat T cells single cultured or co-cultured with HCT116 or LoVo cells, and treated with MTE, which were labeled in top row of blots or bottom row of column chart. (C) ELISA analysis of supernatants from single cultures or co-cultures. In single culture, supernatants of HCT116 or LoVo cells was used to test TGF-β1 secretion, and supernatants of Jurkat T cells was used to test IL-2 and IL-10 secretion levels.*P < 0.05, **P < 0.01.

Figure 4

MTE treatment of CAC mice. CAC mice were generated by AOM/DSS treatment. ‘Control’ represents healthy mice that received saline but not AOM, and drank water but not DSS. ‘Model’ represents AOM/DSS treated mice. ‘LD’, ‘MD’ and ‘HD’ represent low (5 ml/kg), medium (10 ml/kg) and high (20 ml/kg) dose of MTE treatment, respectively. Scale bars, 100 μm. Data in the bar graphs are the mean ± S.D. (A) Images showing tumor nodules in the large intestines of CAC mice. The different groups of mice were compared for the (B) number of tumor nodules, (C) survival (survival days on the X-axis represents the time after dosing), and (D) H&E staining of the colons (E) Immunohistochemistry findings from the tumor nodules. The top row indicates the measured proteins. (F) Quantitation of cells with positive immunohistochemical staining. (G) Serum levels of IL-2, IL-10 and TGF-β1 measured by ELISA. Scale bar: 100 μm. The bar graphs present data as mean ± S.D. *P < 0.05, **P < 0.01.

Figure 5

CT26 subcutaneous tumor model. ‘Model’ represents mice with subcutaneous tumors. ‘LD’, ‘MD’ and ‘HD’ represent mice treated with low (5 ml/kg), medium (10 ml/kg) and high (20 ml/kg) dose of MTE, respectively. (A) Shown are the tumor bearing mice before they were sacrificed and the tumors. Mice in different groups were compared for (B) H&E staining of the tumors and (C) tumor weight. (D) Tumors in the model and HD groups were compared for the expression of different proteins by immunohistochemistry. (E) Survival in the different groups. (F) Quantitation of cells with positive immunohistochemical staining. (G) Serum levels of IL-2, IL-10 and TGF-β1 measured by ELISA. Scale bar: 100 μm. The bar graphs present data as mean ± S.D. *P < 0.05, **P < 0.01.

Figure 6

MTE does not induce toxicity in other organs. Mice were treated with low (LD, 5 ml/kg), medium (MD, 10 ml/kg), or high (HD, 20 ml/kg) dose MTE. ‘Control’ represents healthy mice, and ‘subcutaneous’ represents mice with subcutaneous tumors. (A) H&E staining of the lungs, liver and kidney after MTE treatment. (B) Changes of spleen index after different doses of MTE treatment. The mice in the different groups were compared for serum levels of (C) ALT and (D) AST. Scale bars, 100 μm. Data of columns are shown as the mean ± S.D. **P < 0.01.