Antitumor immunity of low-dose cyclophosphamide: changes in T cells and cytokines TGF-beta and IL-10 in mice with colon-cancer liver metastasis

Abstract

Background: The tumor immune microenvironment is one of the most important prognostic factors in liver metastasis from colorectal cancer. Low-dose cyclophosphamide (CTX) is widely believed to be involved in the modulation of the immune system. However, the underlying mechanism of low-dose CTX remains unknown. This study aimed to investigate the antitumor immunity of low-dose CTX in the treatment of colon-cancer liver metastasis.

Methods: Thirty mice were randomly divided into five groups. After liver metastasis was established in colon-cancer models, mice in the treatment groups were injected with low-dose CTX (20 mg/kg) at different time points. Liver and spleen tissues were examined for T-cell markers via flow cytometry. Interleukin (IL)-10 and transforming growth factor (TGF)-β1 expression levels in liver tissues were analysed by immunohistochemistry. Serum interferon (IFN)-γ and IL-10 levels were detected by enzyme-linked immunosorbent assay. An additional 20 mice were randomly allocated into two groups and the survival times were recorded.

Results: The expression levels of CD4⁺ T cells, CD8⁺ T cells, and IFN-γ were down-regulated, whereas those of IL-10 and TGF-β1 were up-regulated in liver metastasis from colon cancer in mice. Furthermore, the local and systemic microenvironments of the liver were altered, which led to reduced antitumor immune responses and subsequently liver metastasis. However, treatment with low-dose CTX reversed these effects. The survival times of mice treated with low-dose CTX were significantly longer than those of the other groups.

Conclusions: Low-dose CTX exerts its antitumor activity by changing the systemic and local immune microenvironments and enhancing immune regulation in mice. CTX could be used as a drug to prevent and treat liver metastasis from colon cancer.

Keywords: colon cancer; cyclophosphamide; immune microenvironment; liver metastasis.

Figure 1.

The number of mice that developed liver metastasis of colon cancer through splenic tumor-cell injection (5 × 10⁴ CT26 cells) in different treatment groups. The liver metastasis of colon cancer was observed only in two of six mice in the CLM-CTX1, CLM-CTX2, and CLM-CTX3 groups. Macroscopic liver metastasis of colon cancer was observed in all mice in the CLM group. No macroscopic and microscopic liver metastases were observed in the normal group. Mice in the CLM-CTX1 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) post surgery immediately. Mice in the CLM-CTX2 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0 and 7 post surgery. Mice in the CLM-CTX3 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0, 7, and 14 post surgery. The mice of the liver metastasis only (CLM) group (n = 6) were not injected with CTX. CLM, colon-cancer liver metastasis; CLM-CTX, colon-cancer liver metastasis-cyclophosphamide; CTX, cyclophosphamide.

Figure 2.

Establishment of macroscopic and microscopic liver metastases through splenic tumor-cell injection (5 × 10⁴ CT26 cells) in different treatment groups. The liver metastasis of colon cancer was observed only in two of six mice in the CLM-CTX1, CLM-CTX2, and CLM-CTX3 groups. Macroscopic liver metastasis of colon cancer was observed in all mice in the CLM group. No macroscopic and microscopic liver metastases were observed in the normal group. Mice in the CLM-CTX1 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) post surgery immediately. Mice in the CLM-CTX2 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0 and 7 post surgery. Mice in the CLM-CTX3 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0, 7, and 14 post surgery. The mice of the liver-metastasis-only (CLM) group (n = 6) were not injected with CTX. CLM, colon-cancer liver metastasis; CLM-CTX, colon-cancer liver metastasis-cyclophosphamide; CTX, cyclophosphamide.

Figure 3.

Flow-cytometry analysis of the CD4⁺CD25⁺FOXP3⁺ Treg-cell population in the spleen and liver. In the spleen, no significant differences were observed among the five groups with respect to the proportion of CD4⁺CD25⁺FOXP3⁺ Treg cells in the total CD4⁺ T-cell population (both P > 0.05) ((A) and (B)). In the liver, the proportion of CD4⁺CD25⁺FOXP3⁺ Treg cells in the total CD4⁺ T-cell population was significantly higher in the CLM group than in other groups (P = 0.032) ((C) and (D)). No significant differences were observed among the other four groups (both P > 0.05). Mice in the CLM-CTX1 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) post surgery immediately. Mice in the CLM-CTX2 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0 and 7 post surgery. Mice in the CLM-CTX3 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0, 7, and 14 post surgery. The mice of the liver-metastasis-only (CLM) group (n = 6) were not injected with CTX. CLM, colon-cancer liver metastasis; CLM-CTX, colon-cancer liver metastasis-cyclophosphamide; CTX, cyclophosphamide.

Figure 4.

Flow-cytometry analysis of the CD8⁺ T-cell population in the spleen and liver. The number of CD8⁺ T cells in the spleen was significantly lower in the CLM group than in the other groups (P < 0.001), with no significant differences among the CLM-CTX1, CLM-CTX2, CLM-CTX3, and control groups (both P > 0.05) ((A) and (B)). In the liver, the number of CD8⁺ T cells significantly decreased in the CLM group compared with that in the CLM-CTX2 (P = 0.020) and control groups (P = 0.017). No significant differences were observed among the CLM, CLM-CTX1, and CLM-CTX3 groups (P = 0.690 and P = 0.327) ((C) and (D)). Mice in the CLM-CTX1 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) post surgery immediately. Mice in the CLM-CTX2 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0 and 7 post surgery. Mice in the CLM-CTX3 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0, 7, and 14 post surgery. The mice of the liver-metastasis-only (CLM) group (n = 6) were not injected with CTX. CLM, colon-cancer liver metastasis; CLM-CTX, colon-cancer liver metastasis-cyclophosphamide; CTX, cyclophosphamide.

Figure 5.

Flow-cytometry analysis of the CD4⁺ T-cell population in the spleen and liver. In the spleen, the number of CD4⁺ T cells significantly decreased in the CLM group (P < 0.001) and there were no significant differences among the CLM-CTX1, CLM-CTX2, CLM-CTX3, and control groups (both P > 0.05) ((A) and (B)). A similar trend was also observed in the liver (P < 0.001) ((C) and (D)). Mice in the CLM-CTX1 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) post surgery immediately. Mice in the CLM-CTX2 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0 and 7 post surgery. Mice in the CLM-CTX3 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0, 7, and 14 post surgery. The mice of the liver-metastasis-only (CLM) group (n = 6) were not injected with CTX. CLM, colon-cancer liver metastasis; CLM-CTX, colon-cancer liver metastasis-cyclophosphamide; CTX, cyclophosphamide.

Figure 6.

Expression of IL-10 and TGF-β1 in the liver metastases of colon cancer. The expression of IL-10 was negative in the liver tissues of normal mice and extremely high in the CLM group compared with that in the three CLM-CTX groups (P < 0.001) ((A) and (B)). A similar trend was observed with respect to the TGF-β1 expression in the liver tissues (P < 0.001) ((C) and (D)). Mice in the CLM-CTX1 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) post surgery immediately. Mice in the CLM-CTX2 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0 and 7 post surgery. Mice in the CLM-CTX3 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0, 7, and 14 post surgery. The mice of the liver-metastasis-only (CLM) group (n = 6) were not injected with CTX. CLM, colon-cancer liver metastasis; CLM-CTX, colon-cancer liver metastasis-cyclophosphamide; CTX, cyclophosphamide; IOD, integrated optical density.

Figure 7.

Expression of IL-10 and IFN-γ in the serum of metastasis-induced mice. IL-10 levels in the CLM group were significantly higher than those in other groups (P = 0.032). No differences in IL-10 levels were observed among the CLM-CTX and control groups (both P > 0.05) (A). The IFN-γ levels in the CLM group were significantly lower than those in the other groups (P = 0.015) (B). Mice in the CLM-CTX1 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) post surgery immediately. Mice in the CLM-CTX2 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0 and 7 post surgery. Mice in the CLM-CTX3 group (n = 6) were intraperitoneally injected with CTX (20 mg/kg) on Days 0, 7, and 14 post surgery. The mice of the liver-metastasis-only (CLM) group (n = 6) were not injected with CTX. CLM, colon-cancer liver metastasis; CLM-CTX, colon-cancer liver metastasis-cyclophosphamide; CTX, cyclophosphamide.

Figure 8.

Survival time of mice with liver metastases of colon cancer. The survival time of mice in the CLM-CTX group (n = 10) was significantly longer than that of mice in the CLM group (n = 10) (P = 0.001). Mice of the CLM-CTX group were injected with low-dose CTX (20 mg/kg) into the abdomen on the day of surgery to establish liver metastasis of colon cancer, while the CLM-group mice were injected with the same amount of saline. CLM, colon-cancer liver metastasis; CLM-CTX, colon-cancer liver metastasis-cyclophosphamide; CTX, cyclophosphamide.