[Pirfenidone inhibits bladder cancer xenograft growth in mice by regulating regulatory T cells]

Abstract

Objectives: To investigate the inhibitory effect of pirfenidone (PFD) on growth of bladder cancer xenograft and its regulatory effect on Treg cells in tumor-bearing mice.

Methods: Thirty-two C57BL/6 mice bearing ectopic bladder tumors were randomized into control and PFD groups (n=16). In PFD group, PFD was administered orally at the daily dose of 500 mg/kg, and tumor growth and survival of the mice were monitored. After treatment for 21 days, the tumors and vital organs were harvested for analysis. Immunohistochemistry was used to assess CD3, CD4, CD8, and FOXP3 expressions in the tumors. Flow cytometry and RT-qPCR were used to analyze the percentage of CD4⁺CD25⁺FOXP3⁺ Treg cells and IL-2, IL-10, and IL-35 expressions in the tumors and spleens; organ damage of the mice was examined with HE staining.

Results: Compared with the control group, the PFD-treated mice exhibited significantly lower tumor growth rate with smaller tumor volumes at day 21, along with improved survival at day 28. Immunohistochemistry revealed no significant differences in the infiltration of CD3⁺ and CD8⁺ cells between the two groups, but the percentages of CD4⁺ and FOXP3⁺ cells were significantly lower in the tumors of PFD-treated mice. Flow cytometric analysis confirmed a decrease in CD4⁺CD25⁺FOXP3⁺ Treg cells in the tumors from PFD-treated mice, which also had reduced expression levels of IL-2, IL-10 and IL-35 mRNAs in the tumors. No significant differences were found in Treg cell populations or cytokine expressions in the spleen tissues between the two groups. HE staining showed obvious organ damage in neither of the groups.

Conclusions: PFD inhibits bladder cancer growth and enhances survival of tumor-bearing mice possibly by suppressing Treg cells in the tumor microenvironment.

Keywords: bladder cancer; immune microenvironment; pirfenidone; regulatory T cells.

图1

PFD对小鼠膀胱肿瘤的影响 Fig.1 Effect of PFD on tumor growth in the mouse models bearing bladder cancer xenografts. A: Tumor growth rate in control and PFD groups. B: Tumor volume in the two groups after 21 days of PFD treatment. C: Survival rate of the mice in two groups. *P<0.05, **P<0.01.

图2

PFD对肿瘤组织T细胞数量的影响 Fig.2 Effect of PFD on the quantity of T cells in tumor tissues. A: CD3⁺, CD4⁺, CD8⁺ and Foxp3⁺ T cells in bladder cancer tissues detected by immunohistochemistry (Original magnification: ×100). B-E: Quantitative analysis of CD3⁺ (B), CD4⁺ (C), CD8⁺ (D) and Foxp3⁺ (E) T cells. *P<0.05.

图3

PFD对肿瘤组织Treg细胞相关因子的影响 Fig.3 Effect of PFD on Treg cells-associated factors in the tumor tissues. A, B: Flow cytometry for detecting CD4⁺CD25⁺Foxp3⁺ Treg cells in the tumor tissues of control (A) and PFD (B) groups. C: Quantitative analysis of CD4⁺CD25⁺Foxp3⁺ Treg cells in the two groups. D-F: RT-qPCR for detecting mRNA expressions of IL-2 (D), IL-10 (E) and IL-35 (F) in the tumor tissues. *P<0.05.

图4

PFD对脾脏组织CD4⁺CD25⁺FOXP3⁺ Treg细胞的影响 Fig.4 Effect of PFD on Treg cells in the spleen tissues of the tumor-bearing mice. A, B: Flow cytometry for detecting CD4⁺CD25⁺Foxp3⁺ Treg cells in the spleen tissues of control (A) and PFD (B) groups. C: Quantitative analysis of CD4⁺CD25⁺Foxp3⁺ Treg cells in the two groups. D-F: RT-qPCR for detecting mRNA expressions of IL-2 (D), IL-10 (E) and IL-35 (F) in the spleen tissues.

图5

PFD对小鼠内脏器官的影响 Fig.5 Organ toxicity of PFD in the liver, spleen, lung, kidney and heart of the tumor-bearing mice (HE staining, ×100).