Sulindac, a non-steroidal anti-inflammatory drug, mediates breast cancer inhibition as an immune modulator

Abstract

The cooperation of adaptive immunity with pharmacologic therapy influences cancer progression. Though non-steroidal anti-inflammatory drugs (NSAIDs) have a long history of cancer prevention, it is unclear whether adaptive immune system affects the action of those drugs. In present study, we revealed a novel immunological mechanism of sulindac. Our data showed that sulindac had substantial efficacy as a single agent against 4T1 murine breast cancer and prolonged the survival of tumor-bearing mice. However, in the athymic nude mice, sulindac treatment was ineffective. Further in vivo T cell subsets depletion experiments showed that CD8+ T lymphocytes deficiency reversed the anti-tumor effect of sulindac. In addition, sulindac significantly reduced M2 macrophages recruitment, cancer-related inflammation and tumor angiogenesis. Our results advance our understanding of the mechanisms of NSAIDs, and more importantly, this will provide insight into rational drug design or antitumor immunotherapy.

Figure 1. Sulindac inhibited cancer growth and and prolong survival.

(A) 4T1-bearing mice were treated intraperitoneally with 60 mg/kg sulindac for fourteen consecutive days. Tumor volume was tracked and calculated every three days. *P < 0.05. (B) Kaplan-Meyer analysis of mouse survival with a log-rank test. Sulindac leaded to a significant survival advantage. P = 0.001 between two groups.

Figure 2. Adaptive immune system mediated the anti-tumor effect of sulindac.

(A) 4T1 tumor-bearing athymic nude mice were treated intraperitoneally with 60 mg/kg sulindac for fourteen consecutive days. Tumor volume was tracked and calculated every three days. Sulindac was ineffective in immune defective mice. No difference of tumor volume was observed between vehicle and sulindac groups. (B) 4T1-bearing BALB/c mice were treated with sulindac, and depleted of CD4+ and CD8+ T cells with GK1.5 and 2.43 antibodies, respectively. IgG group received isotype antibodies. CD8+ T cells depletion reversed the anti-tumor action of sulindac. *P < 0.05 versus control group.

Figure 3. Sulindac reduced macrophages recruitment in tumor microenvironment.

(A) Seven days after sulindac treatment, CD11b+Gr-1+ MDSCs were evaluated in peripheral blood, spleen and tumors by flow cytometry. (B) Quantification of CD11b + F4/80 + CD206 + M2 macrophages was assessed by flow cytometry. There was a reduced number of M2 macrophages after sulindac treatment. *P < 0.05 versus control group. (C) The levels of S100A8, S100A9, S100A10, CCL-2, CSF-1, CSF-2 and CXCL12 were analyzed by RT-PCR. Macrophage-associated chemokines were reduced by sulindac. **P < 0.01 versus control group.

Figure 4. Sulindac reduced pro-inflammatory and immunosuppressive factors in tumor microenvironment.

(A) The levels of TNFα, iNOS, IL-1β, IL-6 and IL-12 were analyzed by RT-PCR. Sulindac reduced pro-inflammatory factors. *P < 0.05. (B) The levels of TGFβ, arginase-1 and versican were evaluated with RT-PCR. Immunosuppressive factors TGFβ, arginase-1 and versican were decreased by sulindac. *P < 0.05.

Figure 5. Sulindac decreased tumor vasculature in tumor microenvironment.

(A) CD31 staining revealed that sulindac reduced tumor angiogenesis in 4T1 tumor model. Scale bars, 50 μm. (B) Quantification of tumor vessel densities was assessed. *P < 0.05. (C) The level of VEGF and PlGF was evaluated by RT-PCR. Sulindac reduced pro-angiogenic factors VEGF and PlGF. **P < 0.01.