5-aza-2'-deoxycytidine potentiates anti-tumor immunity in colorectal peritoneal metastasis by modulating ABC A9-mediated cholesterol accumulation in macrophages.
5-aza-2'-deoxycytidine potentiates anti-tumor immunity in colorectal peritoneal metastasis by modulating ABC A9-mediated cholesterol accumulation in macrophages.
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作者：
Shi R;Zhao K;Wang T;Yuan J;Zhang D;Xiang W;Qian J;Luo N;Zhou Y;Tang B;Li C;Miao H
背景：5-aza-2'-脱氧胞苷（5Aza）是一种DNA甲基转移酶（DNMT）抑制剂，可以激活肿瘤适应性免疫以抑制肿瘤进展。然而，5Aza调节肿瘤免疫微环境的分子机制仍不完全清楚。方法：研究5Aza在结直肠癌（CRC）腹膜癌病（PC）免疫微环境中的作用。通过流式细胞术、实时 PCR、蛋白质印迹分析和药物亲和力响应靶稳定性 (DARTS) 研究了 5Aza 对巨噬细胞活化的影响。 5Aza 对肿瘤免疫的影响在 CRC 患者的基质巨噬细胞和 T 细胞中得到了验证。结果：5Aza 可以刺激巨噬细胞向 M1 样表型激活，随后激活转移前脂肪组织中的 T 细胞，并最终在免疫活性小鼠模型中抑制 CRC-PC。从机制上讲，5Aza 刺激原代小鼠巨噬细胞形成 M1 样表型，其特征是 p65 磷酸化和 IL-6 表达增加。此外，我们筛选并鉴定了 ATP 结合盒转运蛋白 A9 (ABC A9) 作为 5Aza 的结合靶点。 5Aza 以 ABC A9 依赖性方式诱导胆固醇积累、p65 磷酸化和 IL-6 表达。 NF-κB 的药理学抑制或 IL-6 的基因耗竭消除了 5Aza 在小鼠中的抗肿瘤作用。此外，5Aza 的抗肿瘤作用与传统化疗药物 5-Fu 或 OXP 具有协同增强作用。最后，我们验证了 5Aza 在 CRC 患者基质巨噬细胞和 T 细胞的抗肿瘤免疫中的重编程作用。结论：综上所述，我们的研究结果首次表明，5Aza 通过调节转移前微环境中巨噬细胞依赖性 T 细胞活化来抑制 CRC-PC，同时揭示了 5Aza 调节 ABC A9 相关胆固醇代谢和巨噬细胞活化的 DNA 甲基化独立机制。
Background: 5-aza-2'-deoxycytidine (5Aza), a DNA methyltransferase (DNMT) inhibitor, could activate tumor adaptive immunity to inhibit tumor progression. However, the molecular mechanisms by which 5Aza regulates tumor immune microenvironment are still not fully understood. Methods: The role of 5Aza in immune microenvironment of peritoneal carcinomatosis (PC) of colorectal cancer (CRC) was investigated. The effects of 5Aza on macrophage activation were studied by flow cytometry, real-time PCR, Western blotting assays, and Drug Affinity Responsive Target Stability (DARTS). The effects of 5Aza on tumor immunity were validated in stromal macrophages and T cells from CRC patients. Results: 5Aza could stimulate the activation of macrophages toward an M1-like phenotype and subsequent activation of T cells in premetastatic fat tissues, and ultimately suppress CRC-PC in immune-competent mouse models. Mechanistically, 5Aza stimulated primary mouse macrophages toward to a M1-like phenotype characterized by the increase of p65 phosphorylation and IL-6 expression. Furthermore, we screened and identified ATP-binding cassette transporter A9 (ABC A9) as a binding target of 5Aza. 5Aza induced cholesterol accumulation, p65 phosphorylation and IL-6 expression in an ABC A9-dependent manner. Pharmacological inhibition of NF-κB, or genetic depletion of IL-6 abolished the antitumor effect of 5Aza in mice. In addition, the antitumor effect of 5Aza was synergistically potentiated by conventional chemotherapeutic drugs 5-Fu or OXP. Finally, we validated the reprogramming role of 5Aza in antitumor immunity in stromal macrophages and T cells from CRC patients. Conclusions: Taken together, our findings showed for the first time that 5Aza suppressed CRC-PC by regulating macrophage-dependent T cell activation in premetastatic microenvironment, meanwhile uncovered a DNA methylation-independent mechanism of 5Aza in regulating ABC A9-associated cholesterol metabolism and macrophage activation.