S100A8诱导PD-L1表达参与肿瘤免疫逃逸的机制

Cancer immunotherapy has become highly successful against an array of distinct hematological.and solid metastatic malignancies. Immune checkpoint inhibitors (ICIs) targeting the PD-1, PD-L1, and CTLA-4 induce sustained clinical responses in a sizable minority of cancer patients. To identify proper biomarkers that correlate with response to ICIs therapy, and the underlying mechanisms of the therapy has becoming an urgent task. .PD-L1 and PD-L2 engage PD-1 on T cells and induce PD-1 signaling, which then causes multiple effects in T cells including exhaustion, alterations in glycolytic and mitochondrial metabolism, and reduced cell cycle activity. Tumor cells (or macrophages, dendritic cells) expressing PD-L1 on their surface use the PD-1 pathway to evade an effective antitumor immune response. The regulation factors of PD-L1 expression has been under intensive investigation, which includes extrinsic factors such as inflammatory cytokines, and intrinsic factors, such as genetic mutation of oncogene (e.g. c-Myc, K-RAS, JAK, etc) and epigenetic mechanisms. .We previously dynamically and globally dissect the alteration of key pathways and molecules in the development from colitis to colorectal cancer in the AOM-DSS murine model. Evidence from our gene expression profiling reveals that pro-inflammatory cytokines S100A8/A9 were strikingly upregulated throughout the entire process of AOM-DSS induced inflammation-cancer link. .We reported that S100A8 was induced by inflammation and then promoted colorectal tumorigenesis downstream by activating Id3. S100A8 and S100A9 acted as chemoattractant proteins by recruiting macrophages, promoting the proliferation and invasion of colon cancer cell, as well as spurring the cycle that culminates in the acceleration of cancer metastasis in a nude mouse model. S100A8 regulated colon cancer cell cycle and proliferation by inducing Id3 expression while inhibiting p21. .Administration of a neutralizing anti-S100a9 antibody significantly ameliorated AOM-DSS -induced colitis-related carcinogenesis. Gene expression profiling showed that key pathways known to be involved in colitis-related carcinogenesis process, such as Wnt signaling pathway, PI3K–Akt signaling pathway, cytokine–cytokine receptor interaction, and ECM–receptor interaction pathway, were suppressed after treatment with anti-S100a9 antibody in the AOM-DSS mouse model. .We recently discovered that S100A8 and S100A9 proteins can promote the expression of PD-L1 of murine macrophage cells. The underlying mechanism is related to TLR4-NF-kB pathway, as well as other possibilities. The upregulation of PD-L1 in macrophages can inhibit T cell proliferation, CD8+T cell killing function and inhibit T cell IL4, IL17a, IFN-g secretion. Treatment cells with anti-PD-L1 neutralizing antibody to block PD1-PD-L1 pathways can reverse the inhibition of T cell functions. We also found that S100A8 treated macrophages promoted tumor immune evasion in vivo. .Based on these findings, we suggest a hypothesis in this proposal that the inflammatory mediator S100A8 is an important extrinsic inducer of PD-L1 for macrophages. S100A8 could be a useful biomarker for predicting the outcome of anti-PD-L1 therapy in clinic. Targeting of S100A8 will be a promising strategy to assist immune checkpoint inhibitors. .In this study, we will dissect the molecular mechanisms underlying how S100A8 regulating PD-L1 expression in macrophages. By inducing PD-L1, how S100A8 inhibits anti-tumor responses initiated by macrophages (as antigen-present cells) and T cells (as effect cells) both in vitro and in vivo. We will promote our study in clinic specimens to evaluate the utility for S100A8 levels as biomarker for predicting the outcome of anti-PD-L1 therapy in cancer patients. PDX (Patient-derived xenograft) model will be employed to assess whether targeting S100A8 is helpful for immune checkpoint blockade.

肿瘤检查点抑制剂(anti-PD-1、PD-L1、CTLA4等)成为肿瘤治疗的新希望，寻找能预测其疗效的标志物，及其作用的分子机制成为当务之急。调控PD-L1表达的机制包括细胞外的因素，如炎症因子；和细胞内源性因素，如遗传变异和表观遗传变异。我们前期发现炎症介质钙卫蛋白S100A8、A9在小鼠溃疡性结肠炎-癌变转化全过程中持续高表达，招募巨噬细胞，促进结肠癌细胞增殖和侵袭转移。自制的anti-S100A9的中和抗体能有效治疗该小鼠疾病模型。我们新近发现，S100A8、A9蛋白能够显著升高巨噬细胞的PD-L1表达水平，抑制T细胞增殖与功能，促进肿瘤的免疫逃逸。本项目中，我们将全面分析S100A8诱导巨噬细胞表达PD-L1，及其促进肿瘤免疫逃逸的机制，并利用临床标本和PDX模型探讨它作为预测标志物和治疗靶点的可能性。

我们发现炎症介质S100A8/A9蛋白能够显著升高巨噬细胞PD-L1的表达水平。S100A8对于巨噬细胞PD-L1的诱导，是通过TLR4通路，而非RAGE通路。在其涉及的信号通路中，TLR4、NF-κB、STAT3、ERK、PI3K的抑制剂可以逆转这一现象。在更深层次的机制研究中，我们发现S100A8蛋白能够改变PD-L1启动子区域的组蛋白乙酰化和甲基化修饰丰度，最终导致PD-L1转录大量增加。在功能上，S100A8蛋白促进巨噬细胞表达PD-L1后，可抑制T细胞增殖与CD8+ T细胞的杀伤功能，使用anti-PD-L1抗体可逆转S100A8对T细胞功能的抑制。NOD/SCID小鼠皮下成瘤和巨噬细胞与肿瘤相关的T细胞过继转移实验证实，经S100A8处理后的巨噬细胞，其抑制皮下移植瘤生长的作用被逆转。这个发现揭示了炎症介质S100A8/A9与免疫检查点分子PD-L1的关系，可通过干预肿瘤微环境中的S100A8/A9，增强T细胞肿瘤免疫。. 此外，我们积极寻找靶向S100A8/A9的方法。在先前的研究中我们合成了anti-S100A9抗体，并在小鼠结直肠炎-癌模型中验证出其具有良好的S100A9靶向效果。在本项研究中，我们开发了S100A9特异性靶向的仿生纳米给药系统。通过包裹过表达TLR4的巨噬细胞的膜并利用其炎症归巢效应来特异性地增加纳米颗粒在小鼠肠道炎症部位的聚集。这种新型给药系统显著减轻了小鼠结肠炎的症状，并降低了炎症信号通路关键转录因子和炎症因子的表达。

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