Immunosuppression and Metabolic Rewiring in Tumor-infiltrating Neutrophils

项目总结/摘要 转移性乳腺癌（BC）在很大程度上仍然对免疫检查点阻断（ICB）治疗具有抗性。 具有免疫抑制活性的肿瘤浸润性中性粒细胞（TIN）是肿瘤的主要成分， 微环境（TME）来驱动免疫疗法抗性。免疫抑制剂治疗性衰弱 TIN是一种很有前途的方法，当与免疫疗法结合时，可以产生协同效应。然而，在这方面， TIN的治疗靶向在选择性和安全性方面面临挑战，突出表现为缺乏TIN特异性 目标的最近，我们使用单细胞RNA测序来比较BC模型中的TIN和循环中性粒细胞 并鉴定乌头酸脱羧酶1（Acod 1）在TIN中唯一上调。Acod 1催化 产生衣康酸盐，一种在巨噬细胞中具有抗炎活性的代谢物。但是Acod 1在 中性粒细胞的定义不明确。我们在BC小鼠模型中的初步结果表明，TIN依赖于Acod 1， 维持TME中的存活，并且Acod 1缺失导致TIN浸润和转移减少。仍有 关于Acod 1在TIN中的功能、机制和治疗潜力的重大知识缺口。 我们的中心假设是，Acod 1在浸润转移性BC的TIN中上调，这对于肿瘤的发生是至关重要的。 TIN在TME中持续存在并发挥免疫抑制功能，因此Acod 1消融使TIN衰弱， 有利于抗肿瘤免疫，并使转移性BC对免疫治疗敏感。我们建议实现三个 具体目的：（目的1）研究Acod 1在同基因型和自发性TIN中的促转移功能 鼠乳腺肿瘤模型。(Aim 2）识别上游和下游分子机制 在TIN中Acod 1的上调和功能的基础。(Aim 3）改善转移性BC对 通过Acod 1消融进行免疫治疗并验证临床样品中的ACOD 1表达。 为了实现我们的研究目标，我们开发了全身和嗜中性粒细胞特异性Acod 1 基因敲除小鼠作为BC同基因模型的宿主，TIN的小鼠和人体外细胞模型， 用于基因分析和免疫细胞表型分析的细胞技术， 肿瘤和转移到肺和骨的小鼠，和多重免疫荧光染色技术 适用于使用临床样品的验证研究。我们组建了一支强大的研究团队， 这是一种互补的专门知识，进一步确保了拟议的研究是高度可行的。 在项目完成后，我们希望能够揭示以前未知的功能和机制， 在BC转移中富集的免疫抑制性TIN中的Acod 1，提供了代谢和肿瘤之间的新联系。 TIN的重新连接和免疫调节功能，并产生靶向Acod 1的关键临床前证据 来改善免疫疗法从长远来看，我们设想，我们的研究结果的临床翻译 通过开发Acod 1抑制剂， 重新编程免疫代谢和免疫治疗剂以治愈BC和其他癌症。

PROJECT SUMMARY/ABSTRACT Metastatic breast cancer (BC) remains largely resistant to immune checkpoint blockade (ICB) therapy. Tumor-infiltrating neutrophils (TINs) with immunosuppressive activity represent a major component in the tumor microenvironment (TME) to drive immunotherapy resistance. Therapeutic debilitation of immunosuppressive TINs is a promising approach to elicit synergistic efficacy when combined with immunotherapy. However, therapeutic targeting of TINs faces challenges in selectivity and safety, highlighted by the lack of TIN-specific targets. Recently, we used single-cell RNA sequencing to compare TINs and circulating neutrophils in BC models and identified aconitate decarboxylase 1 (Acod1) to be uniquely upregulated in TINs. Acod1 catalyzes the production of itaconate, a metabolite with anti-inflammatory activity in macrophages. But Acod1 function in neutrophils is poorly defined. Our preliminary results in BC mouse models suggest that TINs rely on Acod1 to sustain survival in the TME, and Acod1 loss leads to reduced TIN infiltration and metastasis. There are still significant knowledge gaps about the function, mechanism and therapeutic potential of Acod1 in TINs. Our central hypothesis is that Acod1 upregulated in TINs that infiltrate metastatic BC is essential for the TINs to persist in the TME and exert the immunosuppressive function, thus Acod1 ablation debilitates TINs, favors anti-tumor immunity, and sensitizes metastatic BC to immunotherapy. We propose to accomplish three Specific Aims: (Aim 1) Validate the pro-metastasis function of Acod1 in TINs in syngeneic and spontaneous murine mammary tumor models. (Aim 2) Identify the upstream and downstream molecular mechanisms underlying the upregulation and function of Acod1 in TINs. (Aim 3) Improve metastatic BC response to immunotherapy by Acod1 ablation and validate ACOD1 expression in clinical samples. To achieve our research goals, we have developed both whole-body and neutrophil-specific Acod1 knockout mice as hosts for BC syngeneic models, both mouse and human in vitro cell models of TINs, single- cell technologies for gene profiling and immune cell phenotyping, injection techniques that generate mammary tumors and metastases to lung and bone in the mice, and multiplex immunofluorescence staining techniques suitable for validation studies using clinical samples. We have assembled a strong research team with complementary expertise, which further ensures that the studies proposed are highly feasible to accomplish. Upon completion of the project, we expect to uncover the previous unknown function and mechanism of Acod1 in immunosuppressive TINs that are enriched in BC metastases, provide novel links between metabolic rewiring and immunoregulatory function of TINs, and generate the key preclinical evidence for targeting Acod1 to improve immunotherapy. In the long term, we envision that the bench-to-bedside translation of our findings through development of Acod1 inhibitors may accelerate the therapeutic application of combining agents that reprogram immunometabolism and immunotherapeutics to the curative treatment of BC and other cancers.