Macrophages in sensing and executing ferroptosis

铁下垂的特征是脂质过氧化和铁依赖。虽然一些肿瘤细胞系对铁下垂诱导剂高度敏感，但原代人类巨噬细胞基本上对这种形式的调节细胞死亡没有反应。实验数据显示，低氧和RSL3增加了巨噬细胞中铁储存蛋白的表达，如重铁蛋白(FTH)、铁输出蛋白(FPN)和铜蓝蛋白(CP)，从而导致铁的氧化和输出。我们假设，储存或输出铁的能力增加，可以保护巨噬细胞免受铁下垂的侵袭。在实验上，我们通过击倒FTH、FPN和/或CP来改变巨噬细胞存储和/或输出铁的能力，以增加它们的铁下垂脆弱性。铁储存和进入铁链细胞死亡途径的能力之间的关系将在低氧和常氧以及经典极化和交替极化的巨噬细胞中进行分析，因为这些条件已知会改变细胞的铁代谢。在肿瘤微环境中，巨噬细胞可能向肿瘤细胞输送铁并支持其生长。我们推测，这种能力使肿瘤细胞对铁性下垂敏感。我们将在3D肿瘤模型中使用野生型巨噬细胞与向肿瘤细胞输送铁的能力减弱的细胞进行比较，以检验我们的假设。根据巨噬细胞增加铁存储从而获得耐缺氧的能力，这在3D肿瘤模型中自然发生，具有FTH、FPN或CP基因敲除的巨噬细胞将用于3D培养，目的不仅是通过铁下垂杀死肿瘤细胞，而且还包括通过铁下垂杀死巨噬细胞。巨噬细胞的专业吞噬能力使它们成为主要细胞，分别移动到、识别和吞噬死亡细胞，以对死亡细胞产生的信号做出反应。这在肿瘤微环境中特别重要，死亡的肿瘤细胞塑造巨噬细胞的表型。目前，铁链细胞如何与巨噬细胞相互作用在很大程度上还不清楚。因此，我们分析了铁链细胞是如何向巨噬细胞传递信号的，并利用脂质组学、代谢组学和蛋白质组学的方法研究了铁链细胞的“Find-Me”、“Eat-Me”和“Do‘t Eat-Me”信号。通过这些方法，我们打算揭开氧化还原信号级联的变化，在铁上腺细胞的分泌体中，最终它们对巨噬细胞的影响。随后将在2D和3D培养中使用具有单核/巨噬细胞浸润的球形和有机类物质来模拟临床前相关的肿瘤模型。我们的目标是研究巨噬细胞如何通过调节铁代谢来调节其对铁下垂的敏感性，以及铁下垂肿瘤细胞如何极化巨噬细胞。了解这些基本机制对于建议将铁性下垂作为一种肿瘤治疗工具至关重要。

Ferroptosis is characterized by lipid peroxidation and its iron dependency. Whereas several tumor cell lines are highly vulnerable to ferroptosis inducers, primary human macrophages are basically inert to this form of regulated cell death. Pilot data show that hypoxia as well as RSL3 increase the expression of iron storage proteins in macrophages such as ferritin heavy (FTH), iron export proteins, i.e. ferroportin (FPN) and ceruloplasmin (CP), which causes iron oxidation and export. We hypothesize that the increased ability to store or export iron protects macrophages from ferroptosis. Experimentally, we alter the ability of macrophages to store and/or export iron by knocking down FTH, FPN, and/or CP to increase their ferroptosis vulnerability. The relationship between iron storage and the ability to enter ferroptotic cell death pathways will be analyzed under hypoxia vs. normoxia as well as for classically vs. alternatively polarized macrophages, because these conditions are known to alter cellular iron metabolism. In the tumor microenvironment, macrophages may deliver iron to tumor cells and support their growth. We hypothesize that this ability sensitizes tumor cells to ferroptosis. We will test our hypothesis in 3D tumor models using wild-type macrophages compared to cells with an attenuated ability to deliver iron to tumor cells. Referring to the ability of macrophages to increase iron storage and thus, to gain ferroptosis resistance under hypoxia, which naturally occurs in 3D tumor models, macrophages with a knockdown of FTH, FPN, or CP will be used in 3D culture with the intention to kill not only tumor cells but also macrophages by ferrroptosis. The professional phagocytotic capacity of macrophages makes them the primary cell to move to, to recognize, and to ingest dying cells, respectively to respond to signals generated by dying cells. This is of particular importance in the tumor microenvironment, where dying tumor cells shape the phenotype of macrophages. At present it is largely unknown how ferroptotic cells interact with macrophages. Thus, we analyze how ferroptotic cells signal towards macrophages and study “find-me”, “eat-me”, and “don’t eat-me” signals of ferroptotic cells by using lipidomics, metabolomics, and proteomics. By these methods, we intend to unravel alterations in redox signaling cascades, in the secretome of ferroptotic cells, and finally their implications on macrophages. This will be followed in 2D and 3D culture using spheroids and organoids with monocytes/macrophage infiltration to mimic a preclinical relevant tumor model. We aim at investigating how macrophages regulate their sensitivity to ferroptosis by adjusting iron metabolism and how ferroptotic tumor cells polarize macrophages. Understanding these basic mechanisms will be critical to propose ferroptosis as a tool for tumor therapy.