Macrophages in sensing and executing ferroptosis

铁缺乏症以脂质过氧化和铁依赖为特征。尽管几种肿瘤细胞系对铁凋亡诱导剂非常脆弱，但原代人巨噬细胞对这种形式的受调节的细胞死亡基本上是惰性的。初步数据显示，缺氧以及RSL 3增加了巨噬细胞中铁储存蛋白的表达，例如重铁蛋白（FTH）、铁输出蛋白，即膜铁转运蛋白（FPN）和血浆铜蓝蛋白（CP），其引起铁氧化和输出。我们推测，增加储存或输出铁的能力保护巨噬细胞免于铁凋亡。实验上，我们通过敲低FTH、FPN和/或CP来改变巨噬细胞储存和/或输出铁的能力，以增加其铁凋亡的脆弱性。将在缺氧与常氧下以及经典极化与交替极化巨噬细胞下分析铁储存与进入铁凋亡细胞死亡途径的能力之间的关系，因为已知这些条件会改变细胞铁代谢。在肿瘤微环境中，巨噬细胞可以将铁输送到肿瘤细胞并支持其生长。我们假设这种能力使肿瘤细胞对铁凋亡敏感。我们将在3D肿瘤模型中使用野生型巨噬细胞与具有减弱的向肿瘤细胞递送铁的能力的细胞进行比较来测试我们的假设。关于巨噬细胞在缺氧下增加铁储存并因此获得铁凋亡抗性的能力，这在3D肿瘤模型中自然发生，具有FTH、FPN或CP敲低的巨噬细胞将用于3D培养，目的是通过铁凋亡不仅杀死肿瘤细胞，而且杀死巨噬细胞。巨噬细胞的专业吞噬能力使它们成为移动、识别和摄取垂死细胞的主要细胞，分别对垂死细胞产生的信号作出反应。这在肿瘤微环境中特别重要，其中垂死的肿瘤细胞塑造巨噬细胞的表型。目前，铁促凋亡细胞如何与巨噬细胞相互作用尚不清楚。因此，我们分析了铁蛋白减少细胞如何向巨噬细胞发出信号，并通过使用脂质组学，代谢组学和蛋白质组学研究铁蛋白减少细胞的“发现我”，“吃我”和“不吃我”信号。通过这些方法，我们打算解开氧化还原信号级联的变化，在促铁细胞的分泌组，并最终对巨噬细胞的影响。这将在2D和3D培养中进行，使用具有单核细胞/巨噬细胞浸润的球状体和类器官来模拟临床前相关肿瘤模型。我们的目的是研究巨噬细胞如何通过调节铁代谢来调节其对铁凋亡的敏感性，以及铁凋亡肿瘤细胞如何吞噬巨噬细胞。了解这些基本机制将是至关重要的，提出作为肿瘤治疗的工具铁凋亡。

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.