作为对抗病原体的第一道防线的一部分，巨噬细胞具有分化成不同表型以及各种不同功能的能力。这些细胞改变特性的过程，通常被称为巨噬细胞极化。这允许它们变成广泛的促炎（M1）或抗炎（M2）亚型，取决于极化刺激物。巨噬细胞表型的失调可能导致不同的病理变化或影响某些疾病的性质，如癌症和动脉粥样硬化。因此，需要更好地了解相关模型中的巨噬细胞表型转换，以阐明其在疾病中的潜在作用。然而，在多细胞环境中跟踪巨噬细胞变化的探针很少。在本研究中，我们基于RAW264.7细胞中的诱导型一氧化氮合酶（iNos）启动子活性生成了一个eGFP报告基因细胞系（RAW:iNos-eGFP）。iNos与巨噬细胞活化到促炎状态有关，与免疫抑制状态下的细胞有所下降。我们通过细胞因子介导的极化验证了报告基因对iNos的准确度，并确认报告基因细胞与母细胞表现相似。然后，我们使用RAW:iNos-eGFP细胞跟踪不同体外乳腺癌模型中的巨噬细胞响应，并使用一种先前报道的嘧啶并（5,4-b）吲哚小分子PBI1，将它们从抗炎表型重新学习为促炎表型。使用两种小鼠乳腺癌细胞株4T1和EMT6，通过条件培养基、二维/单层共培养以及三维球体模型评估了对巨噬细胞的影响。虽然来源于4T1或EMT6细胞的条件培养基以及每种癌细胞与RAW:iNos-eGFP细胞的单层共同培养都导致荧光下降，但趋势和影响程度不同。我们还观察到，与基于4T1或EMT6的球体共同培养的巨噬细胞中iNos-eGFP信号下降。随后，我们展示了这些癌症模型中使用PBI1增强iNos的生产，跟踪增加的荧光信号。总体而言，这项研究证明了这种基于报告基因的方法提供了一种简便的方法，可以研究复杂、多组分环境中的巨噬细胞响应。除了此处所介绍的初始研究外，该平台可以与各种体外模型一起使用，并扩展到体内应用中进行活体成像。 版权所有 © 2023 Mas-Rosario、Medor、Jeffway、Martínez-Montes和Farkas。

As part of the first line of defense against pathogens, macrophages possess the ability to differentiate into divergent phenotypes with varying functions. The process by which these cells change their characteristics, commonly referred to as macrophage polarization, allows them to change into broadly pro-inflammatory (M1) or anti-inflammatory (M2) subtypes, and depends on the polarizing stimuli. Deregulation of macrophage phenotypes can result in different pathologies or affect the nature of some diseases, such as cancer and atherosclerosis. Therefore, a better understanding of macrophage phenotype conversion in relevant models is needed to elucidate its potential roles in disease. However, there are few existing probes to track macrophage changes in multicellular environments. In this study, we generated an eGFP reporter cell line based on inducible nitric oxide synthase (iNos) promoter activity in RAW264.7 cells (RAW:iNos-eGFP). iNos is associated with macrophage activation to pro-inflammatory states and decreases in immune-suppressing ones. We validated the fidelity of the reporter for iNos following cytokine-mediated polarization and confirmed that reporter and parental cells behaved similarly. RAW:iNos-eGFP cells were then used to track macrophage responses in different in vitro breast cancer models, and their re-education from anti- to pro-inflammatory phenotypes via a previously reported pyrimido(5,4-b)indole small molecule, PBI1. Using two mouse mammary carcinoma cell lines, 4T1 and EMT6, effects on macrophages were assessed via conditioned media, two-dimensional/monolayer co-culture, and three-dimensional spheroid models. While conditioned media derived from 4T1 or EMT6 cells and monolayer co-cultures of each cancer cell line with RAW:iNos-eGFP cells all resulted in decreased fluorescence, the trends and extents of effects differed. We also observed decreases in iNos-eGFP signal in the macrophages in co-culture assays with 4T1- or EMT6-based spheroids. We then showed that iNos production is enhanced in these cancer models using PBI1, tracking increased fluorescence. Collectively, this work demonstrates that this reporter-based approach provides a facile means to study macrophage responses in complex, multicomponent environments. Beyond the initial studies presented here, this platform can be used with a variety of in vitro models and extended to in vivo applications with intravital imaging.Copyright © 2023 Mas-Rosario, Medor, Jeffway, Martínez-Montes and Farkas.