Mas-Rosario Javier A, Medor Josue D, Jeffway Mary I, Martínez-Montes José M, Farkas Michelle E
Molecular and Cellular Biology Graduate Program, University of Massachusetts Amherst, Ahmerst, MA, United States.
Department of Biochemistry & Molecular Biology, University of Massachusetts Amherst, Ahmerst, MA, United States.
Front Oncol. 2023 Apr 5;13:1151384. doi: 10.3389/fonc.2023.1151384. eCollection 2023.
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 () promoter activity in RAW264.7 cells (RAW:-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 following cytokine-mediated polarization and confirmed that reporter and parental cells behaved similarly. RAW:-eGFP cells were then used to track macrophage responses in different breast cancer models, and their re-education from anti- to pro-inflammatory phenotypes 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 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:-eGFP cells all resulted in decreased fluorescence, the trends and extents of effects differed. We also observed decreases in -eGFP signal in the macrophages in co-culture assays with 4T1- or EMT6-based spheroids. We then showed that 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 models and extended to applications with intravital imaging.
作为抵御病原体的第一道防线的一部分，巨噬细胞具有分化为功能各异的不同表型的能力。这些细胞改变其特征的过程，通常称为巨噬细胞极化，使它们能够转变为广泛的促炎（M1）或抗炎（M2）亚型，这取决于极化刺激。巨噬细胞表型的失调会导致不同的病理状况或影响某些疾病的性质，如癌症和动脉粥样硬化。因此，需要在相关模型中更好地理解巨噬细胞表型转换，以阐明其在疾病中的潜在作用。然而，现有的用于追踪多细胞环境中巨噬细胞变化的探针很少。在本研究中，我们基于RAW264.7细胞（RAW：-eGFP）中诱导型一氧化氮合酶（iNos）启动子活性生成了一种增强型绿色荧光蛋白（eGFP）报告细胞系。iNos与巨噬细胞激活至促炎状态以及免疫抑制状态的降低相关。我们验证了该报告基因在细胞因子介导的极化过程中的准确性，并确认报告细胞和亲本细胞表现相似。然后，RAW：-eGFP细胞被用于追踪不同乳腺癌模型中的巨噬细胞反应，以及它们通过一种先前报道的嘧啶并（5,4-b）吲哚小分子PBI1从抗炎表型重新编程为促炎表型的过程。使用两种小鼠乳腺癌细胞系4T1和EMT6，通过条件培养基、二维/单层共培养和三维球体模型评估对巨噬细胞的影响。虽然源自4T1或EMT6细胞的条件培养基以及每种癌细胞系与RAW：-eGFP细胞的单层共培养均导致荧光降低，但影响的趋势和程度有所不同。我们还在与基于4T1或EMT6的球体的共培养试验中观察到巨噬细胞中-eGFP信号的降低。然后我们表明，使用PBI1在这些癌症模型中一氧化氮的产生增强，荧光增加。总体而言，这项工作表明这种基于报告基因的方法为研究复杂多组分环境中的巨噬细胞反应提供了一种简便的手段。除了这里介绍的初步研究之外，这个平台可以与各种模型一起使用，并扩展到活体成像应用。
