Quantitative Analysis of Monocyte-Derived Macrophage NFκB Signaling in Cancer Co-culture Models Using Luciferase-Based Biosensing.

IF 5 4区医学 Q3 BIOPHYSICS

Cellular and molecular bioengineering Pub Date : 2025-10-23 eCollection Date: 2025-10-01 DOI:10.1007/s12195-025-00870-1

Colette Li, Megha Anand, Garrett McPheron, Maeve Stiles, Elizabeth Wayne

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引用次数: 0

Abstract

Background and purpose: In this study, we investigate the plasticity of tumor-associated macrophages, which originate from circulating monocytes and are associated with poor cancer prognosis. The differentiation of monocytes into macrophages is a dynamic and spatiotemporal process, as is the resulting macrophages' polarization. However, traditional methods for measuring polarization, such as qPCR and flow cytometry, provide only static information about polarization. To supplement these methods, we present a novel bioluminescent method that allows for time-resolved measurement of NFκB activation in macrophages while in co-culture with cancer cells. By using a monocyte cell line whose NFκB responsive element is labeled with firefly luciferase, we obtain a quantitative and temporal characterization of macrophage polarization in response to tumor-derived signals.

Materials and methods: To quantify the effect of tumor cell signaling THP-1 monocytes encoded with a firefly luciferase NFκB response element were co-cultured with cancer cells. We investigated the impact of the following factors on NFκB signaling cancer cell type (HCT116 or MDA-MB-231), ratio of the number of cancer cells to macrophages in co-culture, and the THP-1 cell differentiation state (monocyte or monocyte-derived macrophage). Bioluminescence was measured over three days. Descriptive features of the bioluminescence response curves were then extracted to compare effects between cancer types.

Results: We observed that the MDA-MB-231 cancer cells induced lower but more persistent NFκB activation in THP-1 monocyte-derived macrophages than was observed in HCT116 co-culture. Higher number of cancer cells (lower macrophage ratio) elicited higher AUC values in HCT116 co-culture compared to low cancer cell conditions. There was no difference between high and low macrophage ratios within the MDA-MB-231 co-culture condition. Moreover, the addition of macrophage differentiation stimuli modulated the NFκB profile in the co-culture. PMA-differentiated macrophages expressed higher and faster peaks of NFκB activation.

Conclusion: Cancer cells can modulate monocyte/macrophage NFκB transcriptional activity, impacting the overall tumor microenvironment. Using NFκB reporter cells, we found that HCT116 colorectal cancer cells induced fast and strong NFκB activation profiles. In contrast, MDA-MB-231 cancer cells elicited lower but more persistent NFκB activation profiles. This study highlights how bioluminescence reporter assays can be used to extract meaningful metrics about monocyte/macrophage behavior during tumor progression. This approach could also be used to understand the crosstalk between cancer cells and monocytes/macrophages that could be useful in a therapeutic of diagnostic context.

Graphical abstract:

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00870-1.

Abstract Image

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基于荧光素酶的生物传感技术定量分析肿瘤共培养模型中单核细胞来源的巨噬细胞NFκB信号。

背景与目的：在本研究中，我们研究肿瘤相关巨噬细胞的可塑性，这些巨噬细胞起源于循环单核细胞，与癌症预后不良有关。单核细胞向巨噬细胞的分化是一个动态的、时空的过程，巨噬细胞的极化也是如此。然而，传统的偏振测量方法，如qPCR和流式细胞术，只能提供关于偏振的静态信息。为了补充这些方法，我们提出了一种新的生物发光方法，可以在与癌细胞共培养时测量巨噬细胞中NFκB活化的时间分辨。通过使用一种单核细胞系，其NFκB反应元件被萤火虫荧光素酶标记，我们获得了巨噬细胞极化对肿瘤来源信号响应的定量和时间表征。材料和方法：将编码萤火虫荧光素酶NFκB应答元件的THP-1单核细胞与癌细胞共培养，量化肿瘤细胞信号传导的影响。我们研究了以下因素对NFκB信号癌细胞类型（HCT116或MDA-MB-231）、共培养中癌细胞与巨噬细胞数量的比例以及THP-1细胞分化状态（单核细胞或单核细胞衍生的巨噬细胞）的影响。三天内测量生物发光。然后提取生物发光反应曲线的描述性特征，以比较不同癌症类型的效果。结果：与HCT116共培养相比，MDA-MB-231癌细胞诱导THP-1单核细胞源性巨噬细胞中NFκB的激活程度较低，但持续时间更长。与低癌细胞条件相比，在HCT116共培养中，较高的癌细胞数量（较低的巨噬细胞比例）引起较高的AUC值。在MDA-MB-231共培养条件下，高、低巨噬细胞比例无差异。此外，巨噬细胞分化刺激的加入调节了共培养中NFκB的谱。pma分化的巨噬细胞表达更高更快的NFκB活化峰。结论：肿瘤细胞可调节单核/巨噬细胞NFκB转录活性，影响肿瘤整体微环境。使用NFκB报告细胞，我们发现HCT116结直肠癌细胞诱导了快速和强烈的NFκB激活谱。相比之下，MDA-MB-231癌细胞引发了较低但更持久的NFκB激活谱。这项研究强调了如何利用生物发光报告法提取有关肿瘤进展过程中单核细胞/巨噬细胞行为的有意义的指标。这种方法也可以用来理解癌细胞和单核细胞/巨噬细胞之间的串扰，这在治疗和诊断方面是有用的。图片摘要：补充资料：在线版本包含补充资料，网址为10.1007/s12195-025-00870-1。

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来源期刊

Cellular and molecular bioengineering 生物-生物物理

CiteScore

5.60

自引率

3.60%

发文量

审稿时长

>12 weeks

期刊介绍： The field of cellular and molecular bioengineering seeks to understand, so that we may ultimately control, the mechanical, chemical, and electrical processes of the cell. A key challenge in improving human health is to understand how cellular behavior arises from molecular-level interactions. CMBE, an official journal of the Biomedical Engineering Society, publishes original research and review papers in the following seven general areas: Molecular: DNA-protein/RNA-protein interactions, protein folding and function, protein-protein and receptor-ligand interactions, lipids, polysaccharides, molecular motors, and the biophysics of macromolecules that function as therapeutics or engineered matrices, for example. Cellular: Studies of how cells sense physicochemical events surrounding and within cells, and how cells transduce these events into biological responses. Specific cell processes of interest include cell growth, differentiation, migration, signal transduction, protein secretion and transport, gene expression and regulation, and cell-matrix interactions. Mechanobiology: The mechanical properties of cells and biomolecules, cellular/molecular force generation and adhesion, the response of cells to their mechanical microenvironment, and mechanotransduction in response to various physical forces such as fluid shear stress. Nanomedicine: The engineering of nanoparticles for advanced drug delivery and molecular imaging applications, with particular focus on the interaction of such particles with living cells. Also, the application of nanostructured materials to control the behavior of cells and biomolecules.