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

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.