Monitoring Macrophage Polarization with Gene Expression Reporters and Bioluminescence Phasor Analysis

Macrophages exhibit a spectrum of behaviors upon activation and are generally classified as one of two types: inflammatory (M1) or anti-inflammatory (M2). Tracking these phenotypes in living cells can provide insight into immune function but remains a challenging pursuit. Existing methods are mostly limited to static readouts or are difficult to employ for multiplexed imaging in complex 3D environments while maintaining cellular resolution. We aimed to fill this void using bioluminescent technologies. Here we report genetically engineered luciferase reporters for the long-term monitoring of macrophage polarization via spectral phasor analysis. M1- and M2-specific promoters were used to drive the expression of bioluminescent enzymes in macrophage cell lines. The readouts were multiplexed and discernible in both 2D and 3D formats with single-cell resolution in living samples. Collectively, this work expands the toolbox of methods for monitoring macrophage polarization and provides a blueprint for monitoring other multifaceted networks in heterogeneous environments.