Macrophages play an important role in tumor therapeutic response. However, current methods cannot image heterogeneities in macrophage activity on a single-cell level over time within intact, living samples. Macrophage metabolism is closely linked to macrophage function, as M1-like macrophages rely on aerobic glycolysis and M2-like macrophages rely on fatty acid oxidation and oxidative phosphorylation. Thus, we propose to develop optical metabolic imaging (OMI) using two-photon microscopy to monitor cell-level changes in macrophage polarization and functional activity. OMI exploits the auto-fluorescence intensities and lifetimes of the metabolic co-enzymes NAD(P)H and FAD, and has been previously used to dynamically image in vivo tumors with cell-level resolution. The optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD) provides a global measure of redox balance within individual cells. The fluorescence lifetimes of NAD(P)H and FAD provide information in protein-binding activities in metabolic reactions. OMI was used to monitor metabolic changes in macrophages on a single-cell level during polarization and after metabolic perturbations. RAW 264.7 macrophages were stimulated towards an M1- or M2-like phenotype, and imaged with OMI at 24, 48, and 72 hours post-polarization. M1- and M2-like macrophages exhibited significant differences (p<0.05) in redox ratio and FAD fluorescence lifetime values at 48 and 72 hours post-polarization. The M1-like and M2-like character of the macrophages were validated with quantitative polymerase chain reaction (qPCR) analysis. Population density modeling of single-cell metabolism established differences in intra-sample heterogeneity across subsets over time. M1- and M2-like macrophages were also treated with 2-deoxy-glucose (glycolysis inhibitor), etomoxir (fatty acid oxidation inhibitor), and cyanide (oxidative phosphorylation inhibitor. Treatment with etomoxir caused a significant decrease in redox ratio in M2-like macrophages (p<0.05) with no significant effect on M1-like macrophages. In contrast, the redox ratio decreased in M1-like macrophages upon inhibition of glycolysis (p<0.05). Comparable to previously published data, inhibition of oxidative phosphorylation in both groups yielded a decreased NAD(P)H mean lifetimes (p<0.05) and increased FAD mean lifetimes (p<0.05). These results establish OMI as a method to image heterogeneity in macrophage populations within intact, living samples. This method could be used to understand macrophage interactions in the microenvironment of intact in vivo tumors.

Citation Format: Tiffany M. Heaster, David L. Elion, Rebecca S. Cook, Melissa C. Skala. Quantitative imaging of metabolic changes in macrophage subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3702. doi:10.1158/1538-7445.AM2017-3702