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Macrophages within the tumor microenvironment (TME) exhibit a spectrum of protumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate two-photon autofluorescence imaging of NAD(P)H and FAD to nondestructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours poststimulation for mouse macrophages (RAW264.7) stimulated with IFNγ or IL4 plus IL13 in 2D culture, confirming that autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse polyoma-middle T virus or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor cocultures exhibited significantly different FAD mean lifetimes and greater migration than monocultures at 24, 48, and 72 hours postseeding. In cocultures with primary human cancer cells, actively migrating monocyte-derived macrophages had greater redox ratios [NAD(P)H/FAD intensity] compared with passively migrating monocytes at 24 and 48 hours postseeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free autofluorescence imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and imaging system provides unique insights into spatiotemporal tumor-immune cross-talk within the 3D TME. SIGNIFICANCE: Label-free metabolic imaging and microscale culture technologies enable monitoring of single-cell macrophage metabolism, migration, and function in the 3D tumor microenvironment.

Autofluorescence Imaging of 3D Tumor–Macrophage Microscale Cultures Resolves Spatial and Temporal Dynamics of Macrophage Metabolism