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

Abstract

Macrophages within the tumor microenvironment (TME) exhibit a spectrum of pro-tumor and anti-tumor 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 non-destructively 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 post-stimulation for mouse macrophages (RAW 264.7) stimulated with IFN-γ or IL-4 plus IL-13 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 PyVMT or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor co-cultures exhibited significantly different FAD mean lifetimes and greater migration than monocultures at 24, 48, and 72 hours post-seeding. In co-cultures with primary human cancer cells, actively-migrating monocyte-derived macrophages had greater redox ratios (NAD(P)H/FAD intensity) compared to passivelymigrating monocytes at 24 and 48 hours post-seeding, reflecting metabolic heterogeneity in this sub-population 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 crosstalk within the 3D TME. SIGNIFICANCE Label-free metabolic imaging and microscale culture technologies enable monitoring of singlecell macrophage metabolism, migration, and function in the 3D tumor microenvironment. Research.

FLIM of NAD(P)H in Lymphatic Nodes Resolves T-Cell Immune Response to the Tumor

“…There are only a few works with optical metabolic imaging of the immune cells so far. For example, in 2020–2022 the group by M. Skala reported on in vitro [ 18 ] and intravital [ 26 , 27 ] studies of macrophages metabolism in normal and cancerous tissue using the intensity-based redox ratio and FLIM of NAD(P)H and FAD. Moreover, in 2021, they demonstrated the ability of autofluorescence imaging to distinguish between quiescent and activated T-cells, as well as between the subsets of CD4+ and CD8+ cells [ 19 ].…”

“…Fluorescence lifetime imaging has been extensively used for metabolic investigations of cancer and other diseases [ 12 , 13 , 14 , 15 , 16 , 17 ]; however, the studies of immune cells using metabolic autofluorescence imaging is a very recent trend, and there are only a few studies so far [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ].…”

Label-free multimodal non-linear optical imaging of three-dimensional cell cultures

“…To study macrophage heterogeneity within the TME, twophoton FLIM imaging was used to assess the redox state in both mono-culture and 3D co-culture [50]. Increased redox ratio was observed in 3D cultures compared to monocultures.…”

Label-free optical imaging of cell function and collagen structure for cell-based therapies

“…The fluorescence intensity is proportional to fluorophore concentration while the fluorescence lifetime is altered due to microenvironmental changes, such as binding state for NADH and FAD [ 14 ]. Fluorescence lifetime is the time that the fluorophore is in the excited state which normally ranges from picoseconds to nano-seconds in duration [ 15 ]. The fluorescence lifetime of NAD(P)H and FAD allows quantification of the enzyme-binding fractions of NAD(P)H and FAD, as well as the lifetimes of the free- and bound-fractions, due to binding-dependent conformational changes of NAD(P)H and FAD that alter the quenching of the fluorophore [ 16 , 17 ].…”