Intravital Metabolic Autofluorescence Imaging Captures Macrophage Heterogeneity Across Normal and Cancerous Tissue

Abstract

Macrophages are dynamic immune cells that govern both normal tissue function and disease progression. However, standard methods to measure heterogeneity in macrophage function within tissues require tissue excision and fixation, which limits our understanding of diverse macrophage function in vivo. Two-photon microscopy of the endogenous metabolic co-enzymes NAD(P)H and flavin adenine dinucleotide (FAD) (metabolic autofluorescence imaging) enables dynamic imaging of mouse models in vivo. Here, we demonstrate metabolic autofluorescence imaging to assess cell-level macrophage heterogeneity in response to normal and cancerous tissue microenvironments in vivo. NAD(P)H and FAD fluorescence intensities and lifetimes were measured for both tissue-resident macrophages in mouse ear dermis and tumor-associated macrophages in pancreatic flank tumors. Metabolic and spatial organization of macrophages were determined by performing metabolic autofluorescence imaging and single macrophage segmentation in mice engineered for macrophage-specific fluorescent protein expression. Tumor-associated macrophages exhibited decreased optical redox ratio [NAD(P)H divided by FAD intensity] compared to dermal macrophages, indicating that tumor-associated macrophages are more oxidized than dermal macrophages. The mean fluorescence lifetimes of NAD(P)H and FAD were longer in dermal macrophages than in tumor-associated macrophages, which reflects changes in NAD(P)H and FAD protein-binding activities. Dermal macrophages had greater heterogeneity in optical redox ratio, NAD(P)H mean lifetime, and FAD mean lifetime compared to tumor-associated macrophages. Similarly, standard markers of macrophage phenotype (CD206 and CD86) assessed by immunofluorescence revealed greater heterogeneity in dermal macrophages compared to tumor-associated macrophages. Ultimately, metabolic autofluorescence imaging provides a novel tool to assess tissue-specific macrophage behavior and cell-level heterogeneity in vivo in animal models.

In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish

“…To isolate autofluorescence signals associated with macrophages from the whole tissue, we used mCherry and green fluorescent protein (GFP) transgenic reporter lines. GFP is suitable to image in conjunction with NAD(P)H, but it excludes the acquisition of FAD because they have overlapping spectra ( Datta et al, 2020 ; Qian et al, 2021 ), while mCherry is compatible for simultaneous imaging with NAD(P)H and FAD ( Heaster et al, 2021 ; Hoffmann and Ponik, 2020 ). The traditional serial acquisition of NAD(P)H and FAD was not suitable for imaging motile cells, such as macrophages, in live larvae.…”

“…Autofluorescense imaging of the endogenous fluorescence of metabolic coenzymes is an attractive approach because it allows for the quantitative analysis of metabolic changes on a single-cell level, while maintaining cells in their native microenvironment. Studies on the metabolic profiles of macrophages in vivo using autofluorescence imaging have been limited, with one study demonstrating that macrophages have distinguishable lifetime signatures from tumor cells in the tumor microenvironment ( Szulczewski et al, 2016 ), and one study demonstrating changes in optical redox ratio, and mean lifetimes ( ) of NAD(P)H and FAD between dermal and tumor macrophages in vivo in mice ( Heaster et al, 2021 ).…”

Label-free imaging of M1 and M2 macrophage phenotypes in the human dermis in vivo using two-photon excited FLIM

“…Fluorescence lifetime imaging (FLIM) employs NAD(P)H and fluorescence decay parameters of cellular compartments as specific indicators of cell types and phenotypes ( Alfonso-García et al, 2016 ; Heaster et al, 2021 ). Combined with two-photon tomography, two-photon excited fluorescence lifetime imaging (TPE-FLIM) allows for label-free and noninvasive imaging of dermal cells.…”

Label-free imaging of macrophage phenotypes and phagocytic activity in the human dermisin vivousing two-photon excited FLIM

“…Also, histopathological analyses of skin biopsies are not well suited for characterizing MΦ functions such as phagocytosis and for long term monitoring of MΦ distribution in the skin. Fluorescence lifetime imaging (FLIM) employs NAD(P)H and fluorescence decay parameters of cellular compartments as specific indicators of cell types and phenotypes (Alfonso-García et al, 2016; Heaster et al, 2021). Combined with two-photon tomography, two-photon excited fluorescence lifetime imaging (TPE-FLIM) allows for label-free and non-invasive imaging of dermal cells.…”

“…Fluorescence lifetime imaging (FLIM) employs NAD(P)H and fluorescence decay parameters of cellular compartments as specific indicators of cell types and phenotypes (Alfonso-García et al, 2016;Heaster et al, 2021). Combined with two-photon tomography, two-photon excited fluorescence lifetime imaging (TPE-FLIM) allows for label-free and non-invasive imaging of dermal cells.…”