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

Abstract

Assessment of T-cell response to the tumor is important for diagnosis of the disease and monitoring of therapeutic efficacy. For this, new non-destructive label-free methods are required. Fluorescence lifetime imaging (FLIM) of metabolic coenzymes is a promising innovative technology for the assessment of the functional status of cells. The purpose of this work was to test whether FLIM can resolve metabolic alterations that accompany T-cell reactivation to the tumors. The study was carried out on C57Bl/6 FoxP3-EGFP mice bearing B16F0 melanoma. Autofluorescence of the immune cells in fresh lymphatic nodes (LNs) was investigated. It was found that fluorescence lifetime parameters of nicotinamide adenine dinucleotide (phosphate) NAD(P)H are sensitive to tumor development. Effector T-cells in the LNs displayed higher contribution of free NADH, the form associated with glycolysis, in all tumors and the presence of protein-bound NADPH, associated with biosynthetic processes, in the tumors of large size. Flow cytometry showed that the changes in the NADH fraction of the effector T-cells correlated with their activation, while changes in NADPH correlated with cell proliferation. In conclusion, FLIM of NAD(P)H in fresh lymphoid tissue is a powerful tool for assessing the immune response to tumor development.

Imaging immunometabolism in situ in live animals

“…Furthermore, T cells could be distinguished from tumor cells within the same tumor, where T cells had higher FAD and lower NAD(P)H mean lifetimes than tumor cells [124] . Another study tracked metabolic changes in T cells within the lymphatic nodes of melanoma-bearing mice over time as the tumor developed [125] . The authors detected an increase in unbound NADH ( α 1 ) and a decrease in protein-bound NADH ( α 2 ).…”

“…The authors detected an increase in unbound NADH ( α 1 ) and a decrease in protein-bound NADH ( α 2 ). This study applied a triple-exponential decay model to calculate the contribution of NADPH ( α 3 ) that was present only in the large tumor-bearing mice [125] . The changes in the NAD(P)H and FAD lifetime characteristics of tumor-associated T cells detected by these groups indicated a shift toward a glycolytic metabolism, consistent with prior work that identified a glycolytic phenotype in T cells [128] .…”

“…In most cases, TCSPC electronics were used for time-domain lifetime imaging, with one study opting for frequency-domain FLIM [111] and estimated lifetimes with the curve-fitting and phasor plot methods. Several intravital proof-of-principles studies have shown that FLIM of NAD(P)H and FAD can also be used to assess the intracellular metabolism of immune cells in situ in a living animal [121][122][123][124][125] . These studies used 2-photon microscopy combined with TCSPC electronics for lifetime imaging and analyzed lifetime decay using the curve-fitting method.…”

“…This is consistent with recent findings in a mouse model of wound healing, which showed that early-phase proinflammatory macrophages required mitochondrial ROS production [127] . Immune cells can be successfully imaged not only in living zebrafish but also in live mouse models [123][124][125] . Lifetime measurements could discern heterogeneity among normal tissue-resident macrophages and differentiate between healthy and tumor-associated macrophages in living mice with pancreatic tumors induced by Panc02 pancreatic cancer cells [123] .…”

Study on the Key Genes and Molecular Mechanisms of IL-27 Promoting Keratinocytes Proliferation Based on Transcriptome Sequencing

“…30 The mitosis process requires double cellular Compared with mitochondrial oxidative phosphorylation, aerobic glycolysis produces more NADPH and synthetic substrates and rapidly generates more ATP to satisfy the needs of proliferating cells. 31,32 GLUT1 is an important protein initiating cellular Glu uptake. It is reported that GLUT1 expression is upregulated in the psoriatic epidermis and correlated with the degree of acanthosis and the percentage of Ki-67 expression.…”