Label-free spatially maintained measurements of metabolic phenotypes in cells

Metabolic reprogramming at a cellular level contributes to many diseases including cancer, yet few assays are capable of measuring metabolic pathway usage by individual cells within living samples. Here, autofluorescence lifetime imaging is combined with single-cell segmentation and machine-learning models to predict the metabolic pathway usage of cancer cells. The metabolic activities of MCF7 breast cancer cells and HepG2 liver cancer cells were controlled by growing the cells in culture media with specific substrates and metabolic inhibitors. Fluorescence lifetime images of two endogenous metabolic coenzymes, reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), were acquired by a multi-photon fluorescence lifetime microscope and analyzed at the cellular level. Quantitative changes of NADH and FAD lifetime components were observed for cells using glycolysis, oxidative phosphorylation, and glutaminolysis. Conventional machine learning models trained with the autofluorescence features classified cells as dependent on glycolytic or oxidative metabolism with 90%-92% accuracy. Furthermore, adapting convolutional neural networks to predict cancer cell metabolic perturbations from the autofluorescence lifetime images provided improved performance, 95% accuracy, over traditional models trained via extracted features. Additionally, the model trained with the lifetime features of cancer cells could be transferred to autofluorescence lifetime images of T cells, with a prediction that 80% of activated T cells were glycolytic, and 97% of quiescent T cells were oxidative. In summary, autofluorescence lifetime imaging combined with machine learning models can detect metabolic perturbations between glycolysis and oxidative metabolism of living samples at a cellular level, providing a label-free technology to study cellular metabolism and metabolic heterogeneity.