Label‐Free Monitoring of Cancer‐Associated Fibroblast Activation Using NAD(P)H Fluorescence Lifetime Imaging

Cancer‐associated fibroblasts (CAFs) are key regulators of tumor progression, yet their activation state is commonly assessed using static, endpoint assays that do not allow dynamic analysis in living cells. Although CAF activation is accompanied by pronounced metabolic remodeling, label‐free approaches that exploit these changes for real‐time monitoring remain limited. Here, we demonstrate that NAD(P)H fluorescence lifetime imaging microscopy (FLIM) can be used to monitor activation‐associated metabolic remodeling. CAFs activated with transforming growth factor beta (TGF‐β) exhibit a reproducible shift toward longer NAD(P)H fluorescence lifetimes compared to non‐activated cells, consistent with changes in the relative contributions of free and protein‐bound NAD(P)H. By combining live‐cell FLIM with α‐smooth muscle actin staining in the same cells, we directly link metabolic signatures to cellular activation state. We further demonstrate the potential of this approach to dynamically monitor CAF activation in live, migrating cells. Together, these results establish NAD(P)H fluorescence lifetime imaging as a label‐free metabolic approach for monitoring CAF activation dynamics, complementing conventional marker‐based methods and enabling continuous monitoring of tumor–stroma interactions.