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

Area of Science:

• Cell Biology
• Cancer Research
• Metabolic Imaging

Background:

• Cancer-associated fibroblasts (CAFs) are crucial for tumor progression.
• Current methods for assessing CAF activation are static and do not allow real-time analysis.
• CAF activation is associated with significant metabolic remodeling, but label-free monitoring tools are limited.

Purpose of the Study:

• To establish NAD(P)H fluorescence lifetime imaging microscopy (FLIM) as a label-free method for real-time monitoring of CAF activation.
• To correlate metabolic changes with CAF activation states.
• To enable dynamic analysis of CAF activation in live cells.

Main Methods:

• Utilized NAD(P)H fluorescence lifetime imaging microscopy (FLIM) to monitor metabolic changes in CAFs.
• Activated CAFs using transforming growth factor beta (TGF-β).
• Combined live-cell FLIM with α-smooth muscle actin staining for direct correlation.

Main Results:

• Activated CAFs showed a reproducible shift toward longer NAD(P)H fluorescence lifetimes compared to non-activated cells.
• This metabolic shift reflects changes in free and protein-bound NAD(P)H.
• Successfully linked metabolic signatures to CAF activation states in live, migrating cells.

Conclusions:

• NAD(P)H FLIM is a viable label-free approach for monitoring CAF activation dynamics.
• This technique complements traditional marker-based assays.
• Enables continuous monitoring of tumor-stroma interactions and CAF behavior in real-time.