Real-Time Monitoring of Glutathione Dynamics In Lung Cancer Cells Following Radiotherapy Using Real Thiol–Based Optical Sensing and Fluorescence Microscopy
Abstract
Purpose
To quantify radiation induced glutathione (GSH) dynamics in A549 lung cancer cells using a ratiometric RealThiol (RT) probe and to assess microscopy and optical sensor platforms for real-time monitoring of redox responses following conventional and FLASH radiotherapy.
Methods
Intracellular GSH levels in A549 cells were measured using the RealThiol acetoxymethyl ester (RT-AM), a ratiometric fluorescent GSH probe (excitation: 405 nm and 488 nm). A calibration curve relating fluorescence ratio to reduced GSH concentration was established. Probe sensitivity to oxidative stress was validated by exposing cells to a range of hydrogen peroxide concentrations and quantifying responses using a plate reader. Radiation-induced GSH oxidation dynamics were assessed following conventional X-ray irradiation (10 Gy) of live cells (2D monolayers and 3D spheroids) by fluorescence microscopy. In parallel, a custom fiber-coupled spectrometer was implemented to monitor ratiometric fluorescence in 3D A549 tumor spheroids, enabling real-time monitoring of GSH redox state. Time dependent fluorescence responses were analyzed across platforms. This framework is being applied in ongoing studies to compare GSH dynamics following conventional and FLASH dose rate irradiation.
Results
RT calibration demonstrated a monotonic relationship between fluorescence ratio and GSH concentration. Hydrogen peroxide treatment induced a dose dependent decrease in GSH, confirming probe responsiveness to oxidative stress. Conventional X-ray irradiation resulted in a time dependent reduction in intracellular GSH. Microscopy-based measurements and optical fiber readouts showed similar trends in GSH depletion. In spheroid cultures, optical sensing revealed delayed and spatially averaged GSH responses relative to monolayer cells.
Conclusion
RT-AM based ratiometric sensing enables quantitative assessment of radiation induced GSH dynamics in tumor models. Establishing the optical fiber platform allows us to monitor GSH during and after irradiation. This study provides a foundation for our ongoing comparative studies of redox responses following conventional and FLASH radiotherapy, supporting mechanistic investigations of dose rate dependent radiobiological effects.