Ferroptosis Pathway Discrimination and Anticancer Therapeutic Screening Enabled by Cascade-Activated Fluorescence Reporting of Cysteine-Viscosity Dynamics

Elucidating pathway-specific ferroptosis mechanisms is crucial for advancing our understanding of cell death regulation; however, this goal has been hindered by the persistent absence of reliable detection techniques and standardized analytical approaches. Mechanistically, ferroptosis is governed by the nexus between Glutathione Peroxidase 4 (GPX4) inactivation and lipid-peroxide-driven membrane destabilization, with cysteine (Cys) as a key regulator for GPX4 synthesis and ferroptosis trigger differentiation. Lipid peroxide accumulation elevates cytoplasmic viscosity, enabling the decryption of ferroptosis-trigger mechanisms by elucidating Cys-viscosity dynamics. For this, we engineer an innovative dual-parameter sensing platform: a conformationally flexible molecular scaffold with triple-emission viscosity sensitivity was functionalized with a Cys-reactive aldehyde group. This probe system features a fluorescence quenching mechanism and then sequentially restores red and blue fluorescence in the presence of Cys and high viscosity. Notably, while distinct ferroptosis pathways induce an observable viscosity increase, the Cys flux profiles exhibit pathway-specific signatures. Direct synthesis blockade displays a remarkable Cys fluctuation (Δ[Cys] = 20%, 4 h) compared to GPX4 inhibition (Δ[Cys] = 21%, 12 h), whereas overload iron demonstrates imperceptible variation (Δ[Cys] = 28%, 36 h latency). Furthermore, the evaluation of Cys-dependent ferroptosis in pancreatic cancer models uncovers therapy response mechanisms. This work highlights the key role of Cys and viscosity kinetics in ferroptosis initiation and execution phases, advancing the fundamental research and precise regulation of ferroptosis.