Quantitative FLIM of Lipid Droplet Polarity with a Deep-Red BODIPY Probe Reveals Signatures of Ferroptosis Resistance

The efficacy of ferroptosis-based cancer therapies is often limited by acquired resistance, but the underlying adaptive mechanisms, particularly those involving lipid droplet (LD) remodeling, remain poorly understood. Existing methods for studying ferroptosis do not quantitatively capture the biophysical changes in LDs that support cell survival. Here, we report BP-Ph-LD, a red-emitting BODIPY probe that enables the quantitative mapping of LD polarity via fluorescence lifetime imaging microscopy (FLIM). Using this probe, we monitored the LD dynamics in breast cancer cells undergoing prolonged treatment with the ferroptosis inducer erastin. The resistant state was characterized by a marked decrease in LD polarity, indicating a metabolic shift toward more saturated and less oxidizable lipids. This shift coincided with spatial segregation of LDs: nonpolar LDs clustered near the nucleus, whereas more polar, peroxide-rich LDs localized at the cell periphery. Morphologically distinct, nonspherical LDs also emerged in resistant cells. In early ferroptosis, in contrast, LD polarity and positioning correlated with mitochondrial integrity, linking biophysical adaptation to organelle protection under oxidative stress. Collectively, these findings identify LD polarity as a functional biomarker for ferroptosis resistance and introduce a quantitative optical tool to investigate biophysical adaptation during drug tolerance in cancer.