An electroneutral fluorescent probe for revealing lipid droplet viscosity heterogeneity in giant cell tumor of bone

Dysregulated lipid droplets (LDs) metabolism is implicated in cancer progression, yet its role in giant cell tumor of bone (GCTB) remains obscure due to the lack of suitable imaging tools for the dense tumor microenvironment. Here, we rationally designed an electroneutral molecular rotor as LDs probe based on twisted intramolecular charge transfer (TICT) mechanism. LD-B1 exhibits 13-fold fluorescence enhancement with increasing viscosity (1–1410 cP), high photostability, and excellent specificity for LDs in live cells. Using this probe, we visualized dynamic LDs fusion and mapped spatial heterogeneity in LDs viscosity within patient-derived GCTB tissues. Notably, tumor cells exhibited significantly higher LDs viscosity than myeloid-derived cells. Single-cell/nucleus RNA sequencing revealed the upregulation of lipid metabolic genes (e.g., NNMT and GPX8) in tumor cells. Furthermore, knockdown of these lipid-associated factors led to a decrease in LDs viscosity, while a lipid tracer assay provided direct evidence of intercellular lipid trafficking from myeloid-derived cells to tumor cells. Together, the findings of this work provide a powerful chemical tool for imaging LDs heterogeneity, and reveal LDs viscosity as a potential metabolic hallmark and therapeutic target in musculoskeletal oncology.