A NIR fluorotag reporter CETIF6a enables bright pan-tumor labeling and functional proteomic profiling.

Tumor-seeking fluorescent dyes enable precise lesion localization by recognizing overexpressed receptors, providing a critical adjunctive technology for cancer histopathology. However, tumor heterogeneity and the poor understanding of targeting mechanisms limit their efficacy. Here we engineer CETIF6a, a click chemistry-compatible heptamethine cyanine dye, for multi-cancer targeting, intraoperative histopathology, and proteome-wide target identification. CETIF6a demonstrates margin delineation across multiple cancer types ( > 90% concordance with H&E staining). Quantitative proteomics reveals that the dye targets 5-15 times more tumor-specific proteins than in paracancerous tissues. Synergistic pan-cancer targeting is achieved through 491 conserved tumor-enriched proteins involved in ribosomal, proteasomal, and metabolic pathways, effectively overcoming heterogeneity. Mechanistic studies confirm that CETIF6a emits bright fluorescence upon covalent binding to targets via nucleophilic substitution at cysteine thiol residues within hydrophobic cavities. The modifiable scaffold of CETIF6a supports both intraoperative tumor diagnosis and functional targets profiling, providing a foundation for systematic probe optimization.