Imaging and Mechanistic Study of EGFR-Targeted Small Molecule Fluorescent Probes in Head and Neck Squamous Cell Carcinoma

Introduction:: EGFR is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), making it a critical target for diagnosis and therapy. We evaluated AFN-pH, a novel EGFR-targeted theranostic probe (an afatinib-based fluorescent small molecule developed by collaborators and previously characterized), focusing on its tumor imaging capability, apoptosis induction, and preliminary mechanism of action in HNSCC models. Methods:: HNSCC tumor cell lines and normal control cells were used to assess AFN-pH binding and uptake via fluorescence imaging, with a competitive inhibition assay using afatinib pre-treatment to examine EGFR-mediated targeting specificity. Apoptosis induction was evaluated by morphological observation of nuclear changes using DAPI staining (a morphological assay with known limitations), complemented by cell viability assays (CCK-8). Key EGFR downstream signaling molecules (PI3K, AKT, KRAS) were analyzed by qPCR and Western blot to investigate the probe’s mechanism of action. Results:: EGFR expression was confirmed to be elevated in HNSCC cells, and correspondingly, AFNpH showed strong binding and internalization in these tumor cells with minimal uptake in normal cells. Pre-treatment with excess afatinib significantly reduced AFN-pH fluorescence in tumor cells, supporting an EGFR-mediated targeting mechanism. AFN-pH exposure induced apoptosis in HNSCC cells, as evidenced by nuclear condensation and fragmentation (DAPI staining) and reduced cell viability. Mechanistically, AFN-pH treatment downregulated key downstream signaling molecules of EGFR (PI3K, AKT, KRAS), similar to the effects of free afatinib, suggesting that the probe triggers apoptosis via inhibition of EGFR-driven pathways. Conclusion:: AFN-pH selectively accumulates in EGFR-overexpressing HNSCC cells and induces apoptosis through suppression of EGFR signaling pathways. These findings suggest that AFN-pH is a promising theranostic probe for HNSCC, integrating fluorescence imaging for diagnosis with targeted therapeutic effects.