Abstract: Objective: To establish a co-culture system of patient-derived organoids (PDOs) from oral squamous cell carcinoma (OSCC) and peripheral blood natural killer (NK) cells, and to explore the cytotoxic effects of NK cells combined with Cetuximab on OSCC, providing preclinical evidence for immunotherapy combined with targeted therapy. Methods: PDOs were established in vitro from 35 OSCC patients, and NK cells were isolated from peripheral blood of 35 healthy volunteers. Both cell types were identified by morphology, immunohistochemistry (IHC), and flow cytometry. A co-culture model of OSCC PDOs and NK cells was established, with different concentrations of Cetuximab (12.5, 25, 50, 100, 200, and 400 μg/mL). The viability of PDOs was detected using the CCK-8, and apoptosis was analyzed by flow cytometry. Results: Twenty-eight OSCC PDOs were successfully established, showing high consistency with primary tumors in morphology and molecular markers. Compared with the PDO monoculture group, the co-culture group with NK cells significantly inhibited PDO growth, and the cytotoxicity increased with the effector-to-target ratio. Cetuximab combined with NK cells significantly enhanced PDO apoptosis, suggesting a synergistic pro-apoptotic effect. Conclusion: Cetuximab synergistically enhances the cytotoxicity of NK cells against OSCC PDOs, possibly through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. This study provides a theoretical and experimental basis for combined immunotherapy and targeted therapy in advanced OSCC.

Key words: oral squamous cell carcinoma, patient-derived organoid, natural killer cell, Cetuximab, co-culture