Chapter 3 │ Page 66 Abstract Recurrent and metastatic head and neck squamous cell carcinoma (R/M HNSCC) remains a major clinical challenge, with limited treatment options and poor prognosis. While platinum-based chemotherapy (CDDP) and immune checkpoint inhibitors (ICIs) form the backbone of current therapies, e icacy is limited by low response rates and severe side e ects. A promising approach to enhance therapeutic outcomes is the induction of immunogenic cell death (ICD), which promotes tumor antigen release and immune activation. As non-thermal plasma (NTP) has been reported as an ICD inducer, our goal was to evaluate the immunogenicity of a novel NTP-CDDP combination strategy. This study was performed in advanced 3D in vitro and in vivo tumor models to pursue clinical introduction. All experiments were performed with a microsecond-pulsed dielectric barrier discharge plasma system. First, we assessed NTP-CDDP e ects in HNSCC spheroids and patient-derived organoids to evaluate tumor kinetics and treatment synergy. We then examined ICD induction via DAMP release and cytokine/chemokine secretion, followed by hierarchical clustering to identify overarching response pathways. Finally, we validated the immunogenic potential of NTP-CDDP treatment using the goldstandard vaccination assay in a syngeneic HNSCC mouse model. Our results demonstrate that the NTP-CDDP combination exerts a strong additive or even synergistic e ect, enhancing tumor cell death across multiple HNSCC models. NTP addition augments ICD hallmarks, while cyto/chemokine responses show distinct signatures depending on treatment exposure. Importantly, our in vivo assessment provide compelling evidence for the immunogenicity of NTP-CDDP therapy, with 37.5% of mice achieving protection against tumor rechallenge. Taken together, our study demonstrates that NTP enhances the immunogenic properties of CDDP, providing a rationally designed strategy to improve tumor
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