Hanne Verswyvel

Chapter 3 │ Page 98 4. DISCUSSION In this research, we explored the potential of NTP in combination with CDDP as novel, immune-triggering therapeutic strategy in the context of R/M HNSCC. Recognizing the limitations of traditional 2D culture systems, we conducted our experiments solely in advanced 3D tumor models, which better replicate tumor heterogeneity, spatial organization and cellular communication, and therapy e ects [51-53]. Through a systematic approach, we assessed tumor kinetics, synergy between NTP and CDDP, ICD induction, cytokine and chemokine signaling, and ultimately, in vivo immunogenicity. We first determined the sensitivity of di erent HNSCC spheroids to both NTP and CDDP (Suppl. Figure 1) before assessing their combination e ects. While all cell lines exhibit comparable sensitivity to NTP, the SCC61 cell line displays significantly greater resistance to CDDP compared to the other two cell lines. To ensure an appropriate therapeutic window for combination treatment, we opted to adjust the CDDP concentration accordingly for each cell line. With these established conditions, we then evaluated tumor spheroid kinetics (Figure 2) and synergy (Figure 3), and demonstrated that the NTP-CDDP combination exerts pronounced additive to synergistic e ects in tumor reduction over time. The observed benefits of combining NTP with CDDP may be attributed to their distinct mechanisms of action, targeting di erent pathways and vulnerabilities in cancer cells; NTP primarily induces rapid cell death and downstream oxidative (ER) stress via ROS overload, while CDDP exerts its e ects through DNA intercalation and crosslinking, leading to irreparable DNA damage [25, 54-56]. By intervening on di erent pathways and molecular levels, combinations like NTP-CDDP may exert a more robust anti-cancer e ect. Targeting multiple cellular pathways is particularly important in aspects like treatment resistance, a major challenge in chemotherapy-based treatments for HNSCC and cancer management in general

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