Chapter 3 │ Page 81 3. RESULTS 3.1. Combination of NTP and CDDP Enhances Cytotoxicity in HNSCC Cells To determine the sensitivity of di erent HNSCC cell lines, including Cal27, SCC22B and SCC61, to the monotherapies first, tumor spheroids were treated with CDDP or NTP under varying conditions (Suppl. Figure 1). For CDDP, the response was determined through a 7-points titration experiment, whereafter two concentrations were selected: (i) CDDPlow, which caused minimal cell death in the spheroids after 24 hours and about 25% cell death 48 hours post-treatment; and (ii) CDDPhigh, which resulted in 25% cell death at 24 hours and 50% at 48 hours post-treatment. The e ect of CDDP varied across cell lines, with SCC61 cells (Suppl. Figure 1F) exhibiting lower sensitivity compared to the Cal27 and SCC22B cell lines. Therefore, cell line-specific working concentrations were selected (Table 4). In contrast, the sensitivity to NTP was more uniform across the cell lines. A treatment regime resulting in approximately 25% cell death after 24 hours was selected due to NTP’s rapid mechanism of action and to maintain an appropriate therapeutic window to evaluate combinatorial e ects (Table 4). These treatment regimens were utilized consistently throughout the project. Table 4. CDDP and NTP conditions determined per cell line. Cal27 SCC22B SCC61 CDDPlow 1.71 μM 0.925 μM 5.85 μM CDDPhigh 5.85 μM 3.16 μM 20 μM NTP 1000 Hz-10 sec 1000 Hz-10 sec 1000 Hz-10 sec To assess the impact of the combination of NTP with CDDP, spheroid viability assessment via ATP luminescence was performed on the three HNSCC cell lines at 24 and 48 hours post-treatment (Figure 2). The addition of NTP significantly enhanced cytotoxicity in both CDDP conditions compared to their respective monotherapy.
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