Hanne Verswyvel

Chapter 3 │ Page 89 Figure 7: Expression of membrane-associated DAMPs after NTP-CDDP treatment. NTP significantly promoted the surface relocation of CRT, HSP70 and HSP90 24 hours post treatment. Bar plots show expression levels normalized to untreated controls. Data presented as mean ± SEM (n = 15, 3 independent repeats). Statistical significance for all pairwise comparisons was determined via linear mixed model analysis with Tukey’s correction for multiple testing. (* p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001). When treatments were combined, NTP was able to further enhance the expression of both HSPs, with the most pronounced changes in the CDDPhigh combination. For ecto-CRT, the combinatorial benefits were more moderated, with the exception of the SCC22B cell line, which exhibited the most pronounced translocation to the surface (Figure 7D; p < 0.001). Moreover, it could be noted that the observed increase in surface DAMP expression in the combination treatments was largely NTP driven, reinforcing its importance in the immunogenicity of cancer cell killing. We progressed in the evaluation of the e ects of NTP on the immunogenicity of CDDP with analyzing the release of ATP and HMGB1 in the supernatant of spheroid cultures (Figure 8). These two key DAMPs signal immunogenic cell death at very distinct time points: i) ATP secretion, a forefront indicator of ICD, was measured at 4 hours and 24 hours post-treatment, while ii) the late-stage marker, HMGB1 release, was assessed at 48 hours and 72 hours. Overall, di erences in ATP secretion were limited, with NTP-including conditions showing a variable increase (up to 4-fold; p ≤ 0.0122) across most conditions (Figure 8A-F). However, treatment e ects were much more pronounced for HMGB1 release, where significant upregulation was observed for nearly all combination treatments across cell lines (Figure 8G-L). Notably, while both monotherapies failed to provoke persistent HMGB1 detection, the combination of both agents stimulated this late-stage DAMP significantly compared to both monotherapies in half of the cases (p ≤ 0.0355), suggesting that HMGB1 is promoted by the combined action of both agents, rather than primarily NTP alone.

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