Chapter 3 │ Page 101 of its secretion. These di erences underscore the distinct biological pathways involved in DAMP activation, aligning with the notion that di erent types of ICD inducers may elicit varying DAMP repertoires [18]. Furthermore, these findings may also be influenced by the methodological limitations of assessing secreted markers. Unlike membrane-associated DAMPs, where we could account for spheroid size post-treatment, this information was lost for secreted molecules, potentially a ecting the interpretation of overall marker release vs marker release per cell present. Our cytokine and chemokine profiling at 48 to 72 hours post therapy (Figure 10, Suppl. Figure 2) revealed moderate treatment e ects, further emphasizing the potential challenge of interpreting secreted immune signals in the context of tumor shrinkage and variable expression levels. To obtain a global picture, we opted for hierarchical clustering on the complete data set, and identified two distinct cyto/chemokine clusters. The first cluster was characterized by upregulation, at variable levels, of several cytokines and chemokines following monotherapy CDDP. Although this was not initially anticipated, our data aligns with previous reports showing that CDDP can enhance the secretion of various signaling molecules, including IL-1β, IL-8, IL-10, and CCL4 [68-71]. However, this upregulation may paradoxically contribute to CDDP resistance [69]. In contrast, the second cluster, which showed downregulation in NTP-treated groups (both as monotherapy and in combination with CDDP), suggested that NTP may induce a distinct molecular response that modifies or overrides CDDP-driven signaling induction. Additionally, clear clustering among cell lines could be appreciated, with Cal27 and SCC61 cells grouping together, whereas the SCC22B cell line exhibited a distinct profile. Strikingly, this outcome is consistent with our previous research, where we reported that these SCC22B cells responded di erently to NTP-induced oxidation, particularly in the context of NK-ligand expression [36]. Given that SCC22B originates from a metastatic lymph node[72], while Cal27 and SCC61 are derived
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