Chapter 3 │ Page 100 spontaneous growth arrest following initial outgrowth. In addition, generating organoids from healthy tissue adjacent to the tumor, which could be a very interesting experimental counterpart or experimental control to the HNSCC PDOs, was not successful despite repeated attempts. These challenges, combined with the long expansion times required, limited our ability to complete certain downstream analyses (e.g. NTP’s selectivity for HNSCC PDOs vs healthy PDOs). As the model becomes more broadly established in our lab, in the future immunological aspects can be explored in PDOs, for instance, by analyzing DAMPs and cytokine/chemokine secretion, or by implementing autologous co-culture experiments with patient-matched PBMCs, as blood samples are being collected alongside the tumor tissue. In that regard, to explore the ICD-exploring potential of the NTP-CDDP combination therapy, we focused on our HNSCC spheroid model and conducted a comprehensive analysis of DAMP release (Figure 6-9). We expanded our investigation beyond the three main hallmarks (CRT, ATP, and HMGB1) [18, 66] and included several HSPs, and progressed with cytokine and chemokine analysis (Figure 10). Given the highly regulated nature of ICD, timing of detection was crucial to capture the dynamic process of DAMP translocation and presentation, and secretion. ATP is actively secreted in a autophagy-dependent manner during the pre-mortem phase[19], similar to CRT and HSP70/90, which are pre-apoptotic markers that translocate to the cancer cell membrane in response to ER stress [21, 24], making them relevant to asses in earlier time points (4 to 24 hours posttreatment). In contrast, HMGB1 is released post-mortem[18, 67], pushing its evaluation at later stages (48 to 72 hours post-treatment). Our results showed that the most pronounced e ects of the combination treatment were observed in membrane-associated DAMPs (CRT and HSP70/90) and in HMGB1 release, while the impact on ATP secretion was comparatively less pronounced. Notably, for ATP, NTP monotherapy appeared to be the primary driver
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