Chapter 6 │ Page 204 complex, incorporating 3D co-cultures, patient-derived organoids, and in vivo tumor follow-up, fluorescence-based and live-imaging techniques remain irreplaceable. However, to ensure the reliability of therapeutic evaluation, future work should make e orts to minimize fluorescence-induced cellular alterations, optimize imaging conditions, and explore alternative labeling methods. For subsequent studies and to prevent experimental inconsistencies we previously encountered in transduced HNSCC spheroids, we addressed these concerns by minimizing ambient light exposure for cells with fluorescent reporters, and optimizing imaging protocols whereby the frequency of live-cell imaging scans is reduced to the minimal. For experiments where endpoint measurements were su icient and continuous monitoring was not critical, we opted to exclude fluorescent reporters altogether and instead employed endpoint luminescence assays. Another emerging facet is that the integration of artificial intelligence and machine learning in image analysis o ers attractive advantages, as the ability to extract biological information from brightfield images could, in some cases, eliminate the need for labeling altogether [8-10]. With the first reports emerging bringing attention to these issues, including the first research chapter of this study, we are on the cutting edge of raising this awareness[11, 12], and more broadly, these findings emphasize the need for more acknowledgment of potential confounding factors in experimental design and data interpretation. By critically assessing methodological choices, we can better bridge the gap between laboratory research and clinical translation, ensuring that preclinical findings accurately reflect therapeutic potential. When the initial anti-tumoral e ects of NTP were established, research e orts expanded to new topics and evolved towards the immunogenic capacities of the therapy, an area in which our laboratory is one of the forerunners [13-16]. Previous studies from our hand and others demonstrated that NTP is a bona fide inducer of immunogenic cell death (ICD), reporting release of damage-associated molecular
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