Chapter 2 │ Page 39 outcome and cellular fate. Low, physiological levels of ROS are known to stimulate cell proliferation and di erentiation, while higher levels are reported to induce cell cycle arrest and even cell death [21-23]. Hence, undesired ROS generation leading to phototoxicity can be a major confounding variable in the experimental evaluation of NTP treatment. This is especially problematic when the fluorescent reporter is stably integrated in the biological sample, where damage can be gradually accumulated over time and repeated cell culturing. Consequently, the potential impact of fluorescence-induced phototoxicity must be carefully evaluated in order to attribute the correct cellular e ects of NTP treatment for di erent applications. In this study, we investigated the e ect of a stable nuclear fluorescent signaling probe, commonly used for live-cell tracking, on phototoxicity and cell response to NTP exposure. We hypothesized that accumulating photodamage can significantly interfere with the cellular sensitivity towards NTP treatment through dysregulation of the cancer cells’ redox balance. Using a virally-transduced model of the head and neck squamous cell carcinoma cell line (SC263), proliferation rates, intracellular ROS levels, and NTP treatment responses were investigated between the parental and fluorescently-transduced cells at low and high cell passages, which correspond to the cell culturing cycles and the extent to which the cells have remained in culture. Furthermore, we evaluated the sources of light (ambient light or laser-induced fluorescence excitation), which were able to interfere with cellular homeostasis. Our results indicate that fluorescent integration upon viral transduction renders cancer cells more sensitive to applied NTP treatment, especially in cells that have been extensively passaged. Remarkably, not only was fluorescence imaging able to introduce NTP sensitization in transduced cancer cells, but ambient light also had a detrimental e ect. Investigation into the underlying mechanism revealed that fluorescently-transduced cancer cells exhibited significantly lower proliferation rates and elevated baseline intracellular ROS levels. These e ects become more pronounced the longer the cells remain in
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