Chapter 2 │ Page 55 consequently live in a permanent state of oxidative stress [40]. It was stated that such cells are the first to reach the threshold of damaging redox imbalance when challenged with exogenous sources of reactive species, thereby rendering them more prone to therapies like NTP [20, 40-42]. Here, we have demonstrated that the more sensitive SC263T cells have elevated intracellular ROS levels after fluorophore incorporation, which becomes more pronounced over time (Figure 5). Hence, inherent elevation of intracellular oxidative stress, caused by the fluorescenceinduced reactive photodamage, could be part of the reason why transduced cancer cells exhibit increased susceptibility to NTP. Of note, a growing body of evidence has indicated that ROS production is intertwined with metabolic changes in cancer cells, and thus, the first studies investigating the link between NTP treatment responsiveness and tumor cell metabolism are emerging [15, 43]. Therefore, it cannot be ruled out that significant metabolic dysregulation, linked to higher intracellular ROS levels, is a contributing factor to the observed changes in NTP vulnerability and this paradigm requires further investigation. Investigation into more specific ROS subtypes, distribution within the cell, and subsequent engagement of defensive antioxidant systems may provide further clarification of the NTP sensitization e ects [44, 45]. The results of the findings presented here highlight several important considerations when designing live-cell imaging experiments to investigate NTP therapy and other redox-based treatments. Since phototoxicity can be accumulated over time, researchers implementing fluorescence live-cell imaging should carefully evaluate when transduced cells diverge from their parental counterparts. This could then be partially avoided by limiting the number of passaging events and reducing the time transduced cells are in culture. Indeed, our cell physiology data complement our findings that high-passaged transduced cells were more sensitive to NTP, as with prolonged passaging, they would incur more photodamage from handling in working environments. In addition, detailed
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