Chapter 2 │ Page 49 cell survival (Appx 2.A). Prolonged exposure to ambient light did not render the parental cells more sensitive to NTP treatment (Figure 4a). However, transduced SC263T showed increased sensitivity towards NTP application after ambient light exposure (Figure 4b), and at the high treatment energy of 2.7 J, caused significantly increased cell death (p = 0.0325). Here, these observations are in accordance with the findings for the SC263T-PH (Figure 3d) prolonged cell culturing and passaging exposed cells to more ambient light compared to their earlier counterparts. Another contributing factor of phototoxicity is repetitive exposure by an excitation laser in time-lapse imaging. To investigate whether this could also a ect cellular physiology, we evaluated intracellular ROS levels in cells following repetitive scanning. Over the course of imaging, the intracellular ROS levels of the SC263T cell line became significantly higher compared to their non-transduced counterparts for both low (p = 0.0444) and high passages (p = 0.0007), which persisted for 48 hours (Figure 4c-d). Moreover, intracellular ROS levels increased more with higher passages; starting from 28 hours, the SC263T-PH cell line exhibited significantly higher intracellular ROS levels than the SC263T-PL cell line (p ≤ 0.05), while no di erences were measured between the low and high passaged, parental cell line (p > 0.05). In summary, our results reveal that fluorescence-induced phototoxicity can originate from di erent sources of light, whereby cellular physiology and treatment response can be a ected. This includes both ambient light in the work environment, as well as laser excitation from time-lapse imaging studies. In the following experiments, we aimed to further investigate the underlying mechanisms of increased NTP sensitivity in transduced cancer cells.
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