Hanne Verswyvel

Chapter 2 │ Page 53 non-transduced cell line (SC263) remained unchanged at low (PL) and high passages (PH), while a significant di erence was observed in the transduced cell line (SC263T). (c) Phase contrast and fluorescence images captured at 10x revealed that the high-passaged, transduced cell line (SC263T-PH) exhibited the highest fluorescence intensity at basal level. (d) Quantification of the mean green fluorescence intensity of the images (GCU) indicate that following transduction, intracellular ROS levels increased and continued to increase with higher passages. Data are represented as mean±SEM of 3 independent repeats (n=1015). Statistical significance was determined using a Two-Way ANOVA and a post hoc Tukey’s multiple comparison test for the proliferation assay; a One-Way ANOVA with a Dunn's multiple comparisons test was used to assess basal intracellular ROS level significance. ** p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001. 4 DISCUSSION AND CONCLUSION In this study, we investigated the cellular e ects of accumulating phototoxicity in living samples after introduction of a stable fluorescent reporter and demonstrated a significant impact on the growth potential and intracellular redox balance. This was accompanied with an increase in sensitivity towards NTP treatment and has broader impacts to other redox-based therapy evaluations using similar analysis techniques. To date, the e ect of fluorescence viral transduction and cellular response to NTP therapy has not yet been investigated. However, we encountered unexpected inconsistencies in our experimental read-outs when using fluorescentlytransduced HNSCC cells, despite maintaining strict culturing conditions. In that regard, we performed a comprehensive study between parental SC263 cells and their transduced counterpart, incorporated with a fluorescent reporter (Figure 1). Based on cancer cell survival quantification (Figure 3b), we observed that the cells most sensitive to NTP were the transduced cells after prolonged passaging (SC263T-PH). This suggested that accumulation of fluorescence-induced phototoxicity is able to modulate cellular susceptibility to NTP and thereby confound experimental results. Further insight into the sources of light that induce fluorescence-associated phototoxicity revealed that both ambient light in the working environment as well as laser excitation in time-lapse experiments, are able

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