Fig. 6. Inhibition of APE1-endonuclease activity exerts toxic effects on patient-derived tumor organoids metabolism and is associated with the p53 mutational status.

(A) Fluorescence confocal microscope imaging of patient-derived organoids. Patient-derived organoids were stained with Ki67 (proliferating cells), OLFM4 (intestinal stem cell), E-Cadherin (intestinal epithelial cells) or Lysozyme (Paneth cells). F-actin was stained with Phalloidin. Nuclei were visualized with DAPI. Scale bars, 50 μm (Ki67 and Lysozyme) and 25 μm (OLFM4 and E-Cadherin). (B) Brightfield representative images of colorectal cancer organoids derived from different patients (P12, P14 and P16). Scale bars, 200 μm. (C) TP53 mutational status in colorectal cancer organoids was assessed by NGS. Patient P12, represented by the green box, harboured a wild-type p53, patient P14, represented by the blue box, beared a stop-gain mutation while patient P16, represented by the orange box, harboured a missense one. (D) Western blot analysis of p53 protein level in CRC patient-derived organoids. The graph shows the densitometric analysis of p53 protein expression in patient-derived organoids. Fold change values relative to P12, arbitrary set to 1, are shown. Values are mean of 3 measurements±SD. (E) Western blot analysis of APE1 protein level in CRC patient-derived organoids. The graph shows the densitometric analysis of APE1 protein expression in patient-derived organoids. Fold change values relative to P12, arbitrary set to 1, are shown. Values are mean of 3 measurements±SD. (F) RealTime-Glo assay on patient-derived tumor organoids. Patient-derived tumor organoids were treated with increasing amounts of Compound #3 (range from 0.5 μM to 5 μM) for 48 h. In graph, the percentage of metabolic activity relative to untreated organoids, arbitrary set to 100%, is represented (n=1). Values are mean of 5 measurements±SD.