Fig. 1: Drug repurposing screen identifies Nifuroxazide (NIF) as a pro-drug specifically bioactivated in cancer stem cells.

A Schematic representation of high content screening strategy. Created in BioRender.com. https://BioRender.com/b62d917. B Representative images of the high-content screening capture from 3 technical replicates. ALDEFLUOR cellular staining (ALDHbr) is represented in green. Nuclei are counterstained in blue (Hoechst). Scale bar: 50 μm. C Z-score analysis of data from the screen. Scatter points represent compounds, the y-axis represents the mean of Z-Score of each cancer cell line. Compounds inducing a reduction of the ALDHbr cell proportion have a negative Z-score. D Tumorsphere forming efficiency (SFE) of cell lines under treatment with Nifuroxazide (NIF) compared to untreated condition (CTRL). n = 6 independent experiments. E Western blot of STAT3 and its downstream protein, cyclin D1 in SUM159 ALDHneg- and ALDHbr-sorted population for NIF-treated and untreated conditions (CTRL). The mean intensities are indicated below each band for each condition from 3 independent experiments. F SUM159 cells exposed to various concentrations of NIF were subjected to clonogenic survival assays and representative images. Scale bar: 2.5 cm. n = 3 independent experiments. G SFE of sgEmpty and sgALDH1A1 SUM159-KRAB, S86-KRAB in NIF-treated cells, estimated in a limiting dilution assay. n = 3 independent experiments. H NIF bio-activation with its derived metabolites M2 and HBH. I SFE of SUM159 and S68 under treatment with M2, HBH compared to untreated condition (CTRL). n = 3 independent experiments. J Tumorsphere forming efficiency (SFE) of sgEmpty and sgALDH1A1 SUM159-KRAB, S86-KRAB under M2 treatment and compared to untreated condition (CTRL). n = 3 independent experiments. In (D, G, I, J) box represents mean ± margin of error (95% Confidence Interval). Statistical significance was calculated using one-sided chi-squared test or t-test. In (F), data are shown as mean ± SEM (Standard Error of Mean), according to two-way ANOVA followed by Sidak multiple range test. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.