Fig. 8

Verteporfin in combination with paclitaxel induces apoptosis-mediated cell death in CSCs from cell lines and patient-derived organoids. a Western blot analyses depict the expression levels of YAP/TAZ, stemness markers (SOX2 and ALDH1A1) and apoptosis markers (cleaved-caspase 3 and cleaved-PARP) in VP-treated MDA-MB-231 mammospheres (n = 3). Markers associated with Hippo signaling pathway are depicted in blue, while markers linked to stemness are represented in pink. b Assessment of the percentage viable cells and c relative cell viability using trypan blue exclusion assay and MTT assay, respectively, in mammospheres after treatment with VP (n = 3). d Representative plots of the percentage apoptotic cells in mammospheres following VP treatment as analyzed by flow-cytometry using Annexin V/PI analysis (n = 3). e Representative plots of apoptotic cell percentage in mammospheres following treatment with VP alone and in combination with paclitaxel, assessed by flow cytometry using Annexin V/PI analysis (n = 3). f Representative plots of the percentage of CD44⁺/CD24⁻ populations in patient-derived spheroids following VP treatment as analyzed by flow-cytometry (n = 5). g Change in ROS levels following VP treatment of patient-derived spheroids were quantified using H2DCFDA assay (n = 5). h Cell viability of untreated and 4 µM VP-treated TNBC patient-derived organoids following paclitaxel exposure. Organoids were pre-treated with VP for 48 h, then exposed to graded doses of paclitaxel. Viability was then quantified after 24 h (n = 5). i Hypothetical model delineating possible mechanistic pathway of YAP/TAZ-mediated regulation of brCSCs on the left, where YAP/TAZ might promote persistence of brCSCs. On the right, an alternative mechanism illustrates the putative cascade that might promote the eradication of the brCSCs upon VP treatment, subsequent to the depletion of YAP/TAZ. This schematic was created using BioRender (https://biorender.com). All protein expressions were normalized against β-tubulin, which served as the internal loading control. The data are presented as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significance was assessed using an unpaired Student’s t-test, and the associated two-tailed p-value is indicated in the bar plots. Compared to the untreated control group: *p < 0.05, **p < 0.01 and ***p < 0.001. 231 MDA-MB-231, 468 MDA-MB-468, VP verteporfin, PAX paclitaxel, CL-CASP3 cleaved-caspase 3, CL-PARP cleaved-PARP, β-TUB β-tubulin, brCSC breast cancer stem cells, ARE anti-oxidant response element, AOs anti-oxidants