Fig. 1. SFX-01 reduces breast CSC activity in primary and metastatic ER+ breast cancer patient derived-samples.

a Mammosphere formation efficiency (MFE) of freshly isolated ER+ early (primary) and metastatic (pleural effusions and ascites) patient-derived samples cultured in the presence of SFX-01 (5 μM) or vehicle control. MFE data for each individual patient sample is represented. MFE was determined on days 7–9 and calculated by dividing the number of mammospheres formed (≥50 μm diameter) by the original number of single cells seeded (500 cells/cm²) and is expressed as the mean percentage of mammosphere formation. b Percentage of ALDH-positive cells in six ER+ metastatic BC patient-derived samples. Cells were grown in low-adherent plates in the presence of SFX-01 (5 μM) or water (control) for 72 h and then subjected to ALDEFLUOR assay. Arrows indicate a reduction greater than 60% compared with control. Representative FACS plots of ALDEFLUOR assay are shown. ALDH-positive cells were discriminated from ALDH-negative cells using the ALDH inhibitor, DEAB. c Percentage of MFE in six ER+ metastatic BC patient-derived samples. Cells were pre-treated in low-adherent plates in the presence of SFX-01 (5 μM) or water (control) for 72 h and then plated for MFE colony assay without drugs. Representative micrographs of mammospheres are shown. d Percentage of MFE of ER+ early (primary) and metastatic patient-derived samples treated with control (water and ethanol), SFX-01 (5 μM), tamoxifen (1 μM) or a combination of SFX-01 with tamoxifen. MFE data are represented as mean percentage ± SEM. *p < 0.05; **p < 0.01.