Fig. 2.

MBZ is toxic to differentiated and stem-cell enriched cultures of triple-negative breast cancers and inhibits their self-renewal capacity. SUM159PT cells were plated on 96-well plates as monolayers (A) or mammosphere cultures enriched for BCICs (B) and treated with increasing doses of MBZ. Cell viability was determined at 24, 48, and 72 hours after MBZ treatment via ATPlite 1step Luminescence Assay, and the relative luminescence units are expressed as percentage of DMSO controls. EC50 values for monolayer and mammospheres were estimated (dashed horizontal lines). (C) SUM159PT monolayer cultures were treated with a single dose of MBZ at the indicated concentrations, and 5 days later the percentage of BCICs was determined based on ALDH1 activity (ALDH1-pos) using the ALDEFLUOR assay. Paired, 2-tailed t test: **P < .001. (D) SUM159PT and MDA-MB-231 cells were plated in serum-free mammosphere media in low-adhesion, 96-well plates, treated with a single dose of MBZ at the indicated concentrations, and allowed to form mammospheres for 2 weeks. The number of mammospheres formed was counted and expressed as a percentage of the number of cells plated. Paired, 2-tailed t test: *P < .01, **P < .001, ***P < .0001. E, ZSG-pos BCICs from SUM159PT-ZsGreen-cODC line were depleted via high-speed FACS and plated on 6-well plates, treated with a single dose of MBZ (0.35 μM), and irradiated 1 hour later. After 5 days, cells were removed and plated in a mammosphere-forming assay in serum-free conditions, and the percentage of dedifferentiated cells able to form mammospheres was determined after 2 weeks. Unpaired, 2-tailed t test: ***P < .0001. Abbreviations: ALDH1 = aldehyde dehydrogenase 1; BCICs = breast cancer-initiating cells; DMSO = dimethyl sulfoxide; FACS = fluorescence activated cell sorting; IR = ionizing radiation; MBZ = mebendazole.