Fig. 7

Mebendazole treatment reduces the cancer stem cell phenotype of TNBC cells. A Quantification of the total number of mammospheres that were formed by MDA-MB-231 cells after 72 h pre-treatment with the indicated doses of MBZ or DMSO. Mean ± SEM; N = 3 independent experiments with n = 3 technical replicates. P values for one-way ANOVA test *< 0.05, **< 0.01. B Representative images of fluorescently tagged MDA-MB-231 mammospheres quantified in (A). C MDA-MB-231 cells were harvested from tumors that were developed in mice that were treated four times per week with 30 mg/kg MBZ in a sesame oil suspension or sesame oil as vehicle control for 30 days, beginning on day 5 post-injection. The total number of mammospheres formed by these cells after 7 days of culture in mammosphere formation media was quantified. Mean ± SEM; n = 6 technical replicates. P values for two-way t test *< 0.05. D Schematic of the experimental setup used for the in vivo limiting dilution assay depicting the location of each injection site with reference to the mouse mammary fat pad. The number of cells injected per site (cell #) is listed. E Tumor incidence (response) at week 6 following the limited dilution and injection of MDA-MB-231 cells (dose) treated as described in 7A. F Mice bearing two MDA-MB-231 tumors (right^R and left^L) were treated with 30 mg/kg of MBZ in a sesame oil suspension (M) or with sesame oil alone (C). The expression of ITGB4 was analyzed in the lysate derived from each tumor. The same tumors were also processed to be injected into recipient nude mice at limiting dilutions (see G). G Tumor incidence at week 4 following secondary transplants of tumor-dissociated cells from mice described in (F). The right column indicates the estimated breast tumor-initiating/stem cell (TIC) frequencies calculated using ELDA. The overall chi-squared test for differences in TIC frequencies between groups is p < 0.05. H Images of tumors resected from nude mice (N = 6) that were injected at limiting dilutions as described in (F, G)