2015;6:14777C95. metabolic Achilles heel will allow us and others to more effectively starve the CSC population. section. Open in a separate window Figure 1 Generating MCF7 DoxyR cellsDoxycycline-resistant (DoxyR) MCF7 cells were generated by serially passaging MCF7 cells, in the presence of increasing step-wise concentrations of Doxycycline (12.5, 25 and 50 M), over a period of 9 weeks. See the section for further details. Unless stated otherwise, MCF7 cells resistant to 25 M Doxycycline were utilized for experiments, such as unbiased proteomics analysis. Doxycycline-treated MCF7 cells were analyzed at each stage for mitochondrial mass. As shown in Figure 2A-2D, Doxycycline-resistant (DoxyR) MCF7 cells show a significant increase in mitochondrial mass (by 1.3- to 1 1.7-fold), as compared to acute treatment with Doxycycline, at the same drug concentration. This overall increase in mitochondrial mass was confirmed by immuno-blot analysis with specific antibodies directed against TOMM20, a well-established marker of mitochondrial mass (Figure ?(Figure2E2E). Open in a separate window Figure 2 MCF7 DoxyR cells exhibit an increase in mitochondrial massA.-D. MCF7 cells were treated with DMSO or Doxycycline for acute (48 h) and chronic stimulation (3 weeks), as specified in < 0.01; (***) < 0.001. D. Representative plots showing increased mitochondrial mass in MCF7 DoxyR cells as compared to MCF7 cells. E. Evaluation of the mitochondrial protein TOMM20 in MCF7 and MCF7 DoxyR cells by western blotting. Side panel shows densitometric analysis of the blots normalized to -actin. Data shown are the mean SEM of 3 independent experiments. Erythrosin B (**) < 0.01. To understand the effects of chronic Doxycycline treatment on cell metabolism, we next performed metabolic flux analysis with the Seahorse XFe96. Interestingly, Figure ?Figure33 illustrates that MCF7-DoxyR cells show a dramatic reduction in oxygen consumption rates (OCR), as compared to matched control MCF7 cells, processed in parallel. As a consequence, ATP levels were severely depleted. Conversely, glycolysis was substantially increased, as measured by the ECAR (extracellular acidification rate) (Figure ?(Figure4).4). Therefore, DoxyR cells are mainly glycolytic. As such, a sub-population of MCF7 cells survive and develop Doxycycline-resistance, by adopting a purely glycolytic phenotype. Open in a separate window Figure 3 Mitochondrial respiration is inhibited in MCF7 DoxyR cellsThe metabolic profile of MCF7 DoxyR cells monolayers chronically treated with increasing concentrations of Doxycycline (12.5 M 50 M), as described in Materials and Methods, was assessed using the Seahorse XF-e96 analyzer. A. Representative tracing of metabolic flux. Dose-dependent significant reduction in basal respiration, proton leak, maximal respiration, ATP levels and spare respiratory capacity were observed Erythrosin B B. Data shown are Erythrosin B the mean SEM of 3 independent experiments performed in sextuplicate. (*) < 0.05; (**) < 0.01; (***) < 0.001. Open in a separate window Figure 4 SRSF2 Glycolysis is increased in MCF7 DoxyR cellsThe metabolic profile of MCF7 DoxyR cells monolayers chronically treated with increasing concentrations of Doxycycline (12.5 M 50 M), as described in Materials and Methods, was assessed using the Seahorse XF-e96 analyzer. A. Representative tracing of metabolic flux. B. Dose-dependent significant increase in glycolysis and decrease in glycolytic reserve as well as glycolytic reserve capacity were observed. Data shown are the mean SEM of 3 independent experiments performed in sextuplicate. (*) < 0.05; (**) < 0.01; (***) < 0.001. Doxycycline-resistant MCF7 cells show an increase in CSC markers, but not in functional CSC activity, as measured using mammosphere assays, proliferation and cell migration ALDH activity and CD44/CD24 levels are routinely used as typical markers to identify breast CSCs [1C7]. Interestingly, MCF7-DoxyR cells show a substantial increase in these two CSC markers, as revealed by FACS analysis (Figure ?(Figure5).5). However, these markers do not reflect CSC activity. To more directly assess functional CSC activity, we used the mammosphere assay. Remarkably, MCF7-DoxyR cells show a > 60% reduction in CSC activity using the mammosphere assay as.