## 4 Discussion  
Most of our current knowledge of how cells react to different kinds of perturbations is based on analysis of the response of an entire cell population. Individual cells may be analyzed e.g., by using flow cytometry as an end response assay, which may display heterogeneity of the studied entity. However, a challenge in biology is to understand the kinetics in the processes of each individual cell that causes the heterogeneity in the end response. Microscopic techniques like confocal microscopy, immunofluorescence microscopy, phase contrast microscopy, and DHM are today used to follow the behavior of live individual cells through time-lapse imaging. All methods have their advantages and disadvantages. Here we have used DHM because of the advantage of low photo toxicity and because it is label-free. The disadvantage of label free microscopy is that there are questions around cell identification. Here we address such questions by analyzing different subpopulations of JIMT-1 breast cancer cells identified by combining DHM with fluorescence microscopy. We developed an assay for the classification of individual cells to subpopulations after long time tracking, where the last frame of a time-lapse with digital holographic images was matched with a fluorescence image from the same field of view. We used this approach to investigate the effect of salinomycin on cell division and cell movement in subpopulations distinguished by their CD24-expression in JIMT-1 breast cancer cells. Salinomycin has been shown to inhibit CSCs in many cancer cell types [8; 9; 10; 11; 12; 13; 14; 15; 16; 17] using different assays. Here we show that the CD24\({}^{-}\) subpopulation was affected within 24 h after addition of salinomycin to the cell culture medium. Cell proliferation was specifically inhibited in the CD24\({}^{-}\) subpopulation resulting in a relative increase of the CD24\({}^{+}\) population. Salinomycin treatment does therefore not appear to induce a phenotypic shift per se regarding CD24. We have previously shown that salinomycin accumulates in the ER of cells within seconds after addition to the medium, resulting in a disturbance of the Ca\({}^{2+}\) transport over the ER membrane caused by the K\({}^{+}\) ionophoric activity of salinomycin [37]. K\({}^{+}\) is needed as a counter ion in Ca\({}^{2+}\) transport over the ER membrane [30]. The disturbed Ca\({}^{+}\) transport results in disturbed Wnt signaling. A number of population-based studies besides our own have shown that a blockade of Wnt/\(\beta\)-catenin signaling suppresses CSCs [38]. By combining DHM and fluorescence microscopy that allow longitudinal single cell tracking and identification, we show a selective effect of salinomycin treatment on the CSC population of JIMT-1 cells. Previously, salinomycin treatment was shown to selectively affect mesenchymal cells, allowing cells with more epithelial characteristics to survive [31]. We have previously shown that 72 h of treatment with salinomycin or salinomycin analogues at their respective IC\({}_{50}\) concentrations increases the number of JIMT-1 cells expressing E-cadherin and \(\beta\)-catenin [30]. Here we further analyze the dynamics in the change of E-cadherin expression. By combining fluorescence labeling of CD24 (breast CSCs are CD24\({}^{-}\)[2; 5; 6]) with labeling of E-cadherin (a marker for epithelial characteristics), we could evaluate if the individual cells shifted towards a phenotype with less CSC characteristics and more epithelial characteristics after salinomycin treatment or if the population distribution changed due to natural selection. A higher E-cadherin expression after 48 h of treatment with 0.5 \(\upmu\)M salinomycin was confirmed in the present study, where CD24\({}^{+}\) cells had the largest proportion of cells with high expression. The increase in E-cadherin expression was caused by a selective inhibition of proliferation of cells expressing low levels of E-cadherin.