| Summary: | A cell line derived from a tumor is a heterogeneous mixture of phenotypically different cells. Such cancer cell lines are used extensively in the search for new anticancer drugs and for investigating their mechanisms of action. Most studies today are population-based, implying that small subpopulations of cells, reacting differently to the potential drug go undetected. This is a problem specifically related to the most aggressive single cancer cells in a tumor as they appear to be insensitive to the drugs used today. These cells are not detected in population-based studies when developing new anticancer drugs. Thus, to get a deeper understanding of how all individual cancer cells react to chemotherapeutic drugs, longitudinal tracking of individual cells is needed. Here we have used digital holography for long time imaging and longitudinal tracking of individual JIMT-1 breast cancer cells. To gain further knowledge about the tracked cells, we combined digital holography with fluorescence microscopy. We grouped the JIMT-1 cells into different subpopulations based on expression of CD24 and E-cadherin and analyzed cell proliferation and cell migration for 72 h. We investigated how the cancer stem cell (CSC) targeting drug salinomycin affected the different subpopulations. By uniquely combining digital holography with fluorescence microscopy we show that salinomycin specifically targeted the CD24<sup>−</sup> subpopulation, i.e., the CSCs, by inhibiting cell proliferation, which was evident already after 24 h of drug treatment. We further found that after salinomycin treatment, the surviving cells were more epithelial-like due to the selection of the CD24<sup>+</sup> cells.
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