## 2 Materials and Methods
### Digital Holographic Time-Lapse Imaging in Combination with Fluorescence and Tracking  
For digital holographic microscopy (DHM), the HoloMonitor(r) M4 (Phase Holographic Imaging AB (PHI), Lund, Sweden), with a motorized stage was used for time-lapse imaging. Images were acquired using the software HStudio(tm). To increase image quality, the standard lid of the Petri dish was replaced with HoloLid(tm) 71 110 (PHI) prior to the start of imaging.  
The imaging was done using two different time setups. First, the method for combining digital holographic microscopy with fluorescence was evaluated using 24 h-time-lapses as illustrated in the upper part of Figure 1A. Cells were seeded in a number of Petri dishes, of which some were used in time-lapse imaging 24-48 h after seeding (i.e., time-lapse/treatment time 0-24 h) and some in time-lapse imaging 48-72 h after seeding (i.e., time-lapse/treatment time 24-48 h). Thus, a 48-h time-span was divided into two consecutive 24-h time-lapses where parallel samples were imaged for the different time-spans. For the other set-up, samples were imaged uninterrupted for 48 h, i.e., from 24-72 h after seeding (i.e., time-lapse/treatment time 0-48), as illustrated in the lower part of Figure 1A.  
The time-lapse images were captured within the marked square using a stitching pattern as shown in Figure 1A. Sixteen images were captured for each square to cover the entire area. Images were captured every 15 min. Thus, for each square, 16 time-lapses were captured with images every 15 min. The experiment was repeated three times with two time-lapses analyzed for each dish, i.e., each figure includes six time-lapses per treatment and time interval.  
At the end of the time-lapses, the cells were fixed in 4% formaldehyde and labeled as described below to allow identification based on CD24, CD44, and E-cadherin expression.  
Figure 1: Schematic view of the experimental set-up. (**A**) Before seeding, an area was marked on the outside of the bottom part of Petri dishes. At time-24 h, cells were seeded in multiple Petri dishes. (�⃝). Salinomycin was added to a final concentration of 0.5 \(\mu\)M 24 h after seeding (⃝). The cells inside the marked area were imaged by digital holography for 24-h or 48-h periods (⃝, Time-lapse 3), with the start of the time-lapses immediately after treatment (⃝, Time-lapse 1) or 24 h after treatment (⃝, Time-lapse 2). After the time-lapse, the cells were fixed and labeled with FITC-conjugated anti-CD44 antibodies and PE-conjugated anti-CD24-antibodies or labeled with Alexa flour 488-labeled anti-E-cadherin and PE-conjugated anti-CD24-antibodies and then imaged in a fluorescence microscope. (**B**) Representative image of cells tracked through a time-lapse of digital holographic images. The colored trails show cell movement during the time-lapse. The tracking is the basis for all longitudinal analysis. (**C**) Representative images used for visual over-lay, which allowed for cell identification in the time-lapse. Left image: digital holography. Upper right image: fluorescence, CD44 staining. Lower right image: fluorescence, CD24 staining. The red box corresponds to the area image by digital holography.