## 3 Results
### Longitudinal Tracking of Effects of Salinomycin on Motility  
From the longitudinal tracking of cells in time-lapses obtained by DHM, it is also possible to extract data regarding cell movement. Thus, we further analyzed our data regarding motility speed and migration directness in 48-h time lapses. We have previously shown that JIMT-1 cells treated with 0.5 uM salinomycin and analyzed with the same longitudinal tracking method exhibited increased motility, but the migration directness was not affected compared to the control [41]. In the software used for this analysis, i.e., Hstudio, the definition of motility is the accumulated distance over the entire tracking time for each cell. The longer the tracking time is, the higher the motility will be, since there is longer time for the cell to move. Since not all cells could be tracked for the entire time-lapse, we chose to use motility speed. The motility speed was calculated by dividing the motility value of the last frame a cell was tracked in (i.e., the max motility for the cell) with the time of tracking for the cell, as this equalizes for the difference in tracking time. Migration is defined as the shortest distance between the starting point of the tracking and the end point of the tracking for each cell. Migration directness is the ratio between migration and motility and it gives information about how much the cells are jumping around to reach an end point. A ratio of 0 means a cell has not moved at all from the starting point, and a ratio of 1 means a cell has moved straight from its origin to its end point. A ratio for migration directness was calculated for every cell for every capture time. Then we calculated the mean of all these values, i.e., the average migration directness. From the fluorescence images, we have sorted the cells into six groups as presented above. However, the cells included in those groups are only the cells present in the last frame of the DHM time-lapse as they are the ones with known expression of CD24, CD44, and E-cadherin. The expression of CD24, CD44, and E-cadherin in cells that leave the frame or divide before the last frame cannot be mapped. The cells included in the fluorescence data were tracked for different time-spans depending on the different time-intervals between cell divisions, depending on both the individual cell and the salinomycin treatment, as can be seen in Figure S2. Max motility speed is the accumulated distance over the time of treatment (max motility) for each cell divided by the time of tracking (Figure 3A,B). Figure 3A,B shows that there was no difference in the max motility speed of cells with unknown fluorescence expression in the control and salinomycin-treated cultures. Figure 3A,B also shows that the max motility speed varied depending on the expression of CD24 and E-cadherin in both control and salinomycin-treated cultures. However, the variation was larger in salinomycin-treated cultures (Figure 3B) compared to the control (Figure 3A) and the highest max motility speed was found in the former. When comparing the max motility speed between control and salinomycin-treated cells based on their expression of E-cadherin only, salinomycin treatment significantly increased the max motility speed in all three E-cadherin groups (low: \(p<0.0001\), mid: \(p<0.01\), and high: \(p<0.0001\)). Comparing the max motility speed between the control and salinomycin-treated cells depending on their expression of CD24 only, there was no significant difference between CD24\({}^{+}\) cells, however for CD24\({}^{-}\) cells salinomycin-treatment significantly increased the max motility speed compared to the control (\(p<0.0001\)). In short, independent of E-cadherin expression, salinomycin treatment caused CD24\({}^{-}\) expressing cells to move faster. Figure 3: Movement behavior of JIMT-1 cells cultured in the absence or presence of 0.5 uM salinomycin. The cells were imaged using time-lapse digital holographic imaging for 48 h starting immediately after treatment.