Figure 3

CXCR2 inhibition reduces dHL-60 cell migration toward PANC-1 tumor spheroids in the “separated” scenario. (a) A representative image showing dHL-60 cells (pseudocolor-coded) in the central channel migrating toward PANC-1 tumor spheroids (brightfield) in the tumor gel channel up to the gel barrier, overlaid with single-cell trajectories over 3 h extracted by TrackMate (ImageJ). Scale bar, 200 µm. The empty gel channel was always loaded with empty gel as a negative control in all conditions and thus not shown for simplicity. (b) An illustration of the calculation of y-FMI, mean velocity, displacement, and directionality of a single-cell trajectory. All trajectories are transformed by setting the start points to (0,0). (c) Representative trajectory plots of migrating dHL-60 cells treated with DMSO vehicle control (i, ii, iv) or 1 μM AZD-5069 (iii) in response to empty gel control (i), tumor spheroids (ii, iii), or 10 nM IL-8 (iv) in the tumor gel channel over 3 h. Numbers in black and in red represent numbers of dHL-60 cells migrating toward (upward) and away from (downward) different conditions, respectively. (d) Bar plot showing the percentage of dHL-60 cells migrating upward toward specified conditions per NTI-chip. N = 9–14 NTI-chips per condition. (e-i) Scatter plots depicting the y-FMI (e), mean velocity (f), maximum velocity (g), displacement (h), and directionality (i) of migrating dHL-60 cells in specified conditions. Each data point represents a single cell and n = 471–944 cells tracked per condition. Bars show mean (red) ± SD (black) with mean values written above the points. At least three independent experiments were performed and results from one representative experiment are shown. ns: ≥ 0.05, **: p < 0.01, ****: p < 0.0001, Brown-Forsythe and Welch ANOVA with Games-Howell's multiple comparisons test.