Galectin-9 depletion reduces DC 3D migration. (A) Left: schematic representation of the 3D collagen migration assay used. Right: representative single-cell tracking paths of WT or gal9 KD moDCs embedded in a 3D collagen matrix. Lines represent the tracking path covered by each cell from their initial position (time = 0 min). Scale bar = 150 µm. (B) Mature WT or gal9 KD moDCs were embedded within a 3D collagen matrix as per (A). The mean cell velocity of a representative donor is shown. (C–E) Mean ± SEM cell velocity (C), MSD (D), and Euclidean distance (E) of four independent donors. 20 cells were analyzed per donor and transfection. Lines in C connect matched WT and gal9 KD moDCs. Graph in E depicts relative Euclidean distance in gal9 KD moDCs after 60 min of tracking with respect to control cells. (F) Individual trajectory plots of WT and gal9 KD moDCs of one representative donor out of four analyzed. End points of tracks are indicated by red dots. The black line indicates the overall movement in x and y direction (µm). (G) Representative single-cell tracking paths of WT or gal9 KD moDCs embedded in a 3D collagen matrix together with a MEL624 malignant melanoma spheroid. Dots indicate cell position at the specified time point, whereas lines represent the tracking path covered by each cell from their initial position (time = 0 min). Scale bar = 150 µm. (H–J) Cell velocity (H), the MSD (I), and the Euclidean distance migrated after 1 h (J) are depicted. Data represent the mean value ± SEM of three independent donors. The Euclidean distance is depicted as the mean value for galectin-9–depleted moDCs relative to the control group after 60 min of tracking. A one-way t test was performed. In (H), lines connect matched WT and gal9 KD moDCs. (K) Collagen matrices from (G) were fixed and stained for actin, and the number of DCs surrounding (left graph) and infiltrated (right graph) in the spheroid was calculated. Measurements were taken at the same tumor spheroid z-plane across conditions. Images are representative of the tumor spheroid and surrounding DCs. Graphs show the mean ± SEM of four independent donors. Unpaired Student’s t test was conducted to compare WT and gal9 KD cells except for E and J, where a one-way t test was performed. *P < 0.05; **P < 0.01; ***P < 0.0001.
Figure 2.

Galectin-9 depletion reduces DC 3D migration. (A) Left: schematic representation of the 3D collagen migration assay used. Right: representative single-cell tracking paths of WT or gal9 KD moDCs embedded in a 3D collagen matrix. Lines represent the tracking path covered by each cell from their initial position (time = 0 min). Scale bar = 150 µm. (B) Mature WT or gal9 KD moDCs were embedded within a 3D collagen matrix as per (A). The mean cell velocity of a representative donor is shown. (C–E) Mean ± SEM cell velocity (C), MSD (D), and Euclidean distance (E) of four independent donors. 20 cells were analyzed per donor and transfection. Lines in C connect matched WT and gal9 KD moDCs. Graph in E depicts relative Euclidean distance in gal9 KD moDCs after 60 min of tracking with respect to control cells. (F) Individual trajectory plots of WT and gal9 KD moDCs of one representative donor out of four analyzed. End points of tracks are indicated by red dots. The black line indicates the overall movement in x and y direction (µm). (G) Representative single-cell tracking paths of WT or gal9 KD moDCs embedded in a 3D collagen matrix together with a MEL624 malignant melanoma spheroid. Dots indicate cell position at the specified time point, whereas lines represent the tracking path covered by each cell from their initial position (time = 0 min). Scale bar = 150 µm. (H–J) Cell velocity (H), the MSD (I), and the Euclidean distance migrated after 1 h (J) are depicted. Data represent the mean value ± SEM of three independent donors. The Euclidean distance is depicted as the mean value for galectin-9–depleted moDCs relative to the control group after 60 min of tracking. A one-way t test was performed. In (H), lines connect matched WT and gal9 KD moDCs. (K) Collagen matrices from (G) were fixed and stained for actin, and the number of DCs surrounding (left graph) and infiltrated (right graph) in the spheroid was calculated. Measurements were taken at the same tumor spheroid z-plane across conditions. Images are representative of the tumor spheroid and surrounding DCs. Graphs show the mean ± SEM of four independent donors. Unpaired Student’s t test was conducted to compare WT and gal9 KD cells except for E and J, where a one-way t test was performed. *P < 0.05; **P < 0.01; ***P < 0.0001.

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