Figure 5.

Disruption of microtubules ameliorates impaired CTL killing in dense collagen matrices. (A–E) Inhibition of microtubule polymerization improves CTL killing in dense collagen. Nocodazole (Noco, 10 µM) was used to disrupt the microtubule network. Images were acquired using ImageXpress (20× objective) at 0 and 12 hours. NALM-6-pCasper and bead-stimulated CTLs (A–C), or SK-Mel-5 pCasper and MART1-specific CTLs (D, E) were used as target and killer cells. Representative experiments from 5 mg/ml are shown in (A, D). (F–J) Nocodazole treatment elevates migration and searching efficiency in dense collagen. CTLs and calcein-loaded target cells (green) were embedded in planer collagen matrix (5 mg/ml) and visualized with cell observer. Migration velocity within the duration of 30 min before conjugating with target cells is shown in (F). Representative migration trajectories are shown in (G). The likelihood for CTLs to find target cells and the time required for CTL to find their first target cell are shown in (H, I). Number of target cells found by one CTL per hour is shown in (J). (K, L) Vinblastin enhances killing efficiency of CTLs in dense matrices. Respective target cells as indicated were embedded in collagen (5 mg/ml). Killing efficiency was determined at 24 hours (K) or 12h (L) after adding CTLs using ImageXpress (20× objective). The ratio between effector cells and targets is 5:1 in (A–C, K). The ratio between effector cells and targets is 1:1 in (D, E, L). One dot represents one donor (B, H, K, n = 3 donors), one independent experiment (E, n = 6 and L, n = 7 experiments), or one cell (F, I, J, n = 4 donors). Scale bars are 50 µm. Results are presented as Mean ± SD. Scale bars are 50 µm. For statistical significance, the paired Student’s t-test (B, E, H) or the Mann-Whitney test (F, I–L) were used.