Figure 6 . NPC43 cell migration behavior in microwells without and with cover. (a) Migration speed of NPC43 cells with PTX and PEG-PTX NPs treatments over 16 h in microwells (a) without cover and (b) with cover. Cell migration trajectories of NPC43 cells over 16 h with PTX added in (c) 50×50 μm2 microwells with cover and (d) 150×150 μm2 microwells with cover. Starting points of cell migration trajectories are (0, 0). One-way ANOVA and Tukey’s post hoc tests, NS – not significant, **p <0.01, and ***p <0.001. Number of NPC43 cells counted is marked in white.
Average migration speed of NPC43 cells on flat surface and in different microwells are as shown in Supporting Table S3. As shown inFigure. 6a , without any treatment, the average migration speeds of cells on the flat surface and in different microwells without covers were similar with speed over 0.3 μm/min. After the NPC43 cells were treated with PTX or PEG-PTX NPs for 16 h, the average migration speed decreased significantly. Especially, the average migration speed was only about 0.1 μm/min after NPC43 cells were treated with PTX. The average migration speed of cells treated with PEG-PTX NPs was faster compared to cells treated with PTX NPs because PTX was more effective than PEG-PTX NPs to treat NPC43 cells in 16 h. After the cells were treated with PEG-PTX NPs in 150×150 μm2 microwells for 16 h, the average migration speed was 0.16±0.04 μm/min. But the average migration speed was 0.13±0.04 μm/min for cells in 50×50 μm2 microwells with the same treatment, indicating that NPC43 cells treated with PEG-PTX NPs possessed a faster speed in larger microwells. Furthermore, the average migration speed was also calculated in microwells with covers as shown in Figure. 6b. The cover decreased the migration speed because the cover inhibited the exchange of nutrients which made the cells unhealthy. For control groups, the average migration speeds were about 0.20 μm/min in all microwells with covers, which were slower than those in microwells without covers with speed over 0.3 μm/min. In microwells with covers, NPC43 cells treated with PTX still possessed the slowest speed among other groups including control without any treatment and NPC43 cells treated with PEG-PTX NPs. However, for the NPC43 cells treated with PTX, there was no difference for the average migration speed in microwells without and with covers as shown in Supporting Figure. S10a. The reason might be the additional confinement by the cover limited the supply of PTX into the microwells and slightly decreased the cytotoxicity, but PTX was still toxic enough to decrease the migration speed. It was a balance of PTX toxicity and the effect of 3D confinement.
After the NPC43 cells were treated with PTX, t0.1 for microwells with covers was larger than t0.1 for microwells without covers as shown in Figure. 5 and Supporting Figure. S12-S13. For the NPC43 cells treated with PEG-PTX NPs in microwells with covers, the average speed was lower than those in microwells without covers, especially in 150×150 μm2 microwells as shown in Supporting Figure. S10b. For the NPC43 cells in 150×150 μm2 microwells without covers, PEG-PTX NPs needed a longer time to release PTX and inhibit cell movement, with t0.1 of 12.9 h as shown in Supporting Figure. S9a. In addition, the cover decreased the migration speed in the first 5 h. So it was a combination effect of nanomedicine and the confinement that caused the decrease in the migration speed. The trajectories of NPC43 cells without any drug treatment and treated with PTX in 50×50 and 150×150 μm2 microwells are shown in Supporting Figure. S11 and Figure. 6c-d, respectively. As shown in Supporting Figure. S11, in microwells with covers, the moving range of NPC43 cells without any drug treatment was smaller than that in microwells without covers. But there was no difference for the moving range of NPC43 cells without any drug treatment in microwells with different sizes. After the NPC43 cells were treated with PTX as shown in Figure. 6c-d, the moving range was reduced. In addition, the moving range of cells in 150×150 μm2microwells was slightly bigger than that in 50×50 μm2microwells as shown in Figure. 6b-c. Cells without any treatment were healthy and possessed normal speed to move and change directions. In complete confinement with covers on top of microwells, the speed of cells decreased and cells could not move as far as cells in microwells without covers. After cells were treated with PTX, cells were too unhealthy to move or change directions. In smaller microwells, it was easier for the NPC43 cells treated with PTX to touch the sidewalls of the microwells, and the cells could not move further, leading to a smaller moving range.