N-cad inhibits PHGG migration on ECM but stimulates migration on neurons and astrocytes. (A and B) Representative images and quantification of cumulative migration distance for control or N-cad shRNA PBT-05 spheroids plated on laminin for 24 h (C and D) Representative images and quantification of invasion in 3D Matrigel for 96 h. (E and F) Images and quantification of migration on neurons for 72 h. PBT-05 spheroids were labeled with cell-permeable Green CMFDA fluorescent dye before transferring to tdTomato-expressing cerebellar neurons that had differentiated along aligned nanofibers. (G and H) Images and quantification of migration on mouse astrocytes for 48 h. PBT-05 cells were detected with human-specific anti-vimentin antibodies. Actin was detected with phalloidin. (I and J) Images and quantification of migration on astrocyte-derived decellularized ECM for 24 h. (A, C, E, and G) Dashed circles represent spheroid at 0 h. Scale bars, 200 μm. (B, D, F, H, and J) Error bars indicate mean ± SEM. Ordinary one-way ANOVA Holm-Šídák’s multiple comparisons tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (B)N = 27–29 spheroids, three experiments. (D)N = 13–16 spheroids, four experiments. (F)N = 9 spheroids, three experiments. (H)N = 16–18 spheroids, three experiments. (J)N = 9–15 spheroids, two experiments.