Intercellular N-cad homotypic interactions slow PHGG migration on ECM and speed migration on neurons and astrocytes. (A) N-cad in intercellular contacts between migrating PBT-05 cells, labeled with Green CMFDA, and cerebellar neurons expressing tdTomato. Migration for 72 h before fixation and staining with N-cad antibody. xy images are maximum intensity projections from multiple z-planes. Insets show enlarged xy, xz, and yz images. Arrowheads indicate N-cad at cell–cell contacts. Scale bars, 20 and 5 μm (inset). (B) Western blot analysis for Ncad^WT-mCherry or Ncad^W161A-mCherry expression in PBT-05 cells. β-Tubulin is shown as a loading control. (C) Cumulative migration distances on neurons for 72 h. (D) Cumulative migration distance on laminin for 24 h. (E) N-cad in the environment stimulates PHGG migration. Migration on poly-D-lysine or N-cad extracellular domain (ECD)-Fc surfaces for 24 h. (F and G) Astrocyte N-cad stimulates PHGG migration. (F) Western blot shows N-cad siRNA depletes N-cad from mouse astrocytes. β-Tubulin is shown as a loading control. (G) Cumulative migration distances on control or N-cad-depleted astrocytes for 48 h. (H) Model diagram: migration on ECM is slowed by N-cad-mediated glioma-glioma interactions but migration on neurons or astrocytes is accelerated by N-cad-mediated glioma binding to surrounding neural cells. (C, D, and G) Unpaired t test. (E) Ordinary one-way ANOVA Šídák’s multiple comparisons test. Error bars show mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001. (C)N = 8–11 spheroids, three experiments. (D)N = 14–16 spheroids, three experiments. (E)N = 5 spheroids, two experiments. (G)N = 13–16 spheroids, three experiments. Source data are available for this figure: SourceData F3.