Medulloblastoma spheroids respond to Slit2. (a) Model of the tumor spheroid cocultured with human embryonic kidney (HEK) or Slit2 aggregate in a collagen gel. Arrow denotes placement of HEK or Slit2 aggregate. (b) Still photograph of Cell Tracker labeled UW3 medulloblastoma spheroid implanted 550 μm from a Slit2 aggregate after 72 h. Arrow denotes placement of Slit2 aggregate. Scale bar = 250 μm. (c–j) Proximal and distal quadrant distribution of UW3 (c–d), Daoy (e–f) medulloblastoma cells and C6 (g–h), U251 (i–j) glioma cells cocultured with HEK control aggregates (c, e, g, i) or Slit2 aggregates (d, f, h, j) after 72 (UW3 and Daoy) and 48 h(C6 and U251). Error bars represent s.e.m. Asterisks denote significance at P < 0.05(*) and P < 0.01(**).

Slit2 inhibits individual medulloblastoma cell rate. (a) Still photographs depicting individual UW3 cell invasion from spheroids cocultured with human embryonic kidney (HEK) (upper panels) or Slit2 aggregates (lower panels) over 20 h. Arrows denote individual cell tracking. Scale bar = 50 μm (b) Image of ‘ruffling’ cell in UW3-Slit2 cocultures. Arrows denote individual cell tracking. Scale bar = 50 μm. (c) Mean invasion rate of individual UW3 cells cocultured with HEK (n = 35) or Slit2 aggregates (n = 32). Error bars represent s.e.m. Asterisks indicate a significant difference at P < 0.001 (***).

Sustained Slit2 delivery using sodium alginate bead microencapsulation inhibits medulloblastoma cell invasion. (a) Schematic showing the position of sodium alginate beads relative to the tumor spheroid. Arrow represents placement of sodium alginate bead. (b) Western blot analysis of Slit2 expression (~200 kDa) in conditioned medium of encapsulated Slit2 cells over 6 days. Lanes 1 and 2 represent conditioned medium from human embryonic kidney (HEK) and Slit2 monolayer cultures as negative and positive controls, respectively. Lanes 3–5 represent conditioned medium from Slit2 beads on days 1, 3 and 6 in culture. Lanes 6–7 represent conditioned medium from HEK beads on days 3 and 6. (c–e) Photographs of UW3 spheroids surrounded by (c) four empty beads, (d) four beads encapsulating HEK cells and (e) four beads encapsulating Slit2 cells. Scale bar = 250 μm. (f–h) Quantification of total invasion by (f) UW3 spheroids, (g–h) C6 and U251 glioma spheroids implanted with empty, HEK or Slit2 beads. Error bars represent s.e.m. Asterisks indicate significance at P < 0.05, P < 0.01** and P < 0.001***. (i–k) Confocal images of UW3 spheroids (DiI labeled-red) confronted with human fetal astrocyte aggregates (DiO labeled-green) in the presence of (i) HEK sodium alginate beads or (j) Slit2 sodium alginate beads after 5 days. Scale bar 250 μm. Results are representative of three independent trials. (k) Quantification of the number of medulloblastoma cells infiltrating astrocyte aggregates after 5 days.

RoboN rescues Slit2 inhibition of UW3 medulloblastoma cell invasion using sodium alginate bead microencapsulation. (a) Western blot analysis of Slit2 and RoboN expression (~200 kDa and ~100 kDa, respectively) in conditioned medium of encapsulated mixed Slit2-human embryonic kidney (HEK) and Slit2-RoboN cells at days 3 and 6. Conditioned medium from Slit2 (lane 1) and RoboN (lane 2) monolayer cultures were used as positive controls for Slit2 and RoboN, respectively. (b) Quantification of UW3 total invasion when cocultured with HEK, Slit2-HEK or Slit2-RoboN beads. Error bars represent s.e.m. Asterisks indicate significance at P < 0.05*, and P < 0.01**.

Reverse transcription-polymerase chain reaction (RT–PCR) analysis of Robo1 and Slit2 expression. (a) Robo1, Slit2 and GAPDH mRNA in medulloblastoma and glioma cell lines. Human fetal astrocytes (HFA) and mouse neonatal cerebellum (C P4 and C P8) were screened as positive controls. (b) Robo1, Slit2 and GAPDH mRNA in medulloblastoma (MD), meningioma (MN) and pilocytic astrocytoma (PA) primary human tumors. (c) Robo1, Slit2 and β-actin mRNA in glioblastoma multiforme (GBM) tumors and normal brain (NB). (d) Slit2 decreases activated Cdc42-GTP in medulloblastoma cells. Cdc42, Rac1 and RhoA loading controls levels were also estimated by Western blot analysis. Results are representative of three independent experiments.