On day 24 of culture, organoids were removed from Matrigel by incubation with Dispase (Sigma) at 37°C and transferred to individual wells of a GravityTRAP ULA Plate (PerkinElmer). Labeled GBM cells were dissociated with Accutase and added to the organoid plate in neural maintenance medium at a concentration of 1000 cells per well. Plates were centrifuged at 100g for 3 min before returning to the incubator. After 2 days, Organoids were live stained with 100 nM SiR-actin (Spirochrome). The following day, organoids were harvested, fixed in 2% paraformaldehyde for 30 min, and embedded in Matrigel for immobilization. Tissue clearing was performed following the fructose/urea/α-thioglycerol (FRUIT) protocol as described.18 (link) Immunohistochemistry and control invasion assays using MCF10AT spheroids grown in Matrigel or SH-SY5Y spheroids were performed as described in the Supplementary Methods. Confocal images were acquired on an LSM780 Axio Observer confocal laser scanning microscope (Zeiss) and analyzed using custom scripts in ImageJ and MatLab, as detailed in the Supplementary Methods.