BCP-ALL cells robustly engraft into cerebral organoids as compared with healthy CD34⁺ HSPCs. (A-B) Cerebral organoids were cocultured with 10 000 CFSE-stained cells, either cord blood–isolated CD34⁺ HSPCs or leukemia cells, for 14 days. Enlarged images of cell clusters are shown by white squares (ROI). Scale bars: 25 μm and 250 μm. (C-D) Cerebral organoids were cocultured with 10 000 CFSE-stained leukemia cells or CD34⁺ HSPCs. After 14 days of coculture period, the total cell counts within the invaded organoids were quantitatively assessed by enumerating CFSE-positive cells in each organoid, using Imaris image processing software. Statistical analysis was conducted to compare the HSPCs, K562, and KCL22 controls to every other condition using the unpaired 2-tailed t test (n = 3 replicates). (E) Visualization of leukemia cell distribution within organoids using Matplotlib-based image analysis. 3D representation illustrates the relative distribution of leukemia cells within the organoid structure. Distances exceeding 10 μm are considered boundaries for deep invasion. (F) Depicted are number of cells that have invaded beyond 10-μm depth in the organoid 3D space (n = 3 replicates, unpaired 2-tailed t test).