Deep invasion of TCF3::PBX1⁺ BCP-ALL cells into cerebral organoids occurs after 14 days of coculture. Cerebral organoids were cultured with (10 000) CFSE-stained TCF3::PBX1⁺ PDX cells. (A-C) Maximum intensity projections (MIP) are shown on the left-hand column, showing the DAPI (4’,6-diamidino-2-phenylindole) and monomeric tag red fluorescent protein tandem colocalizing with the cells’ CFSE signal. Orthogonal views on the right-hand column indicate the position of cells (green dots) by visualizing the organoid in 3 dimensions (XY, ZY, and ZX). Areas of leukemia cells found on the surface or within the organoid itself are shown in the orthogonal viewer and enhanced by the region of interest (ROI) image. (D) Subsurface CFSE-stained leukemia cells localize with other neuronal cell types. (D-E) Collapsed z-stack and merge of CFSE, DAPI, and anti-MAP2 staining are shown. Magnified images provide enhanced details of the leukemia cells colocalizing with the organoid volume (ROI), indicating that the leukemic cells sit in a complex network of neurons and their axonal/dendritic connections. Scale bars: 10 μm, 50 μm, and 500 μm.