TCF3::PBX1⁺ leukemia cells upregulate AP-1 transcription factor upon infiltration into cerebral organoids. (A-B) Volcano plot showing significantly (false discovery rate [FDR] ˂ 0.05; log2(FC) < −1 or log2(FC) ˃ 1) upregulated or downregulated genes from RNA sequencing data of (A) cerebral organoid–infiltrated 697 and (B) Kasumi 2 (K2) cells compared with the noninfiltrated fraction. (C-D) 697 and K2 cell lines isolated from organoid cocultures were stained for FOSB and FOS and measured using flow cytometry. The mean fluorescence intensity (MFI) is plotted for both infiltrating (leukemia cells inside organoids) and suspension (noninfiltrated) cells. Statistical analysis was performed using the unpaired 2-tailed t test (FOSB, n = 3; FOS, n = 4). (E) 697 cells transduced with either the AP-1 construct (697-AP1) or a corresponding negative control (697-Neg) were cocultured in the presence (+ORG) or absence (−ORG) of organoids, respectively. Cells (10 000) were seeded from each condition in triplicate. Live cell imaging revealed an early and consistent rise in green fluorescent protein (GFP) fluorescence exclusively within the 697–AP-1 cells when cocultured with organoids. 697 wild-type cells (697 WT culture) and normal culturing media were included as nonfluorescent controls. Significance between 697–AP-1 + ORG vs 697–AP-1 − ORG was calculated using 2-way analysis of variance (697 AP1 + ORG vs 697 AP1 − ORG, n = 3, P < .0001. (F) The total track length covered by 697 cells, with or without T-5224 treatment was measured by time-lapse imaging for 20 hours, and analysis was performed via TrackMate plugin. The results depict the total track length covered by the cells. Statistical analysis was performed using the unpaired 2-tailed t test (dimethyl sulfoxide [DMSO] vs 5 μM, n = 3, P < .0001). FC, fold change.