Loss of MLL3 drives early neoplastic evolution in a TP53/CDKN2A DKO oral organoid model. (A) A schematic plot, generated using BioRender, showing the development of CRISPR-edited, cross-species organoid models for oral neoplastic evolution (TP53/CDKN2A^DKO) and subsequent knockdown of MLL3 expression. (B) qRT-PCR verification of the knockdown of MLL3 mRNA expression (n = 3 biological replicates). (C) Representative images of organoids from indicated groups at 40× magnification, scale bar = 20 μm. (D) Quantification of organoid size, as measured by ImageJ. (E) WST-1 assay to detect cell proliferation rate of organoids (n = 3 biological replicates). (F and G) Representative images, scale bar = 50 μm (F) and quantification of IF staining for Ki-67 in human organoids (n = 3 biological replicates) (G). (H) Individual tumor growth curves of control (TP53/CDKN2A^DKO + scramble sgRNA) versus MLL3-knockout organoids (TP53/CDKN2A^DKO + MLL3 knockout) (n = 5 biological replicates). (I) Kaplan–Meier plot showing the survival of mice from the two groups (n = 5 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001.