Myoepithelial cell abundance regulates the extent of Twist1⁺ luminal cell dissemination. (A) Twist1-inducible luminal cells were isolated from CMV::rtTA;TRE-Twist1;mT/mG;K14::Actin-GFP mice, and normal K14⁺ (mostly myoepithelial) cells were isolated from mT/mG;K14::Actin-GFP mice via FACS using cell type–specific fluorescent reporters. Twist1-inducible luminal cells (red) were aggregated with normal myoepithelial cells (green) at different ratios to form clusters, and aggregated clusters were cultured in 3D Matrigel. (B) The number of disseminated Twist1⁺ cells per organoid significantly decreased with an increasing number of normal myoepithelial cells. (C–C′′′) Addition of doxycycline (dox) to aggregated organoids induced luminal-specific Twist1 expression. (D and D′) Myoepithelial coverage increased and dissemination decreased with the addition of more myoepithelial cells. Arrowheads indicate disseminated cells. n, total number of organoids; r, number of biological replicates. Data were analyzed by two-tailed nonparametric Kruskal–Wallis multiple comparisons test: **, P < 0.01; ****, P < 0.0001. Data are presented as box plots, with error bars representing the 5th to 95th percentile.