E-cad is required for Twist1-induced single cell dissemination. (A) Organoids from CMV::rtTA;TRE-Twist1 mice were divided into three groups for treatment with lentiviral shRNA against Luc or E-cad (two clones). Puromycin was used to select for transduced cells. Organoids were monitored for dissemination after Twist1 induction. (B) E-cad shRNA induced loss of E-cad protein and reductions in αE-catenin and β-catenin. Whole cell lysate samples were loaded for equal protein based on BCA analysis. (C–E) In FGF2-containing medium with doxycycline, E-cad KD organoids disseminated significantly fewer cells than Luc KD organoids (Video 8). Red arrowheads in C and D indicate disseminated cells. (F) Disseminated cells per organoid were quantified from movies after 100 h of Twist1 induction. E-cad KD significantly reduced single cell dissemination. Box-and-whisker plots are drawn with the box extending from the 25th to 75th percentiles and whiskers at the 10th and 90th percentiles. n, number of time-lapse movies; r, number of biological replicates. P < 0.0001 between Luc shRNA and E-cad shRNA #1 or #2; P = 0.014 between E-cad shRNA #1 and #2 (negative binomial generalized estimating equations model). (G) E-cad KD organoids extended collective chains of cells into the matrix (orange arrowheads; Video 9; G’ and G’’). (H and I) Cells within collective chains stained positive for Twist1 and luminal (K8⁺) and/or myoepithelial (K14⁺) cytokeratins. Bars: (C–E and G) 20 µm; (H and I) 10 µm.