Depletion of α-SMA leads to an increase in cell dissemination. (A) Organoids isolated from K8::Cre-ER;R26::LSL-rtTA;TRE-Twist1;mT/mG mice were treated with lentiviral shRNA against a nontargeting (NT) control and against three different clones of α-SMA (myoepithelial smooth muscle gene). Organoids were treated with tamoxifen (Tam) to induce rtTA expression and cultured in doxycycline (Dox) to induce Twist1 expression in the luminal cell compartment. After Twist1 expression, organoids were monitored for dissemination. (B) Organoids were divided into nontargeting and SMA knockdown treatment groups. The number of disseminated cells per organoid was significantly higher in SMA knockdown organoids compared with the nontargeting control (two- to threefold difference) across five biological replicates. (C) SMA protein levels quantified from IF images confirmed a decrease in SMA expression in SMA knockdown organoids. (D) Laminin-332 protein levels quantified from IF images did not significantly differ between SMA knockdown organoids and controls. (E–F′) IF indicated retention of K14, SMA and Laminin-332 in control organoids. (G–H′) IF indicated retention of K14, loss of SMA, and retention of Laminin-332 in SMA knockdown organoids. Red and white arrowheads indicate disseminated cells. (I) Protruding Twist1⁺ luminal cells were observed to invade past the SMA-depleted myoepithelium and were able to disseminate (arrowheads). n, total number of organoids; r, number of biological replicates. Data were analyzed by two-tailed nonparametric Kruskal–Wallis multiple comparisons test: ***, P < 0.001; ****, P < 0.0001. Data are presented as box plots, with error bars representing the 5th to 95th percentile. Myoepithelial cells in time-lapse videos are pseudocolored in green in I.