Epithelial eversion, a collective rearrangement from apical-in to apical-out polarity, is initiated by a6b4 integrins and sustained by increased cell proliferation and anchorage-independence

Epithelial spheroids can transition from normal, apical-in polarity to apical-out polarity through a collective rearrangement of cells, a process that we and others have termed eversion. We hypothesized that eversion is an integrin-dependent process, where specific integrin-ECM interactions could facilitate the collective cellular migration required for eversion. We observed that epithelial cells lacking either a6 or b4 integrins exhibit lumen collapse when treated with RhoA activator, but do not develop apical-out polarity. Similarly, a6 blocking antibody or culturing spheroids in collagen (lacking laminin) also inhibited the formation apical-out polarity, indicating that laminin-binding a6b4 integrins facilitate eversion. Next, we examined the role of cell proliferation and anchorage independence in maintaining apical-out polarity of everted spheroids. Inhibition of cell proliferation (with DNA synthesis inhibitor aphidicolin) or inhibition of anchorage-independence (with FAK inhibitor 14) were sufficient to restore apical-in polarity to RhoA treated spheroids, indicating that both proliferation and anchorage-independence maintain spheroids in an apical-out state. We also observed that apical-out spheroids can revert to apical-in polarity through apoptotic cavitation of cells located in the center of the spheroids. Lastly, through RNA sequencing we demonstrate that apical-out spheroids have unique gene expression profiles. This study provides new mechanistic insights into the biochemical and biophysical mechanisms that drive eversion and maintain apical-out polarity. This work supports the concept that changing from apical-in to apical-out polarity be an important marker for phenotypic switch in epithelial cells.