Supplementary Material for: Cortical Actin Alteration at the Matrix-Side Cytoplasm in Lung Adenocarcinoma Cells and Its Significance in Invasion

<p><b><i>Objectives:</i></b> Cortical actin is a thin layer of filamentous (F-)actin that lies beneath the plasma membrane, and its role in pathophysiology remains unclear. We investigated the subcellular localization of cortical actin by the histopathological and experimental studies of lung adenocarcinomas. <b><i>Materials and Methods:</i></b> The subcellular localization of cortical actin was studied in surgically resected lung adenocarcinomas tissues and in 3-dimensionally cultured lung adenocarcinoma A549 cells. <b><i>Results:</i></b> In normal type II alveolar cells and the bronchiolar epithelium, cortical actin was localized to the apical-side cytoplasm. In invasive adenocarcinoma cells, cortical actin was frequently localized to the matrix side. The degree of cortical actin localized to the matrix side was associated with the loss of basement membrane and a poor prognosis. In A549 cell spheroids cultured in a type I collagen and basement membrane extract Matrigel™ mixed gel, cortical F-actin was localized to the matrix side with phosphorylated myosin light chain. Super-resolution and electron microscopy results suggest that compact wrinkling of the plasma membrane by myosin-mediated F-actin contraction is an explanation for cortical actin accumulation at the matrix side. The myosin II inhibitor blebbistatin suppressed the 3-dimensional collective migration of A549 cells induced by constitutively active Cdc42 and MT1-MMP. <b><i>Conclusion:</i></b> Cortical actin accumulation at the matrix-side cytoplasm of cancer cells occurs in invasive lung adenocarcinomas and it possibly participates in the migration of cancer cells through myosin-mediated contraction.</p