| dc.description.abstract | Epithelial cells line surfaces of the human body. Since they define the boundary between internal and external environments, these cells maintain a polarity, created by localizing proteins at opposite ends of each cell, in order to facilitate transport of substances between these environments. Basal proteins, which face the external environment, and apical proteins, which face the internal environment, define this polarity. When grown in vitro using a 3D culture, epithelial cells group together to form hollow, fluid-filled spheroids of cells called acini. These acini reflect in vivo polarity with basal proteins facing the 3D matrix and the apical proteins facing the fluid-filled interior. In some cancers, the polarity of epithelial cells becomes inverted which may play a role in cancer metastasis. Previous research projects investigated the process responsible for everting the acini and found a variety of mechanisms. This research project builds on past discovery of using RhoA activation to initiate acinar eversion. Activating RhoA increases the activity of myosin, resulting in higher contractility in the cell. This higher contractility was found to disrupt the mechanical equilibrium of the acini, resulting in breakage, breach, and eversion of acini’s polarity as they collapse and flip inside-out. To further investigate this mechanism involving RhoA, this project documents the procedures to form and image acini using previously untested cell lines made from cancerous lung epithelial cells: 344SQ_shCTRL, 344SQ_shZEB1, and 393P_ZEB1. It was found that 344SQ_shCTRL and 344SQ_ZEB1 acini formed lumens. In making the acini, changes to the protocol included adjusting methods for avoiding imaging complications with thick Matrigel layers, shortening the time period allotted for lumen development, and increasing the concentration of RhoA activator used in experimentation. Initial experimental and fixed staining results collected demonstrate the exciting opportunity for many future research projects utilizing acini such as investigating lumen nuclei orientation, quantifying the factors impacting lumen development, and continuing investigation with acinar eversion. | |
