Quantitative Analysis of Actin Cytoskeleton's Morphological Changes during EMT in Lung Cancer and Pre-Cancer Cell Lines

Abstract

Lung cancer, a highly metastatic disease, is the world's leading cause of cancer death. Epithelial to mesenchymal Transition (EMT), a key element of the metastatic process in lung cancer, is accompanied by corresponding changes in cellular mechanical properties, principally resulting from actin stress fiber formation. The details of the micro and macro rearrangement processes involved in this cytoskeletal reorganization is not yet fully understood. Using standard transfection/ staining and imaging protocols, we identified a novel cellular phenotype during the EMT process. This interim phenotype was characterized by a disoriented (nest-like) stress fiber arrangement in contrast to the highly aligned (semi-parallel) cytoskeleton in the final mesenchymal cell. The formation of this nest-like pattern was consistent amongst different EMT induction techniques and concentrations. Drug treatment assays suggested that inhibition of the PI3K-Akt signaling pathway prevents the formation of the final semi-parallel phenotype from the nest-like phenotype. To quantify the actin cytoskeleton remodeling, we utilized computational extraction of actin stress fibers from wide-field and confocal microscopic images. We developed a computational tool that extracts the actin stress fibers as straight lines, quantified through their locations, lengths and relative orientations. This tool further statistically processes the orientation information to generate the Orientational Order Parameter (OOP), which serves as a figure of merit to classify cells into nest-like cytoskeletal phenotype and the semi-parallel cytoskeletal phenotype. This figure of merit could possibly be utilized for other cytoskeletal remodeling processes such as fibrosis, apoptosis, cell division and migration.