Membrane Cytoskeletal Changes during In-Situ to Invasive Progression of Breast Cancer Cells Observed by Multi-Scale Diffusion Analysis of Transmembrane Proteins

Abstract

When cells change from stationary growth to migration stage, such as during the epithelial to mesenchymal transition (EMT), it is expected that their mechanics change as well as the membrane - cytoskeleton interactions. Such changes are thought to occur during the differentiation of cancers to more invasive types [Choi et al 2013, Human Pathology], during normal development of a tissue, or wound-healing.We show how change of membrane cytoskeletal features can be quantified in single cells, by measuring the diffusion of fluorescently tagged transmembrane (TM) proteins level using camera-based FCS (bimFCS) technique. Data is analyzed in two populations of diffusion states: fast, in between cytoskeleton fences, and slow, due to hopping effect over them. Our technique makes quantification of average fence spacing and the protein-fence interaction strength possible for the ROI observed.Using bimFCS, we analyzed the diffusion behavior of TM proteins in TM12 (epithelial) and TM12T (mesenchymal) breast cancer cell membranes. Cytoskeletal features (average fence spacing, protein-fence interaction strength) of invasive type TM12T cells show clear differences from the ones of in situ type TM12 cells.We also observed changes during the EMT in single cell level, after wound simulating scratches by pipette-tip. Over a period of 24 hours wound-healing experiment on stage, at physiological temperature, we observed critical changes of cytoskeletal features by comparing the slow and fast diffusion coefficients for differentiating TM12 cells during the process.