Mechanical Ways to Study Molecular Structure of Pericellular Layer—AFM Indentation and Ringing Mode

Atomic force microscopy (AFM) has emerged as a powerful tool for investigating the mechanical properties of cells, particularly in the context of malignancy. Numerous studies have reported a softening of various cancer cells compared to their nonmalignant counterparts across multiple cell types. However, the majority of these AFM studies have overlooked the pericellular layer, which can significantly influence the measured cell rigidity and potentially obscure valuable information about the physical properties of this layer. Furthermore, it has been demonstrated that the pericellular layer can substantially change during cell progression towards cancer. Up to now, it is not clear what molecular changes in the pericellular layer are associated with progression towards cancer.<br/>Here, we use two AFM techniques that are sensitive to the presence of the pericellular layer: AFM indentation technique processed through the brush model and AFM Ringing mode that allows imaging of the distribution of mechanical properties over the cell surface. Two cell lines, human bladder epithelial nonmalignant (HCV29) and cancerous (TCCSUP) cells were studied here. To translate the physical information, which is obtained within these two modalities, into biochemical terms more familiar to the cell biology community, we use heparinase and neuraminidase enzymatic treatments. These treatments selectively remove specific molecular components of the pericellular layer. We discussed the observed correlation between the removal of these specific molecular components and the observed changes in both mechanical properties of the pericellular layer and their distributions across the cell surface. We also compared these two methods in terms of their capability to distinguish between treated and nontreated cells.