INVESTIGATION OF CELL-MICROENVIRONMENT INTERACTIONS BY ATOMIC FORCE MICROSCOPY TECHNIQUES

The role of forces is fundamental in a wide variety of biological processes, such as cell adhesion, migration, proliferation and differentiation. The ability of cells to perceive the nanotopographical features of the surrounding microenvironment (i.e. the extracellular matrix, ECM), called mechanotransduction, is mediated by specific trans-membrane proteins, called integrins, clustered together in Integrin Adhesion Complexes (IAC). Unraveling which mechanical and nanoscale morphological properties of the ECM determine the IAC composition and tune specific cellular response, is particularly challenging. Works have shown that biocompatible nanostructured thin films, grown by assembling zirconia nanoparticles (ns-ZrO2) on a substrate by Supersonic Cluster Beam Deposition technique, possess a morphological disorder on nanometer scale with structural features that mimic topographical properties of the ECM. Experiments performed with the neuron-like cell line PC12 demonstrated a link between the nanotopography of the ns-ZrO2 films and mechanotransductive events, which eventually foster neuronal differentiation. During my three years of PhD, I have developed novel approach based on Atomic Force Microscopy (AFM) to quantify cell sensing of nanotopographical features of the microenvironment, represented by ns-ZrO2 films reproducing the nanostructured surface of the ECM. Using custom ns-ZrO2–coated colloidal probes, we have carried out a quantitative analysis of adhesion strength and distribution of IACs by AFM-based adhesive force spectroscopy. We deposited ns-ZrO2 on custom AFM colloidal probes5. Bringing these nanostructured colloidal probes into contact with the body of living PC12 cells, it was possible to measure the strength, number and distribution of the IAC bonds by AFM force spectroscopy6, for different morphological properties of the interface. Furthermore, I used these functionalized probes to characterize the role of the surface pericellular layer, known as the Glycocalyx, in relation to the cell capability to react to external stimuli. Eventually, along with my personal research project, I had the opportunity to participate to two external collaboration with the Istituto Nazionale dei Tumori and with Istitute of Nuclear Physiscs from the Polish academy of Science. These collaborations aimed to exploit the knowledge in the mechanobiolgy field to study the mechanical implications of cells and tissues in cancer development and survival.