THE INFLUENCE OF THE MICROENVIRONMENT OF HEALTHY AND TUMORAL CELLS

Cells use the extracellular matrix (ECM), a part of their microenvironment, as a scaffold for their anchorage, and interact with it in a reciprocal way. The ECM is mainly composed of a three-dimensional network of collagen and crosslinked proteins, such as fibronectin and laminin; ECMs from various tissues can have strong differences in their composition and biophysical properties. The study of reciprocal interactions between cells and their microenvironment pertains to the fields of mechanobiology and mechanotransduction. Different techniques are used to study cellular interactions, among which Atomic Force Microscopy (AFM), for its versatility and robustness, stands as a reliable biosensor. This thesis reports in three main chapters the study of the cell-microenvironment interplay mainly through adhesion force spectroscopy techniques based on AFM. The main objective of this work was to develop novel native ECM probes to investigate early mechanotransductive cellular interactions by means of AFM-based force spectroscopy measurements. ECM probes reproduce faithfully the structural and compositional complexity of the ECM; selection of patient derived ECMs and cells allows to perform personalized experiments with potential applications in the clinic. Murine bladder ECM probes have been produced using a combination of decellularization procedures and Laser Microdissection (LMD) and tested against tumoral rat cell line. As reference, we also performed force spectroscopy experiments using AFM spherical probes functionalized with the two main ECM proteins, laminin and fibronectin. Native ECM probes have been successfully produced, characterized and tested. The integrin-related adhesive pattern measured in the force spectroscopy experiments depends on the mechanical and chemical properties of the native ECM used to produce the probe, which demonstrates the specificity of this approach. Finally, as cellular intercommunication relies on forces in the order of pN, we assessed the reliability of different calibration methods (standard thermal noise, Sader’s, and Laser Doppler Vibrometry).