Investigating the mechanics of tumour: from single cell mechanics to the biophysical interplay between cells and ECM

Abstract

This PhD project has focused the attention on the mechanical characterization of cancer cells and their surroundings. It is well known that the mechanical properties of cells and extracellular matrix (ECM), especially stiffness, play an important role in many biological processes such as cell growth, migration, division and differentiation. The pathological state of a cell implicates the alteration of the cytoskeletal structure and, consequently, of its functions, determining a variation of cell and ECM mechanical properties. In particular, the aim of this work is to investigate how cancer progression changes cell and ECM mechanical properties in vitro and ex vivo conditions. In the first experimental studies, particle tracking microrheology and Atomic Force Microscopy (AFM) techniques were performed to compare the mechanical properties of murine normal and virus-transformed cell lines cultured on glass. The first goal of the work was the identification of several biophysical parameters to discriminate between tumour and healthy cells. They have been useful to understand how virus transformation influence cell physiological processes and mechanical properties and, as a consequence, to identify the existence of a relationship between biological functions and cell mechanics. We observed that the effects of virus induced-transformation are the intensification of cell proliferation, the enhanced capability of transformed cells to migrate, the reduced adhesion capability, the reduction of cell cytoskeletal organization and the increased cell deformability. Successively, taking into account the results collected on the single murine cells, we moved to the characterization of human lung cells with different metastatic potential. Also in this case, combining the analyses of phenotypic characteristics and the biophysical properties of the cells, in particular elasticity, we were able to discriminate benign from cancer cells and, among them, to distinguish the grade of aggressiveness. Thus, we achieved the first milestone of this work with the definition of a new and accurate biomarker of cell metastatic potential. The second goal of the work concerned the investigation of the crosstalk between cancer cells and the surrounding ECM, through the study of ex vivo human biopsy tissues, removed from patients affected by lung adenocarcinoma. To this aim a new technique, based on multiple particle tracking (MPT) has been developed. To perform, at the same time, the mechanical classification of cells and ECM of each sample and a comparison with the healthy equivalent for the entire pool of patients, the ECM structure and morphology of cancer and healthy tissues were investigated and compared. Moreover, results and mechanical phenotypes were correlated to the stage and the grade of cancer, previously classified by the classic immunodiagnostic method. The cancerous transformation of tissues had a remarkable effect on the dynamics of the tracer beads and contributes a sort of symmetric modification of the mechanical properties of the cells and ECM. Indeed, compared to the healthy tissues, particles introduced into the cells of adenocarcinoma tissues increase their motion. Otherwise, unlike healthy tissues, the reduced motion of the beads probing the surrounding ECM suggests that cell in tumour tissues reside in a stiffer matrix. These increased mechanical properties of ECM are associated to an enhancement of collagen cross-linking, also confirmed through the structural and morphological analyses of tissue biopsies. The obtained mechanical properties of cells and their surrounding ECM from MPT represents a reliable indicator of the malignant transformation process and we believe that it can be used in combination with the classical immunohistochemistry-based diagnostic tools to obtain a more effective and precise diagnosis of the cancer.