Probing the determinants of cellular elasticity by AFM

Cells within tissues continuously encounter mechanical challenges to which they respond by remodelling their cytoskeleton. Cellular interactions as well as the microenvironment regulate the cell’s mechanical response under physiological and pathological conditions. An overview of the functional aspects associated with cell cytoskeleton, extracellular matrix (ECM) components and mechanical properties in health and disease is provided in Chapter 1. Atomic force microscopy (AFM) provides the ability to image, measure and mechanically manipulate major cytoskeletal and ECM components that contribute to the mechanical properties of cells and tissues, as will be presented in Chapter 2. Intermediate filaments (IFs) are one of the three main cytoskeletal components, and are essential determinants of cell shape and cytoskeletal integrity. In particular, they are considered the key responsive elements to extreme forces and deformations in living cells. However, their contribution to physiological nanoscale forces has received little attention. This has prompted us to employ a combined AFM/optical microscopy approach to examine the contribution of the vimentin IF network to the mechanical response of rat fibroblasts at high sensitivity and spatial resolution under near-physiological conditions (Chapter 3). To specifically target and modulate the vimentin network, fibroblasts were transfected with GFP-desmin variants. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. These findings demonstrated that specific alterations in the IF filament structure and architecture have a direct impact on the nanomechanical properties of cells. Despite its unique potential for probing cellular nanomechanics under physiological conditions (Chapters 2 and 3), AFM has so far not been applied to three-dimensional (3D) culture models or intact tumor tissues, which more accurately reflect the in vivo characteristics of cancer. As a first step towards this goal, we have systematically investigated nanomechanical changes associated with tumorigenic transformation and hypoxia in 3D culture models (Chapter 4). AFM stiffness maps from 3D spheroids showed that tumor spheroids were softer than their normal counterparts. In particular, the core was significantly softer than peripheral regions. To test whether nanomechanical properties could be measured in complex, organized tissues, we extended our studies to intact mammary tumor tissues ex vivo. The nanomechanical response of mammary tissues from a transgenic mouse model for human breast carcinoma and from human patient biopsies is presented in Chapter 5. AFM measurements revealed a gradual softening from the periphery to the core in human and murine cancer tissues while stromal tissue at the tumor periphery was stiffer than the underlying tumor. Comparison of the nanomechanical signature of human tissue samples with the corresponding histopathological diagnosis suggested a high ratio of soft versus stiff regions to be an indication of a more aggressive phenotype. Taken together, data presented in this thesis provide evidence that the nanomechanical properties may be used as a diagnostic marker for tumorigenesis.