Reliable, standardized measurements for cell mechanical properties.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.

Coupled mechanical mapping and interference contrast microscopy reveal viscoelastic and adhesion hallmarks of monocyte differentiation into macrophages.

Monocytes activated by pro-inflammatory signals adhere to the vascular endothelium and migrate from the bloodstream to the tissue ultimately differentiating into macrophages. Cell mechanics and adhesion play a crucial role in macrophage functions during this inflammatory process. However, how monocytes change their adhesion and mechanical properties upon differentiation into macrophages is still not well understood. In this work, we used various tools to quantify the morphology, adhesion, and viscoelasticity of monocytes and differentiatted macrophages. Combination of atomic force microscopy (AFM) high resolution viscoelastic mapping with interference contrast microscopy (ICM) at the single-cell level revealed viscoelasticity and adhesion hallmarks during monocyte differentiation into macrophages. Quantitative holographic tomography imaging revealed a dramatic increase in cell volume and surface area during monocyte differentiation and the emergence of round and spread macrophage subpopulations. AFM viscoelastic mapping showed important stiffening (increase of the apparent Young's modulus, E0) and solidification (decrease of cell fluidity, ß) on differentiated cells that correlated with increased adhesion area. These changes were enhanced in macrophages with a spread phenotype. Remarkably, when adhesion was perturbed, differentiated macrophages remained stiffer and more solid-like than monocytes, suggesting a permanent reorganization of the cytoskeleton. We speculate that the stiffer and more solid-like microvilli and lamellipodia might help macrophages to minimize energy dissipation during mechanosensitive activities. Thus, our results revealed viscoelastic and adhesion hallmarks of monocyte differentiation that may be important for biological function.

Multi-Step Extracellular Matrix Remodelling and Stiffening in the Development of Idiopathic Pulmonary Fibrosis.

The extracellular matrix (ECM) of the lung is a filamentous network composed mainly of collagens, elastin, and proteoglycans that provides structural and physical support to its populating cells. Proliferation, migration and overall behaviour of those cells is greatly determined by micromechanical queues provided by the ECM. Lung fibrosis displays an aberrant increased deposition of ECM which likely changes filament organization and stiffens the ECM, thus upregulating the profibrotic profile of pulmonary cells. We have previously used AFM to assess changes in the Young's Modulus (E) of the ECM in the lung. Here, we perform further ECM topographical, mechanical and viscoelastic analysis at the micro- and nano-scale throughout fibrosis development. Furthermore, we provide nanoscale correlations between topographical and elastic properties of the ECM fibres. Firstly, we identify a softening of the ECM after rats are instilled with media associated with recovery of mechanical homeostasis, which is hindered in bleomycin-instilled lungs. Moreover, we find opposite correlations between fibre stiffness and roughness in PBS- vs bleomycin-treated lung. Our findings suggest that changes in ECM nanoscale organization take place at different stages of fibrosis, with the potential to help identify pharmacological targets to hinder its progression.