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.

Mechanical Way To Study Molecular Structure of Pericellular Layer.

Atomic force microscopy (AFM) has been used to study the mechanical properties of cells, in particular, malignant cells. Softening of various cancer cells compared to their nonmalignant counterparts has been reported for various cell types. However, in most AFM studies, the pericellular layer was ignored. As was shown, it could substantially change the measured cell rigidity and miss important information on the physical properties of the pericellular layer. Here we take into account the pericellular layer by using the brush model to do the AFM indentation study of bladder epithelial bladder nonmalignant (HCV29) and cancerous (TCCSUP) cells. It allows us to measure not only the quasistatic Young's modulus of the cell body but also the physical properties of the pericellular layer (the equilibrium length and grafting density). We found that the inner pericellular brush was longer for cancer cells, but its grafting density was similar to that found for nonmalignant cells. The outer brush was much shorter and less dense for cancer cells. Furthermore, we demonstrate a method to convert the obtained physical properties of the pericellular layer into biochemical language better known to the cell biology community. It is done by using heparinase I and neuraminidase enzymatic treatments that remove specific molecular parts of the pericellular layer. The presented here approach can also be used to decipher the molecular composition of not only pericellular but also other molecular layers.

Dynamic mechanical analysis of suspended soft bodies via hydraulic force spectroscopy.

The rheological characterization of soft suspended bodies, such as cells, organoids, or synthetic microstructures, is particularly challenging, even with state-of-the-art methods (e.g. atomic force microscopy, AFM). Providing well-defined boundary conditions for modeling typically requires fixating the sample on a substrate, which is a delicate and time-consuming procedure. Moreover, it needs to be tuned for each chemistry and geometry. Here, we validate a novel technique, called hydraulic force spectroscopy (HFS), against AFM dynamic indentation taken as the gold standard. Combining experimental data with finite element modeling, we show that HFS gives results comparable to AFM microrheology over multiple decades, while obviating any sample preparation requirements.

Discriminating bladder cancer cells through rheological mechanomarkers at cell and spheroid levels.

The stiffening or softening of cancers observed in nanoindentation experiments has been recognized as a marker of cancer-related changes. In bladder cancers, continuous stretching/destretching is observed due to its functionality, indicating that shear forces dominate the mechanical response of these cells. Thus, nanoindentation and microrheological measurements conducted in parallel allow for a fully reliable mechanomarker of cancer progression. Here, bladder cancer cell lines, i.e., non-malignant cell cancer of the ureter (HCV29), bladder carcinoma (HT1376), and transitional cell carcinoma (T24), were studied. Nanoindentation and microrheological experiments were conducted on individual cells, cell monolayers, and spheroids that were formed using non-adherent surface plates. The results show that nanoindentation experiments can only differentiate between non-malignant HCV29 (stiffer) and cancerous HT1376 and T24 (softer) cells. Applying microrheology recognizes the type of grade 3 bladder cancers (carcinoma HT1376 or transitional cell carcinoma T24 cells). We showed that actin filaments are a vital element defining the rheological properties of spheroids. Differences in mechanical properties of cell monolayers could be associated with thick actin bundles and intercellular connections, with some extracellular matrix (ECM) contributing to the stiffening of such monolayers. Our findings demonstrate that a complete image of how cancer cells respond to mechanical stress (compressive and shear forces) can only be obtained after microrheological measurements using the transition frequency separating elastic and viscous regimes as a non-labeled biomarker of bladder cancer progression.

Traction force microscopy - Measuring the forces exerted by cells.

Cells generate mechanical forces (traction forces, TFs) while interacting with the extracellular matrix or neighbouring cells. Forces are generated by both cells and extracellular matrix (ECM) and transmitted within the cell-ECM or cell-cell contacts involving focal adhesions or adherens junctions. Within more than two decades, substantial progress has been achieved in techniques that measure TFs. One of the techniques is traction force microscopy (TFM). This review discusses the TFM and its advances in measuring TFs exerted by cells (single cells and multicellular systems) at cell-ECM and cell-cell junctional intracellular interfaces. The answers to how cells sense, adapt and respond to mechanical forces unravel their role in controlling and regulating cell behaviour in normal and pathological conditions.