Biology subjects

Holuigue, H.

Publications and source records attributed to Holuigue, H..

3 recordsLinked to original sources

bioRxiv2023

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 (Youngs 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.

biophysics↗

bioRxiv2022

Novel native extracellular matrix probes to target patient- and tissue- specific cell-microenvironment interactions by force spectroscopy

Atomic Force Microscopy (AFM) is successfully used for the quantitative investigation of the cellular mechanosensing of the microenvironment. To this purpose, several force spectroscopy approaches aim at measuring the adhesive forces between two living cells and also between a cell and a suitable reproduction of the extracellular matrix (ECM), typically exploiting tips suitably functionalised with single components (e.g. collagen, fibronectin) of the ECM. However, these probes only poorly reproduce the complexity of the native cellular microenvironment and consequently of the biological interactions. We developed a novel approach to produce AFM probes that faithfully retain the structural and biochemical complexity of the ECM; this was achieved by attaching to an AFM cantilever a micrometric slice of native decellularised ECM, which was cut by laser microdissection. We demonstrate that these probes preserve the morphological, mechanical, and chemical heterogeneity of the ECM. Native ECM probes can be used in force spectroscopy experiments aimed at targeting cell-microenvironment interactions. Here, we demonstrate the feasibility of dissecting mechanotransductive cell-ECM interactions in the 10 pN range. As proof-of-principle, we tested a rat bladder ECM probe against the AY-27 rat bladder cancer cell line. On the one hand, we obtained reproducible results using different probes derived from the same ECM regions; on the other hand, we detected differences in the adhesion patterns of distinct bladder ECM regions, such as submucosa and detrusor, in line with the disparities in composition and biophysical properties of these ECM regions. Our results demonstrate that native ECM probes, produced from patient-specific regions of organs and tissues, can be used to investigate cell-microenvironment interactions and early mechanotransductive processes by force spectroscopy. This opens new possibilities in the field of personalised medicine.

biophysics↗

bioRxiv2022

Force sensing on cells and tissues by atomic force microscopy

Biosensors are aimed to detect tiny physical and chemical stimuli in biological systems. Physical forces are ubiquitous, being implied in all cellular processes, including cell adhesion, migration, and differentiation. Given the strong interplay between cells and their microenvironment, the extracellular matrix (ECM), the structural and mechanical properties of the ECM play an important role in the transmission of external stimuli to single cells within the tissue. Vice versa, also cells themselves use self-generated forces to probe the biophysical properties of the ECM. ECM mechanics influences cell fate, regulates tissue development and show peculiar features in health and disease conditions of living organisms. Force sensing in biological systems is therefore crucial to dissect and understand complex biological processes, such as mechanotransduction. Atomic Force Microscopy (AFM), which can both sense and apply forces at the nanoscale, with sub-nanoNewton sensitivity, represents an enabling technology and a crucial experimental tool in biophysics and mechanobiology. In this work, we report on the application of AFM to study of biomechanical fingerprints of different components of biological systems, such as the ECM, the whole cell, and cellular components, like the nucleus and the glycocalyx. We show that physical observables like the (spatially resolved) Youngs modulus of elasticity of ECMs or cells, and the effective thickness and stiffness of the glycocalyx, can be quantitatively characterised by AFM. Their modification can be correlated to changes of the microenvironment, physio-pathological conditions, or gene regulation.

biophysics↗