Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells

This work was supported in part by a Marie Curie CIG grant (PCIG14-GA-2013-631011 CSKFingerprints)

A beginner's guide to atomic force microscopy probing for cell mechanics

European Commission . Grant Number: CIG14-2013-631011 Dunhill Medical Trust . Grant Number: R454/111

Vimentin Plays a Crucial Role in Fibroblast Ageing by Regulating Biophysical Properties and Cell Migration

Ageing is the result of changes in biochemical and biophysical processes at the cellular level that lead to progressive organ decline. Here we focus on the biophysical changes that impair cellular function of human dermal fibroblasts using donors of increasing age. We find that cell motility is impaired in cells from older donors, which is associated with increased Young’s modulus, viscosity, and adhesion. Cellular morphology also displays parallel increases in spread area and cytoskeletal assembly, with a threefold increase in vimentin filaments alongside a decrease in its remodelling rate. Treatments with withaferin A or acrylamide show that cell motility can be modulated by regulating vimentin assembly. Crucially, decreasing vimentin amount in cells from older individuals to levels displayed by the neonatal donor rescues their motility. Our results suggest that increased vimentin assembly may underlay the aberrant biophysical properties progressively observed at the cellular level in the course of human ageing and propose vimentin as a potential therapeutic target for ageing-related diseases

Study of fibroblasts activation kinetics and identification of fibroblasts subpopulations in physiological and pathological conditions

Fibroblasts undergo significant morphological and functional changes in response to specific environmental cues, adopting a novel phenotype when sustaining transformative activation in wound healing and cancerous processes. The aim of this project is to characterize cytoskeletal reorganization in both control fibroblasts’ (CFs) and cancer-associated fibroblasts’ (CAFs) activation. To achieve this, we propose a novel method based on the extraction of biophysical biomarkers from epifluorescence images of the cytoskeleton of individual fibroblasts, obtained from a patient with lung cancer and activated using TGF-β. These biophysical outputs were also used to identify CAFs subpopulations. While non-tumoral fibroblasts experience larger morphological changes characterized by an increase in area and the acquisition of a robust network of actin fibers, CAFs exhibited sustained larger areas throughout the process regardless of TGF- β administration, amongst other cytoskeletal transformations. The application of logistic regression has allowed for a classification between CFs and CAFs of 81% accuracy, highlighting the differences in the cytoskeleton of these cell types. Furthermore, the intragroup analysis provided by unsupervised clustering has enabled the identification of five clusters for non- activated CAFs, which converge at 72 hours post-activation into two clusters.This project has been carried out thanks to the resources and facilities available at the Unit of Biophysics of the Faculty of Medicine of University of Barcelona within the project PID2020-116808RB-I00 funded by the Spanish Ministry of Sciences, Innovation and Universities

Study of fibroblasts activation kinetics and identification of fibroblasts subpopulations in physiological and pathological conditions

Fibroblasts undergo significant morphological and functional changes in response to specific environmental cues, adopting a novel phenotype when sustaining transformative activation in wound healing and cancerous processes. The aim of this project is to characterize cytoskeletal reorganization in both control fibroblasts’ (CFs) and cancer-associated fibroblasts’ (CAFs) activation. To achieve this, we propose a novel method based on the extraction of biophysical biomarkers from epifluorescence images of the cytoskeleton of individual fibroblasts, obtained from a patient with lung cancer and activated using TGF-β. These biophysical outputs were also used to identify CAFs subpopulations. While non-tumoral fibroblasts experience larger morphological changes characterized by an increase in area and the acquisition of a robust network of actin fibers, CAFs exhibited sustained larger areas throughout the process regardless of TGF- β administration, amongst other cytoskeletal transformations. The application of logistic regression has allowed for a classification between CFs and CAFs of 81% accuracy, highlighting the differences in the cytoskeleton of these cell types. Furthermore, the intragroup analysis provided by unsupervised clustering has enabled the identification of five clusters for non- activated CAFs, which converge at 72 hours post-activation into two clusters.This project has been carried out thanks to the resources and facilities available at the Unit of Biophysics of the Faculty of Medicine of University of Barcelona within the project PID2020-116808RB-I00 funded by the Spanish Ministry of Sciences, Innovation and Universities

Extracellular fluid viscosity enhances liver cancer cell mechanosensing and migration

The extracellular fluid (ECF) is a crowded environment containing macromolecules that determine its characteristic density, osmotic pressure, and viscosity, which greatly differ between tissues. Precursors and products of degradation of biomaterials enhance ECF crowding and often increase its viscosity. Also, increases in ECF viscosity are related to mucin-producing adenocarcinomas. However, the effect of ECF viscosity on cells remains largely unexplored. Here we show that viscosity-enhancing polymer solutions promote mesenchymal-like cell migration in liver cancer cell lines. Also, we demonstrate that viscosity enhances integrin-dependent cell spreading rate and causes actin cytoskeleton re-arrangements leading to larger cell area, nuclear flattening, and nuclear translocation of YAP and β-catenin, proteins involved in mechanotransduction. Finally, we describe a relationship between ECF viscosity and substrate stiffness in determining cell area, traction force generation and mechanotransduction, effects that are actin-dependent only on ≤ 40 kPa substrates. These findings reveal that enhancing ECF viscosity can induce major biological responses including cell migration and substrate mechanosensing

Lifeact-GFP alters F-actin organization, cellular morphology and biophysical behaviour

Live-imaging techniques are at the forefront of biology research to explore behaviour and function from sub-cellular to whole organism scales. These methods rely on intracellular fluorescent probes to label specific proteins, which are commonly assumed to only introduce artefacts at concentrations far-exceeding routine use. Lifeact, a small peptide with affinity for actin microfilaments has become a gold standard in live cell imaging of the cytoskeleton. Nevertheless, recent reports have raised concerns on Lifeact-associated artefacts at the molecular and whole organism level. We show here that Lifeact induces dose-response artefacts at the cellular level, impacting stress fibre dynamics and actin cytoskeleton architecture. These effects extend to the microtubule and intermediate filament networks as well as the nucleus, and ultimately lead to altered subcellular localization of YAP, reduced cell migration and abnormal mechanical properties. Our results suggest that reduced binding of cofilin to actin filaments may be the underlying cause of the observed Lifeact-induced cellular artefacts

Assessment of the nano-mechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy.

Nano-indentation techniques might be better equipped to assess the heterogeneous material properties of plaques than macroscopic methods but there are no bespoke protocols for this kind of material testing for coronary arteries. Therefore, we developed a measurement protocol to extract mechanical properties from healthy and atherosclerotic coronary artery tissue sections. Young's modulus was derived from force-indentation data. Metrics of collagen fibre density were extracted from the same tissue, and the local material properties were co-registered to the local collagen microstructure with a robust framework. The locations of the indentation were retrospectively classified by histological category (healthy, plaque, lipid-rich, fibrous cap) according to Picrosirius Red stain and adjacent Hematoxylin & Eosin and Oil-Red-O stains. Plaque tissue was softer (p < 0.001) than the healthy coronary wall. Areas rich in collagen within the plaque (fibrous cap) were significantly (p < 0.001) stiffer than areas poor in collagen/lipid-rich, but less than half as stiff as the healthy coronary media. Young's moduli correlated (Pearson's ρ = 0.53, p < 0.05) with collagen content. Atomic force microscopy (AFM) is capable of detecting tissue stiffness changes related to collagen density in healthy and diseased cardiovascular tissue. Mechanical characterization of atherosclerotic plaques with nano-indentation techniques could refine constitutive models for computational modelling

New Bioengineering Breakthroughs and Enabling Tools in Regenerative Medicine.

PURPOSE OF REVIEW: In this review, we provide a general overview of recent bioengineering breakthroughs and enabling tools that are transforming the field of regenerative medicine (RM). We focus on five key areas that are evolving and increasingly interacting including mechanobiology, biomaterials and scaffolds, intracellular delivery strategies, imaging techniques, and computational and mathematical modeling. RECENT FINDINGS: Mechanobiology plays an increasingly important role in tissue regeneration and design of therapies. This knowledge is aiding the design of more precise and effective biomaterials and scaffolds. Likewise, this enhanced precision is enabling ways to communicate with and stimulate cells down to their genome. Novel imaging technologies are permitting visualization and monitoring of all these events with increasing resolution from the research stages up to the clinic. Finally, algorithmic mining of data and soft matter physics and engineering are creating growing opportunities to predict biological scenarios, device performance, and therapeutic outcomes. SUMMARY: We have found that the development of these areas is not only leading to revolutionary technological advances but also enabling a conceptual leap focused on targeting regenerative strategies in a holistic manner. This approach is bringing us ever more closer to the reality of personalized and precise RM