Migration through physical constraints is enabled by MAPK-induced cell softening via actin cytoskeleton re-organization.

Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffness were characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics.This article has an associated First Person interview with the first author of the paper.

Rapid evaporative ionization mass spectrometry (intelligent knife) for point-of-care testing in acute aortic dissection surgery.

OBJECTIVES: Rapid evaporative ionization mass spectrometry (REIMS) can discriminate aneurysmal from normal aortic tissue. Our objective in this work was to probe the integrity of acute dissection tissue using biomechanical, biochemical and histological techniques and demonstrate that REIMS can be used to discriminate identified differences. METHODS: Human aortic tissue was obtained from patients undergoing surgery for acute aortic dissection. Biomechanical, biochemical and histological assessment was carried out to probe mechanical properties and elastin, collagen and glycosaminoglycan composition of the tissue. Monopolar electrocautery was applied to samples and surgical aerosol aspirated and analysed by REIMS to produce mass spectral data. RESULTS: Tissue was obtained from 10 patients giving rise to 26 tissue pieces: 10 false lumen (FL), 10 dissection flap and 6 true lumen samples. Models generated from biomechanical and biochemical data showed that FL tissue was distinct from true lumen and dissection flap tissue. REIMS identified the same pattern being able to classify tissue types with 72.4% accuracy and 69.3% precision. Further analysis of REIMS data for FL tissue suggested patients formed 3 distinct clusters. Histological and biochemical assessment revealed patterns of extracellular matrix degradation within the clusters that are associated with altered tissue integrity identified using biomechanical testing. CONCLUSIONS: Structural integrity of the FL in acute Type A dissection could dictate future clinical distal disease progression. REIMS can detect differences in tissue integrity, supporting its development as a point-of-care test to guide surgical intraoperative decision-making.

Bicuspid valve aortopathy is associated with distinct patterns of matrix degradation.

OBJECTIVE: To explore the micromechanical, biochemical, and microstructural differences between bicuspid aortic valve aneurysm (BAV-A) and tricuspid aortic valve idiopathic degenerative aneurysm (DA), compared with normal aorta. METHODS: Aortic tissue was obtained from patients undergoing aneurysmal repair surgery (BAV-A; n = 15 and DA; n = 15). Control tissue was obtained from aortic punch biopsies during coronary artery bypass graft surgery (n = 9). Nanoindentation was used to determine the elastic modulus on the medial layer. Glycosaminoglycan, collagen, and elastin levels were measured using biochemical assays. Verhoeff Van Gieson-stained cross-sections were imaged for elastin microstructural quantification. RESULTS: The elastic modulus was more than 20% greater for BAV-A relative to control and DA (signifying a loss of compliance). No significance difference between control and DA were observed. Collagen levels for BAV-A (36.9 ± 7.4 µg/mg) and DA (49.9 ± 10.9 µg/mg) were greater compared with the control (30.2 ± 13.1 µg/mg). Glycosaminoglycan and elastin levels were not significant between the groups. Elastin segments were uniform throughout the control. Aneurysmal tissues had less elastin segments close to the intima and adventitia layers. Both BAV-A and DA had elastin segments compacted in the media; however, elastin segments were highly fragmented in DA. CONCLUSIONS: BAV-A has a greater loss of aortic wall compliance relative to DA and the control. Although elastin levels were equal for all groups, spatial distribution of elastin provided a unique profile of matrix degradation for BAV-A. Elastin compaction within the media of BAV-A may have resulted from the altered hemodynamic pressure against the wall, which could explain for the stiffness of the tissue.

Idiopathic degenerative thoracic aneurysms are associated with increased aortic medial amyloid.

Objective: To explore the relationship of aortic medial amyloid with biochemical and micromechanical properties of the aortic wall in aneurysm patients. Methods: Human aortic tissues removed during aneurysm surgery from tricuspid (idiopathic degenerative aneurysm, DA) and bicuspid valve (BAV) patients were subjected to oscillatory nanoindentation experiments to determine localised mechanical properties of the tissue (shear storage modulus, G´ and shear loss modulus, G˝). Collagen, elastin, matrix metalloproteinase 2 and glycosaminoglycans concentrations were determined, along with relative levels of aortic medial amyloid-related factors (medin, milk fat globule-EGF factor 8, oligomers and fibrils). Measurements were combined with clinical data and statistical analyses performed. Results: The DA cohort can be divided based on their phenotype. One group shared similar characteristics with BAV patients, termed bicuspid like phenotype-tricuspid valve. The second group had high amyloid oligomer species present with a significantly lower G´ (p = .01), indicative of reduced elastic response of the tissue, termed amyloid-rich. Conclusions: We identified a group of DA patients with high amyloid oligomers and altered micromechanical and structural properties of the vessel wall. We propose these findings as a cause for aneurysm formation in these patients. Amyloid is not found in BAV patients, suggesting at least two distinct mechanisms for pathogenesis.

A one-step procedure to probe the viscoelastic properties of cells by Atomic Force Microscopy.

The increasingly recognised importance of viscoelastic properties of cells in pathological conditions requires rapid development of advanced cell microrheology technologies. Here, we present a novel Atomic Force Microscopy (AFM)-microrheology (AFM2) method for measuring the viscoelastic properties in living cells, over a wide range of continuous frequencies (0.005 Hz ~ 200 Hz), from a simple stress-relaxation nanoindentation. Experimental data were directly analysed without the need for pre-conceived viscoelastic models. We show the method had an excellent agreement with conventional oscillatory bulk-rheology measurements in gels, opening a new avenue for viscoelastic characterisation of soft matter using minute quantity of materials (or cells). Using this capability, we investigate the viscoelastic responses of cells in association with cancer cell invasive activity modulated by two important molecular regulators (i.e. mutation of the p53 gene and Rho kinase activity). The analysis of elastic (G'(ω)) and viscous (G″(ω)) moduli of living cells has led to the discovery of a characteristic transitions of the loss tangent (G″(ω)/G'(ω)) in the low frequency range (0.005 Hz ~ 0.1 Hz) that is indicative of the capability for cell restructuring of F-actin network. Our method is ready to be implemented in conventional AFMs, providing a simple yet powerful tool for measuring the viscoelastic properties of living cells.

Nanomechanics and ultrastructure of the internal mammary artery adventitia in patients with low and high pulse wave velocity.

The collagen-rich adventitia is the outermost arterial layer and plays an important biomechanical and physiological role in normal vessel function. While there has been a lot of effort to understand the role of the medial layer on arterial biomechanics, the adventitia has received less attention. In this study, we hypothesized that different ultrastructural and nanomechanical properties would be exhibited in the adventitia of the internal mammary artery (IMA) in patients with a low degree of arterial stiffening as compared to those with a high degree of arterial stiffening. Human IMA biopsies were obtained from a cohort of patients with arterial stiffening assessed via carotid-femoral PWV. Patients were grouped as low PWV (8.5 ±â€¯0.7 ms-1, n = 8) and high PWV (13.4 ±â€¯3.0 ms-1, n = 9). Peakforce QNM atomic force microscopy (AFM) was used to determine the nanomechanical and morphological properties of the IMA. The nano-scale elastic modulus was found to correlate with PWV. We show for the first time that nano-scale alterations in adventitial collagen fibrils in the IMA are evident in patients with high PWV, even though the IMA is not involved in the carotid-femoral pathway. Our approach provides new insight into systemic structure-property changes in the vasculature, and also provides a method of characterizing small biopsy samples to predict the development of arterial stiffening. STATEMENT OF SIGNIFICANCE: Arterial stiffening occurs as part of the natural aging process and is strongly linked to cardiovascular risk. Although arterial stiffening is routinely measured in vivo, little is known about how localised changes in artery structure and biomechanics contributes to in vivo arterial stiffening. This study focusses on the role of the outermost layer of arteries, the adventitia, in arterial stiffening. The study provides data on nano-scale changes in collagen fibril structure and mechanical properties in the adventitia and shows how it relates to in vivo stiffness measurements in the vascular system. This is the first study to link in vivo arterial stiffening with nanomechanical changes in artery biopsy samples. Hence, this approach could be used to develop new diagnostic methods for vascular disease.

Polarized cell motility induces hydrogen peroxide to inhibit cofilin via cysteine oxidation.

Mesenchymal cell motility is driven by polarized actin polymerization [1]. Signals at the leading edge recruit actin polymerization machinery to promote membrane protrusion, while matrix adhesion generates tractive force to propel forward movement. To work effectively, cell motility is regulated by a complex network of signaling events that affect protein activity and localization. H2O2 has an important role as a diffusible second messenger [2], and mediates its effects through oxidation of cysteine thiols. One cell activity influenced by H2O2 is motility [3]. However, a lack of sensitive and H2O2-specific probes for measurements in live cells has not allowed for direct observation of H2O2 accumulation in migrating cells or protrusions. In addition, the identities of proteins oxidized by H2O2 that contribute to actin dynamics and cell motility have not been characterized. We now show, as determined by fluorescence lifetime imaging microscopy, that motile cells generate H2O2 at membranes and cell protrusions and that H2O2 inhibits cofilin activity through oxidation of cysteines 139 (C139) and 147 (C147). Molecular modeling suggests that C139 oxidation would sterically hinder actin association, while the increased negative charge of oxidized C147 would lead to electrostatic repulsion of the opposite negatively charged surface. Expression of oxidation-resistant cofilin impairs cell spreading, adhesion, and directional migration. These findings indicate that H2O2 production contributes to polarized cell motility through localized cofilin inhibition and that there are additional proteins oxidized during cell migration that might have similar roles.

Poly(N-acryloylmorpholine): a simple hydrogel system for temporal and spatial control over cell adhesion.

N-acryloylmorpholine (NAM) was photo-polymerized to produce the homopolymer poly(N-acryloylmorpholine) (PNAM). PNAM behaves as a physical hydrogel in aqueous solvents, doubling its dry weight over a 2 h period before undergoing dissolution following a second order exponential decay profile. In vitro cellular experiments using mouse myoblasts showed that PNAM acts as an effective spatial cell barrier for 38 h, with slow migration of cells into the PNAM area occurring between 45 and 73 h after cell seeding. At 80 h myoblasts fully occupied the area initially blocked by PNAM. Immunofluorescent staining of myoblasts adjacent to PNAM showed normal cytoskeletal structure and well developed focal adhesions indicating limited PNAM toxicity. This study shows that PNAM is an easy to synthesize physical hydrogel that acts as a temporal and spatial barrier to cell adhesion.