Probing mechanical properties to study cancer cell migration

To best comprehend cellular behaviour and how it determines cell migration in metastatic cancer, the research described here has focused on cell mechanics. The signalling pathway involving Rho-associated kinase (ROCK) has emerged as being the main regulator for the cellular cytoskeleton and actomyosin contractility that play key roles in metastatic cancer formation. In this thesis, an examination is made of how the cellular properties intertwine as ROCK is overexpressed. In research towards being able to measure and describe the viscoelastic properties of a cell that are associated with cell mechanics, over a wide range of timescales, a novel AFM force indentation data analysis method was applied. In particular, as part of this study, pancreatic ductal adenocarcinoma (PDAC) cells were overexpressed with ROCK, and the influence of ROCK activity on cell’s elastic and viscoelastic properties were quantified. It was found that when ROCK activity was overexpressed in cells, their elasticity decreased while their viscosity remained unchanged. These properties had a direct correlation with the activity of ADF/cofilin - the proteins downstream of ROCK. This meant that with overexpression, more stable actin bundles were present along with their inward stresses generated by the actomyosin contraction. This is consistent with an increased level of compressive forces within cells. Collective compressive forces between cell-cell are associated with the packing of cells that decreases cellular response. To further understand the role of ROCK activity in cancer invasion, a microfluidic device was created to mimic cell migration through tissue. The device consists of precisely defined microchannels with dimensions chosen to hinder and confine the cells in a manner similar to that found in a physiological environment. It was found that overexpressed ROCK1 cells in the confinement had notable decrease in cell size and motility. Along with this decrease in mechanical properties, observations also gave rise to questions about the connection between these properties that remain to be answered

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

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

Macro- and Micro-mechanical Properties of the Ovine Aorta: Correlation with Regional Variations in Collagen, Elastin and Glycosominoglycan Levels

Aortic diseases are a significant cardiovascular health problem and occur in different ways across the vascular tree. Investigation of the mechanical properties of the aorta is important for better understanding of aortic diseases. In this study, the biomechanical and biochemical properties of the ovine aorta have been comprehensively mapped across different regions from the ascending to the abdominal aorta. We have determined the mechanical properties at the macro- (via tensile testing) and at the micro-scale (via oscillatory nanoindentation). Uniaxial tensile testing was conducted on circumferential strips for the ascending, upper thoracic region and upper abdominal region to determine physiological elastic modulus, tangent modulus at 0.5 strain, and the maximum elastic modulus. Nanoindentation was conducted on the medial layer (tissue cross-section) and intimal and adventitial face (longitudinal orientation) to determine the shear storage (G′) and shear loss modulus (G″). All of the measured mechanical properties increased with distance from the heart. For example, G′ increased by 237.1% and 275.3% for the intimal face and adventitial face, respectively. In parallel, collagen, glycosaminoglycans (GAG) and elastin levels were also measured across the entire length of the ovine aorta. The mechanical properties correlated with increasing collagen, and decreasing GAG and elastin. Collagen increased by 147.2% whereas GAG (−120.3%) and elastin decreased (−78.2%). These findings have relevance for developing mechanistic insight into aortic aneurysms and dissections

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

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

Bicuspid valve aortopathy is associated with distinct patterns of matrix degradation

Objective To explore the micromechanical, biochemical and microstructural differences between BAV-A (bicuspid aortic valve aneurysm) and TAV (tricuspid aortic valve) idiopathic degenerative aneurysm (DA), compared to 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 by-pass graft surgery (CABG; n=9). Nanoindentation was used to determine the elastic modulus (E) on the medial layer. Glycosaminoglycan (GAG), collagen and elastin levels were measured using biochemical assays. Verhoeff Van Gieson-stained cross-sections were imaged for elastin microstructural quantification. Results E was over 20% higher 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±7.4μg/mg) and DA (49.9±10.9μg/mg) were higher compared to the control (30.2±13.1μg/mg). GAG 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. Conclusion 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 haemodynamic 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