Senescent stroma induces nuclear deformations in cancer cells via the inhibition of RhoA/ROCK/myosin II-based cytoskeletal tension

The presence of senescent cells within tissues has been functionally linked to malignant transformations. Here, using tension-gauge tethers technology, particle-tracking microrheology, and quantitative microscopy, we demonstrate that senescent associated secretory phenotype (SASP) derived from senescent fibroblasts impose nuclear lobulations and volume shrinkage on malignant cells, which stems from the loss of RhoA/ROCK/myosin II-based cortical tension. This loss in cytoskeletal tension induces decreased cellular contractility, adhesion, and increased mechanical compliance. These SASP-induced morphological changes are in part mediated by lamin A/C. These findings suggest that SASP induces a defective outside-in mechanotransduction, from actomyosin fibers in the cytoplasm to the nuclear lamina, thereby triggering a cascade of biophysical and biomolecular changes in cells that associate with malignant transformations.

Effects of Visfatin on Intracellular Mechanics and Catabolism in Human Primary Chondrocytes through Glycogen Synthase Kinase 3β Inactivation

Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant attention because of the clinical finding of the positive correlation between its serum/synovial level and OA progression. However, the precise mechanism involved is still unclear. This study determined the effect of visfatin on intracellular mechanics and catabolism in human primary chondrocytes isolated from patients. The intracellular stiffness of chondrocytes was analyzed by the particle-tracking microrheology method. It was shown that visfatin damages the microtubule and microfilament networks to influence intracellular mechanics to decrease the intracellular elasticity and viscosity via glycogen synthase kinase 3β (GSK3β) inactivation induced by p38 signaling. Further, microtubule network destruction in human primary chondrocytes is predominantly responsible for the catabolic effect of visfatin on the cyclooxygenase 2 upregulation. The present study shows a more comprehensive interpretation of OA development induced by visfatin through biochemical and biophysical perspectives. Finally, the role of GSK3β inactivation, and subsequent regulation of intracellular mechanics, might be considered as theranostic targets for future drug development for OA.

Measuring the mechanical properties of apoptotic cells using particle tracking microrheology

Numerical models developed to study high frequency ultrasound scattering during apoptosis require knowledge of mechanical properties of cells. Particle Tracking Microrheology (PTM) is a technique for studying the mechanical properties of soft materials. By tracking the Brownian movement of particles embedded in a material, its mechanical properties can be extracted. In this thesis, PTM is used to measure the relative changes in the viscoelasticity of apoptotic PC3 cells. PTM was first validated in purely viscous and viscoelastic phantoms. It was found to work well in viscous phantoms, but was limited to only measuring relative changes of the viscoelasticity of viscoelastic materials. After validation, PTM measurements in cells showed that the elastic and viscous modulus increased by over 50 Pa and 20 Pa respectively over the course of the treatment. Preliminary development of another technique known as Two-Point Particle Tracking Microrheology (TPM) is also presented in this thesis.

Measuring the mechanical properties of apoptotic cells using particle tracking microrheology

Numerical models developed to study high frequency ultrasound scattering during apoptosis require knowledge of mechanical properties of cells. Particle Tracking Microrheology (PTM) is a technique for studying the mechanical properties of soft materials. By tracking the Brownian movement of particles embedded in a material, its mechanical properties can be extracted. In this thesis, PTM is used to measure the relative changes in the viscoelasticity of apoptotic PC3 cells. PTM was first validated in purely viscous and viscoelastic phantoms. It was found to work well in viscous phantoms, but was limited to only measuring relative changes of the viscoelasticity of viscoelastic materials. After validation, PTM measurements in cells showed that the elastic and viscous modulus increased by over 50 Pa and 20 Pa respectively over the course of the treatment. Preliminary development of another technique known as Two-Point Particle Tracking Microrheology (TPM) is also presented in this thesis.

Measurement of viscoelastic properties of treated and untreated cancer cells using passive microrheology

Experiments have shown that there is an increase in ultrasound backscatter from cells during cell death. Since cell scattering depends on the mechanical property variations, one step towards a better understanding of the phenomenon involves measuring the cells' viscoelastic properties. Two promising techniques used for such studies are particle tracking microrheology (1P) and two-point microrheology (2P). The main aim of this work is to develop and test the ability of both to measure changes in viscous and elastic moduli of breast cancer cells during chemotherapeutic treatments. First, the viscosities of glycerol-water mixtures measured using microrheology were found to be within 5% of rheometer values. The viscous and elastic moduli of 4% and 6% poly(ethylene oxide) solutions were successfully measured at 30°C and 37°C. For MCF-7 cells, a 10-fold increase in the elastic modulus was observed using 1P, without a corresponding increase in the viscous modulus. Thus, it was shown that MCF-7 cells undergo an increase in stiffness during apoptosis.

Measurement of viscoelastic properties of treated and untreated cancer cells using passive microrheology

Experiments have shown that there is an increase in ultrasound backscatter from cells during cell death. Since cell scattering depends on the mechanical property variations, one step towards a better understanding of the phenomenon involves measuring the cells' viscoelastic properties. Two promising techniques used for such studies are particle tracking microrheology (1P) and two-point microrheology (2P). The main aim of this work is to develop and test the ability of both to measure changes in viscous and elastic moduli of breast cancer cells during chemotherapeutic treatments. First, the viscosities of glycerol-water mixtures measured using microrheology were found to be within 5% of rheometer values. The viscous and elastic moduli of 4% and 6% poly(ethylene oxide) solutions were successfully measured at 30°C and 37°C. For MCF-7 cells, a 10-fold increase in the elastic modulus was observed using 1P, without a corresponding increase in the viscous modulus. Thus, it was shown that MCF-7 cells undergo an increase in stiffness during apoptosis.

Investigating longitudinal changes in the mechanical properties of MCF-7 cells exposed to paclitaxol using particle tracking microrheology

Evidence suggests that compression and shear wave elastography are sensitive to the mechanical property changes occuring in dying cells following chemotherapy, and can hence be used to monitor cancer treatment response. A qualitative and quantitative understanding of the mechanical changes at the cellular level would allow to better infer how these changes affect macroscopic tissue mechanical properties and therefore allow the optimization of elastographic techniques (such as shear wave elastography) for the monitoring of cancer therapy. We used intracellular particle tracking microrheology (PTM) to investigate the mechanical property changes of cells exposed to paclitaxol, a mitotic inhibitor used in cancer chemotherapy. The average elastic and viscous moduli of the cytoplasm of treated MCF-7 breast cancer cells were calculated for frequency ranges between 0.2 and 100 rad s–1 (corresponding to 0.03 and 15.92 Hz, respectively). A significant increase in the complex shear modulus of the cell cytoplasm was detected at 12 h post treatment. At 24 h after drug exposure, the elastic and viscous moduli increased by a total of 191.3 Pa (>8000×) and 9 Pa (~9×), respectively for low frequency shear modulus measurements (at 1 rad s–1). At higher frequencies (10 rad s–1), the elastic and viscous moduli increased by 188.5 Pa (~60×) and 1.7 Pa (~1.1×), respectively. Our work demonstrates that PTM can be used to measure changes in the mechanical properties of treated cells and that cell elasticity significantly increases by 24 h after chemotherapy exposure.

Investigating longitudinal changes in the mechanical properties of MCF-7 cells exposed to paclitaxol using particle tracking microrheology

Evidence suggests that compression and shear wave elastography are sensitive to the mechanical property changes occuring in dying cells following chemotherapy, and can hence be used to monitor cancer treatment response. A qualitative and quantitative understanding of the mechanical changes at the cellular level would allow to better infer how these changes affect macroscopic tissue mechanical properties and therefore allow the optimization of elastographic techniques (such as shear wave elastography) for the monitoring of cancer therapy. We used intracellular particle tracking microrheology (PTM) to investigate the mechanical property changes of cells exposed to paclitaxol, a mitotic inhibitor used in cancer chemotherapy. The average elastic and viscous moduli of the cytoplasm of treated MCF-7 breast cancer cells were calculated for frequency ranges between 0.2 and 100 rad s–1 (corresponding to 0.03 and 15.92 Hz, respectively). A significant increase in the complex shear modulus of the cell cytoplasm was detected at 12 h post treatment. At 24 h after drug exposure, the elastic and viscous moduli increased by a total of 191.3 Pa (>8000×) and 9 Pa (~9×), respectively for low frequency shear modulus measurements (at 1 rad s–1). At higher frequencies (10 rad s–1), the elastic and viscous moduli increased by 188.5 Pa (~60×) and 1.7 Pa (~1.1×), respectively. Our work demonstrates that PTM can be used to measure changes in the mechanical properties of treated cells and that cell elasticity significantly increases by 24 h after chemotherapy exposure.