Studying fluctuating trajectories of optically confined passive tracers inside cells provides familiar active forces.

In recent years, there has been a growing interest in studying the trajectories of microparticles inside living cells. Among other things, such studies are useful in understanding the spatio-temporal properties of a cell. In this work, we study the stochastic trajectories of a passive microparticle inside a cell using experiments and theory. Our theory is based on modeling the microparticle inside a cell as an active particle in a viscoelastic medium. The activity is included in our model from an additional stochastic term with non-zero persistence in the Langevin equation describing the dynamics of the microparticle. Using this model, we are able to predict the power spectral density (PSD) measured in the experiment and compute active forces. This caters to the situation where a tracer particle is optically confined and then yields a PSD for positional fluctuations. The low frequency part of the PSD yields information about the active forces that the particle feels. The fit to the model extracts such active force. Thus, we can conclude that trapping the particle does not affect the values of the forces extracted from the active fits if accounted for appropriately by proper theoretical models. In addition, the fit also provides system properties and optical tweezers trap stiffness.

Comparison of thermal and athermal dynamics of the cell membrane slope fluctuations in the presence and absence of Latrunculin-B.

Conventionally, only the normal cell membrane fluctuations have been studied and used to ascertain membrane properties like the bending rigidity. A new concept, the membrane local slope fluctuations was introduced recently (Vaippullyet al2020Soft Matter167606), which can be modelled as a gradient of the normal fluctuations. It has been found that the power spectral density (PSD) of slope fluctuations behave as (frequency)-1while the normal fluctuations yields (frequency)-5/3even on the apical cell membrane in the high frequency region. In this manuscript, we explore a different situation where the cell is applied with the drug Latrunculin-B which inhibits actin polymerization and find the effect on membrane fluctuations. We find that even as the normal fluctuations show a power law (frequency)-5/3as is the case for a free membrane, the slope fluctuations PSD remains (frequency)-1, with exactly the same coefficient as the case when the drug was not applied. Moreover, while sometimes, when the normal fluctuations at high frequency yield a power law of (frequency)-4/3, the pitch PSD still yields (frequency)-1. Thus, this presents a convenient opportunity to study membrane parameters like bending rigidity as a function of time after application of the drug, while the membrane softens. We also investigate the active athermal fluctuations of the membrane appearing in the PSD at low frequencies and find active timescales of slower than 1 s.

Comparison of translational and rotational modes towards passive rheology of the cytoplasm of MCF-7 cells using optical tweezers.

A colloidal particle placed inside the cell cytoplasm is enmeshed within a network of cytoskeletal fibres immersed in the cytosolic fluid. The translational mode is believed to yield different rheological parameters than the rotational mode, given that these modes stretch the fibers differently. We compare the parameters for Michigan Cancer Foundation-7 (MCF-7) cells in this manuscript and find that the results are well comparable to each other. At low values of 0 Hz viscosity, the rotational and translational viscoelasticity matches well. However, discrepancies appear at higher values which may indicate that the cytoskeletal modes involved in rotation and translation of the particle are getting invoked. We also show that the 0 Hz viscosity increases as the cell ages under the conditions of constant room temperature of 25°C on the sample chamber.

Anisotropic 3D confinement of MCF-7 cells induces directed cell-migration and viscoelastic anisotropy of cell-membrane.

Tumor-associated collagen signature-3 (TACS-3) is a prognostic indicator for breast cancer survival. It is characterized by highly organized, parallel bundles of collagen fibers oriented perpendicular to the tumor boundary, serving as directional, confining channels for cancer cell invasion. Here we design a TACS-3-mimetic anisotropic, confined collagen I matrix and examine the relation between anisotropy of matrix, directed cellular migration, and anisotropy of cell membrane-the first direct contact between TACS-3 and cell-using Michigan Cancer Foundation-7 (MCF-7) cells as cancer-model. Using unidirectional freezing, we generated ∼50µm-wide channels filled with collagen I. Optical tweezer (OT) microrheology shows that anisotropic confinement increases collagen viscoelasticity by two orders of magnitude, and the elastic modulus is significantly greater along the direction of anisotropic confinement compared to that along the orthogonal direction, thus establishing matrix anisotropy. Furthermore, MCF-7 cells embedded in anisotropic collagen I, exhibit directionality in cellular morphology and migration. Finally, using customized OT to trap polystyrene probes bound to cell-membrane (and not to ECM) of either free cells or cells under anisotropic confinement, we quantified the effect of matrix anisotropy on membrane viscoelasticity, both in-plane and out-of-plane, vis-à-vis the membrane. Both bulk and viscous modulus of cell-membrane of MCF-7 cells exhibit significant anisotropy under anisotropic confinement. Moreover, the cell membrane of MCF-7 cells under anisotropic confinement is significantly softer (both in-plane and out-of-plane moduli) despite their local environment being five times stiffer than free cells. In order to test if the coupling between anisotropy of extracellular matrix and anisotropy of cell-membrane is regulated by cell-cytoskeleton, actin cytoskeleton was depolymerized for both free and confined cells. Results show that cell membrane viscoelasticity of confined MCF-7 cells is unaffected by actin de-polymerization, in contrast to free cells. Together, these findings suggest that anisotropy of ECM induces directed migration and correlates with anisotropy of cell-membrane viscoelasticity of the MCF-7 cells in an actin-independent manner.

Calcium-channel-blockers exhibit divergent regulation of cancer extravasation through the mechanical properties of cancer cells and underlying vascular endothelial cells.

Cardiovascular and cancer illnesses often co-exist, share pathological pathways, and complicate therapy. In the context of the potential oncological role of cardiovascular-antihypertensive drugs (AHD), here we examine the role of calcium-channel blocking drugs on mechanics of extravasating cancer cells, choosing two clinically-approved calcium-channel blockers (CCB): Verapamil-hydrochloride and Nifedipine, as model AHD to simultaneously target cancer cells (MCF7 and or MDA231) and an underlying monolayer of endothelial cells (HUVEC). First, live-cell microscopy shows that exposure to Nifedipine increases the spreading-area, migration-distance, and frequency of transmigration of MCF-7 cells through the HUVEC monolayer, whereas Verapamil has the opposite effect. Next, impedance-spectroscopy shows that for monolayers of either endothelial or cancer cells, Nifedipine-treatment alone decreases the impedance of both cases, suggesting compromised cell-cell integrity. Furthermore, upon co-culturing MCF-7 on the HUVEC monolayers, Nifedipine-treated MCF-7 cells exhibit weaker impedance than Verapamil-treated MCF-7 cells. Following, fluorescent staining of CCB-treated cytoskeleton, focal adhesions, and cell-cell junction also indicated that Nifedipine treatment diminished the cell-cell integrity, whereas verapamil treatment preserved the integrity. Since CCBs regulate intracellular Ca2+, we next investigated if cancer cell's exposure to CCBs regulates calcium-dependent processes critical to extravasation, specifically traction and mechanics of plasma membrane. Towards this end, first, we quantified the 2D-cellular traction of cells in response to CCBs. Results show that exposure to F-actin depolymerizing drug decreases traction stress significantly only for Nifedipine-treated cells, suggesting an actin-independent mechanism of Verapamil activity. Next, using an optical tweezer to quantify the mechanics of plasma membrane (PM), we observe that under constant, externally-applied tensile strain, PM of Nifedipine-treated cells exhibits smaller relaxation-time than Verapamil and untreated cells. Finally, actin depolymerization significantly decreases MSD only for Verapamil treated cancer-cells and endothelial cells and not for Nifedipine-treated cells. Together, our results show that CCBs can have varied, mechanics-regulating effects on cancer-cell transmigration across endothelial monolayers. A judicious choice of CCBs is critical to minimizing the pro-metastatic effects of antihypertension therapy.

Detection of sub-degree angular fluctuations of the local cell membrane slope using optical tweezers.

Normal thermal fluctuations of the cell membrane have been studied extensively using high resolution microscopy and focused light, particularly at the peripheral regions of a cell. We use a single probe particle attached non-specifically to the cell-membrane to determine that the power spectral density is proportional to (frequency)-5/3 in the range of 5 Hz to 1 kHz. We also use a new technique to simultaneously ascertain the slope fluctuations of the membrane by relying upon the determination of pitch motion of the birefringent probe particle trapped in linearly polarized optical tweezers. In the process, we also develop the technique to identify pitch rotation to a high resolution using optical tweezers. We find that the power spectrum of slope fluctuations is proportional to (frequency)-1, which we also explain theoretically. We find that we can extract parameters like bending rigidity directly from the coefficient of the power spectrum particularly at high frequencies, instead of being convoluted with other parameters, thereby improving the accuracy of estimation. We anticipate this technique for determination of the pitch angle in spherical particles to high resolution as a starting point for many interesting studies using the optical tweezers.

Measurement of viscoelastic properties of the cellular cytoplasm using optically trapped Brownian probes.

Measurement of the viscoelastic properties of a cell using microscopic tracer particles has been complicated given that the medium viscosity is dependent upon the size of the measurement probe leading to reliability issues. Further, a technique for direct calibration of optically trapped particles in vivo has been elusive due to the frequency dependence and spatial inhomogeneity of the cytoplasmic viscosity, and the requirement of accurate knowledge of the medium refractive index. Here, we employ a recent extension of Jeffery's model of viscoelasticity in the microscopic domain to fit the passive motional power spectra of micrometer-sized optically trapped particles embedded in a viscoelastic medium. We find excellent agreement between the 0 Hz viscosity in MCF7 cells and the typical values of viscosity in literature, between 2 to 16 mPa sec expected for the typical concentration of proteins inside the cytoplasmic solvent. This bypasses the dependence on probe size by relying upon small thermal displacements. Our measurements of the relaxation time also match values reported with magnetic tweezers, at about 0.1 s. Finally, we calibrate the optical tweezers and demonstrate the efficacy of the technique to the study of in vivo translational motion.

Collagen-I/silk-fibroin biocomposite exhibits microscalar confinement of cells and induces anisotropic morphology and migration of embedded fibroblasts.

Microstructural anisotropy of tumor-associated matrix correlates with invasion of cancer cells into the surrounding matrix during metastasis. Here, we report the fabrication and characterization of a three-dimensional (3D) silk-fibroin/collagen-I bio-composite based cell-culture model that exhibits microstructural and biochemical anisotropy. Using RGD-deficient silk-fibroin fibers to confine collagen-I gelation, we develop a silk-fibroin/collagen-I (SFC) bio-composite in a one-step process allowing control over the microstructural and biochemical anisotropy and the pore-size. Two forms of the SFC bio-composite are reported: a sandwich (Sfc ) configuration amenable to live-cell microscopy and an unsupported membrane (Mfc ) for use as a scaffold. Both microscalar and macroscalar mechanical properties of the SFC bio-composite are characterized using atomic force microscope (AFM)-based indentation and tensile-testing. We find that the modulus of stiffness of both Sfc and Mfc can be controlled and falls in the physiological range of 5-20 kPa. Furthermore, the modulus of stiffness of Mfc exhibits a ~200% increase in axial direction of microstructure, as compared to lateral direction. This implies a highly anisotropic mechanical stiffness of the microenvironment. Live-cell morphology and migration studies show that both the morphology and the migration of NIH-3 T3 fibroblasts is anisotropic and correlates with microstructural anisotropy. Our results show that SFC bio-composite permits proliferation of cells in both Sfc and Mfc configuration, promotes cell-migration along the major axis of anisotropy and together with morphological and migration data, suggest a potential application of both the composite configurations as a biomimetic scaffold for tissue engineering applications.

Synthesis and viscoelastic characterization of microstructurally aligned Silk fibroin sponges.

Silk fibroin (SF) is a model candidate for use in tissue engineering and regenerative medicine owing to its bio-compatible mechanochemical properties. Despite numerous advances made in the fabrication of various biomimetic substrates using SF, relatively few clinical applications have been designed, primarily due to the lack of complete understanding of its constitutive properties. Here we fabricate microstructurally aligned SF sponge using the unidirectional freezing technique wherein a novel solvent-processing technique involving Acetic acid is employed, which obviates the post-treatment of the sponges to induce their water-stability. Subsequently, we quantify the anisotropic, viscoelastic response of the bulk SF sponge samples by performing a series of mechanical tests under uniaxial compression over a wide range of strain rates. Results for these uniaxial compression tests in the finite strain regime through ramp strain and ramp-relaxation loading histories applied over two orders of strain rate magnitude show that microstructural anisotropy is directly manifested in the bulk viscoelastic solid-like response. Furthermore, the experiments reveal a high degree of volume compressibility of the sponges during deformation, and also evince for their remarkable strain recovery capacity under large compressive strains during strain recovery tests. Finally, in order to predict the bulk viscoelastic material properties of the fabricated and pre-characterized SF sponges, a finite strain kinematics-based, nonlinear, continuum model developed within a thermodynamically-consistent framework in a parallel investigation, was successfully employed to capture the viscoelastic solid-like, transversely isotropic, and compressible response of the sponges macroscopically.

A microfluidic device to select for cells based on chemotactic phenotype.

In the search for biomarkers of metastasis, attention has been largely placed on ensemble-averaged measurements that screen for molecules or genes. However, individual molecular changes do not always result in disease, and population-based measurements can mask the molecular signatures of the cells responsible for disease. Here, we describe a device that selects for cells based on chemotactic behavior rather than based on molecular differences, enabling the most aggressive cells to be studied independently from the heterogeneous population.

ß-Catenin serves as a clutch between low and high intercellular E-cadherin bond strengths.

A wide range of invasive pathological outcomes originate from the loss of epithelial phenotype and involve either loss of function or downregulation of transmembrane adhesive receptor complexes, including Ecadherin (Ecad) and binding partners ß-catenin and α-catenin at adherens junctions. Cellular pathways regulating wild-type ß-catenin level, or direct mutations in ß-catenin that affect the turnover of the protein have been shown to contribute to cancer development, through induction of uncontrolled proliferation of transformed tumor cells, particularly in colon cancer. Using single-molecule force spectroscopy, we show that depletion of ß-catenin or the prominent cancer-related S45 deletion mutation in ß-catenin present in human colon cancers both weaken tumor intercellular Ecad/Ecad bond strength and diminishes the capacity of specific extracellular matrix proteins-including collagen I, collagen IV, and laminin V-to modulate intercellular Ecad/Ecad bond strength through α-catenin and the kinase activity of glycogen synthase kinase 3 (GSK-3ß). Thus, in addition to regulating tumor cell proliferation, cancer-related mutations in ß-catenin can influence tumor progression by weakening the adhesion of tumor cells to one another through reduced individual Ecad/Ecad bond strength and cellular adhesion to specific components of the extracellular matrix and the basement membrane.

Arterial stiffening precedes systolic hypertension in diet-induced obesity.

Stiffening of conduit arteries is a risk factor for cardiovascular morbidity. Aortic wall stiffening increases pulsatile hemodynamic forces that are detrimental to the microcirculation in highly perfused organs, such as the heart, brain, and kidney. Arterial stiffness is associated with hypertension but presumed to be due to an adaptive response to increased hemodynamic load. In contrast, a recent clinical study found that stiffness precedes and may contribute to the development of hypertension although the mechanisms underlying hypertension are unknown. Here, we report that in a diet-induced model of obesity, arterial stiffness, measured in vivo, develops within 1 month of the initiation of the diet and precedes the development of hypertension by 5 months. Diet-induced obese mice recapitulate the metabolic syndrome and are characterized by inflammation in visceral fat and aorta. Normalization of the metabolic state by weight loss resulted in return of arterial stiffness and blood pressure to normal. Our findings support the hypothesis that arterial stiffness is a cause rather than a consequence of hypertension.

The Arp2/3 complex mediates multigeneration dendritic protrusions for efficient 3-dimensional cancer cell migration.

Arp2/3 is a protein complex that nucleates actin filament assembly in the lamellipodium in adherent cells crawling on planar 2-dimensional (2D) substrates. However, in physiopathological situations, cell migration typically occurs within a 3-dimensional (3D) environment, and little is known about the role of Arp2/3 and associated proteins in 3D cell migration. Using time resolved live-cell imaging and HT1080, a fibrosarcoma cell line commonly used to study cell migration, we find that the Arp2/3 complex and associated proteins N-WASP, WAVE1, cortactin, and Cdc42 regulate 3D cell migration. We report that this regulation is caused by formation of multigeneration dendritic protrusions, which mediate traction forces on the surrounding matrix and effective cell migration. The primary protrusions emanating directly from the cell body and prolonging the nucleus forms independent of Arp2/3 and dependent on focal adhesion proteins FAK, talin, and p130Cas. The Arp2/3 complex, N-WASP, WAVE1, cortactin, and Cdc42 regulate the secondary protrusions branching off from the primary protrusions. In 3D matrices, fibrosarcoma cells as well as migrating breast, pancreatic, and prostate cancer cells do not display lamellipodial structures. This study characterizes the unique topology of protrusions made by cells in a 3D matrix and show that these dendritic protrusions play a critical role in 3D cell motility and matrix deformation. The relative contribution of these proteins to 3D migration is significantly different from their role in 2D migration.

Collagen prolyl hydroxylases are essential for breast cancer metastasis.

The presence of hypoxia and fibrosis within the primary tumor are two major risk factors for metastasis of human breast cancer. In this study, we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Finally, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies and show that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer.

Hypoxia-inducible factor 1 (HIF-1) promotes extracellular matrix remodeling under hypoxic conditions by inducing P4HA1, P4HA2, and PLOD2 expression in fibroblasts.

Extracellular matrix (ECM) composition, organization, and compliance provide both architectural and chemical cues that modulate tissue structure and function. ECM produced by stromal fibroblasts plays a key role in breast cancer invasion and metastasis, which are also stimulated by intratumoral hypoxia. Here, we demonstrate that hypoxia-inducible factor 1 (HIF-1) is a critical regulator of ECM remodeling by fibroblasts under hypoxic conditions. HIF-1 activates expression of genes encoding collagen prolyl (P4HA1 and P4HA2) and lysyl (PLOD2) hydroxylases. P4HA1 and P4HA2 are required for collagen deposition, whereas PLOD2 is required for ECM stiffening and collagen fiber alignment. Together P4HA1, P4HA2, and PLOD2 mediate remodeling of ECM composition, alignment, and mechanical properties in response to hypoxia. HIF-1-dependent ECM remodeling by hypoxic fibroblasts induces changes in breast cancer cell morphology, adhesion, and motility that promote invasion and metastasis.

Procollagen lysyl hydroxylase 2 is essential for hypoxia-induced breast cancer metastasis.

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix (ECM) in the metastatic process may lead to the development of improved therapies to treat patients with cancer. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, hypoxia-inducible factor 1 (HIF-1) activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We show that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs.