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.

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.

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.

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.