Extracellular fluid viscosity enhances cell migration and cancer dissemination.

Cells respond to physical stimuli, such as stiffness1, fluid shear stress2 and hydraulic pressure3,4. Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer5. However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology.

The importance of water and hydraulic pressure in cell dynamics.

All mammalian cells live in the aqueous medium, yet for many cell biologists, water is a passive arena in which proteins are the leading players that carry out essential biological functions. Recent studies, as well as decades of previous work, have accumulated evidence to show that this is not the complete picture. Active fluxes of water and solutes of water can play essential roles during cell shape changes, cell motility and tissue function, and can generate significant mechanical forces. Moreover, the extracellular resistance to water flow, known as the hydraulic resistance, and external hydraulic pressures are important mechanical modulators of cell polarization and motility. For the cell to maintain a consistent chemical environment in the cytoplasm, there must exist an intricate molecular system that actively controls the cell water content as well as the cytoplasmic ionic content. This system is difficult to study and poorly understood, but ramifications of which may impact all aspects of cell biology from growth to metabolism to development. In this Review, we describe how mammalian cells maintain the cytoplasmic water content and how water flows across the cell surface to drive cell movement. The roles of mechanical forces and hydraulic pressure during water movement are explored.

On the energy efficiency of cell migration in diverse physical environments.

In this work, we explore fundamental energy requirements during mammalian cell movement. Starting with the conservation of mass and momentum for the cell cytosol and the actin-network phase, we develop useful identities that compute dissipated energies during extensions of the cell boundary. We analyze 2 complementary mechanisms of cell movement: actin-driven and water-driven. The former mechanism occurs on 2-dimensional cell-culture substrate without appreciable external hydraulic resistance, while the latter mechanism is prominent in confined channels where external hydraulic resistance is high. By considering various forms of energy input and dissipation, we find that the water-driven cell-migration mechanism is inefficient and requires more energy. However, in environments with sufficiently high hydraulic resistance, the efficiency of actin-polymerization-driven cell migration decreases considerably, and the water-based mechanism becomes more efficient. Hence, the most efficient way for cells to move depends on the physical environment. This work can be extended to higher dimensions and has implication for understanding energetics of morphogenesis in early embryonic development and cancer-cell metastasis and provides a physical basis for understanding changing metabolic requirements for cell movement in different conditions.

Response of collagen matrices under pressure and hydraulic resistance in hydrogels.

Extracellular matrices in animal tissue are hydrogels mostly made of collagen. In these matrices, collagen fibers are hierarchically assembled and cross-linked to form a porous and elastic material, through which migrating cells can move by either pushing through open matrix pores, or by actively digesting collagen fibers. The influence of matrix mechanical properties on cell behavior is well studied. Less attention has been focused on hydraulic properties of extracellular matrices, and how hydrodynamic flows in these porous hydrogels are influenced by matrix composition and architecture. Here we study the response of collagen hydrogels using rapid changes in the hydraulic pressure within a microfluidic device, and analyze the data using a poroelastic theory. Major poroelastic parameters can be obtained in a single experiment. Results show that depending on the density, porosity, and the degree of geometric confinement, moving micron-sized objects such as cells can experience substantially increased hydraulic resistance (by as much as 106 times) when compared to 2D environments. Therefore, in addition to properties such as mechanical stiffness, the fluidic environment of the cell is also likely to impact cell behavior.

Transition from Actin-Driven to Water-Driven Cell Migration Depends on External Hydraulic Resistance.

Cells in vivo can reside in diverse physical and biochemical environments. For example, epithelial cells typically live in a two-dimensional (2D) environment, whereas metastatic cancer cells can move through dense three-dimensional matrices. These distinct environments impose different kinds of mechanical forces on cells and thus potentially can influence the mechanism of cell migration. For example, cell movement on 2D flat surfaces is mostly driven by forces from focal adhesion and actin polymerization, whereas in confined geometries, it can be driven by water permeation. In this work, we utilize a two-phase model of the cellular cytoplasm in which the mechanics of the cytosol and the F-actin network are treated on an equal footing. Using conservation laws and simple force balance considerations, we are able to describe the contributions of water flux, actin polymerization and flow, and focal adhesions to cell migration both on 2D surfaces and in confined spaces. The theory shows how cell migration can seamlessly transition from a focal adhesion- and actin-based mechanism on 2D surfaces to a water-based mechanism in confined geometries.

Electromechanics and Volume Dynamics in Nonexcitable Tissue Cells.

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell's lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment. Specifically, we demonstrate that a sudden cell depolarization using the whole-cell patch clamp results in a 50% increase in cell volume, whereas hyperpolarization results in a slight volume decrease. We find that cell volume can be partially controlled by changing the chloride or the sodium/potassium concentrations in the extracellular environment while maintaining a constant external osmotic pressure. Depletion of external chloride leads to a volume decrease in suspended HN31 cells. Introducing cells to a high-potassium solution causes volume increase up to 50%. Cell volume is also influenced by cortical tension: actin depolymerization leads to cell volume increase. We present an electrophysiology model of water dynamics driven by changes in membrane potential and the concentrations of permeable ions in the cells surrounding. The model quantitatively predicts that the cell volume is directly proportional to the intracellular protein content.

Going with the Flow: Water Flux and Cell Shape during Cytokinesis.

Cell shape changes during cytokinesis in eukaryotic cells have been attributed to contractile forces from the actomyosin ring and the actomyosin cortex. Here we propose an additional mechanism where active pumping of ions and water at the cell poles and the division furrow can also achieve the same type of shape change during cytokinesis without myosin contraction. We develop a general mathematical model to examine shape changes in a permeable object subject to boundary fluxes. We find that hydrodynamic flows in the cytoplasm and the relative drag between the cytoskeleton network phase and the water phase also play a role in determining the cell shape during cytokinesis. Forces from the actomyosin contractile ring and cortex do contribute to the cell shape, and can work together with water permeation to facilitate cytokinesis. To influence water flow, we osmotically shock the cell during cell division, and find that the cell can actively adapt to osmotic changes and complete division. Depolymerizing the actin cytoskeleton during cytokinesis also does not affect the contraction speed. We also explore the role of membrane ion channels and pumps in setting up the spatially varying water flux.

Cell mechanics: a dialogue.

Under the microscope, eukaryotic animal cells can adopt a variety of different shapes and sizes. These cells also move and deform, and the physical mechanisms driving these movements and shape changes are important in fundamental cell biology, tissue mechanics, as well as disease biology. This article reviews some of the basic mechanical concepts in cells, emphasizing continuum mechanics description of cytoskeletal networks and hydrodynamic flows across the cell membrane. We discuss how cells can generate movement and shape changes by controlling mass fluxes at the cell boundary. These mass fluxes can come from polymerization/depolymerization of actin cytoskeleton, as well as osmotic and hydraulic pressure-driven flow of water across the cell membrane. By combining hydraulic pressure control with force balance conditions at the cell surface, we discuss a quantitative mechanism of cell shape and volume control. The broad consequences of this model on cell mechanosensation and tissue mechanics are outlined.

The Coda of the Transient Response in a Sensitive Cochlea: A Computational Modeling Study.

In a sensitive cochlea, the basilar membrane response to transient excitation of any kind-normal acoustic or artificial intracochlear excitation-consists of not only a primary impulse but also a coda of delayed secondary responses with varying amplitudes but similar spectral content around the characteristic frequency of the measurement location. The coda, sometimes referred to as echoes or ringing, has been described as a form of local, short term memory which may influence the ability of the auditory system to detect gaps in an acoustic stimulus such as speech. Depending on the individual cochlea, the temporal gap between the primary impulse and the following coda ranges from once to thrice the group delay of the primary impulse (the group delay of the primary impulse is on the order of a few hundred microseconds). The coda is physiologically vulnerable, disappearing when the cochlea is compromised even slightly. The multicomponent sensitive response is not yet completely understood. We use a physiologically-based, mathematical model to investigate (i) the generation of the primary impulse response and the dependence of the group delay on the various stimulation methods, (ii) the effect of spatial perturbations in the properties of mechanically sensitive ion channels on the generation and separation of delayed secondary responses. The model suggests that the presence of the secondary responses depends on the wavenumber content of a perturbation and the activity level of the cochlea. In addition, the model shows that the varying temporal gaps between adjacent coda seen in experiments depend on the individual profiles of perturbations. Implications for non-invasive cochlear diagnosis are also discussed.

Flow-Driven Cell Migration under External Electric Fields.

Electric fields influence many aspects of cell physiology, including various forms of cell migration. Many cells are sensitive to electric fields, and they can migrate toward a cathode or an anode, depending on the cell type. In this Letter, we examine an actomyosin-independent mode of cell migration under electrical fields. Our theory considers a one-dimensional cell with water and ionic fluxes at the cell boundary. Water fluxes through the membrane are governed by the osmotic pressure difference across the cell membrane. Fluxes of cations and anions across the cell membrane are determined by the properties of the ion channels as well as the external electric field. Results show that without actin polymerization and myosin contraction, electric fields can also drive cell migration, even when the cell is not polarized. The direction of migration with respect to the electric field direction is influenced by the properties of ion channels, and are cell-type dependent.

Electrodialysis treatment of rhamnolipids hydrolysate and its waste water for use as water-soluble fertilizer.

Rhamnolipids can serve as a precursor for rhamnose production, but using ion exchange resin in purifying rhamnolipids hydrolysate results in excessive high-salinity wastewater, making the process environmentally and economically unfeasible. This study introduced electrodialysis technology as an alternative for purifying rhamnolipids hydrolysate, significantly reducing wastewater to less than 5 % compared to the resin method. To achieve zero wastewater discharge, the electrodialysis-treated wastewater was repurposed into a water-soluble fertilizer containing 7.1 g/L of rhamnolipids, 11.4 g/L of fatty acid, 2.4 g/L of amino acid, and 8.2 g/L of potassium. Unlike traditional fertilizers, the nutritional components with rhamnolipids showed remarkable potential in enhancing tomato plant growth, flowering, and fruit quality. Taken together, the electrodialysis treatment of rhamnolipids hydrolysate largely reduced the water volume, the economic cost, and took a full use of the final wastewater as efficient water-soluble fertilizers, making it applicable for large-scale rhamnose production.

Confinement controls the directional cell responses to fluid forces.

Our understanding of how fluid forces influence cell migration in confining environments remains limited. By integrating microfluidics with live-cell imaging, we demonstrate that cells in tightly-but not moderately-confined spaces reverse direction and move upstream upon exposure to fluid forces. This fluid force-induced directional change occurs less frequently when cells display diminished mechanosensitivity, experience elevated hydraulic resistance, or sense a chemical gradient. Cell reversal requires actin polymerization to the new cell front, as shown mathematically and experimentally. Actin polymerization is necessary for the fluid force-induced activation of NHE1, which cooperates with calcium to induce upstream migration. Calcium levels increase downstream, mirroring the subcellular distribution of myosin IIA, whose activation enhances upstream migration. Reduced lamin A/C levels promote downstream migration of metastatic tumor cells by preventing cell polarity establishment and intracellular calcium rise. This mechanism could allow cancer cells to evade high-pressure environments, such as the primary tumor.

Chimeric nanobody-decorated liposomes by self-assembly.

Liposomes as drug vehicles have advantages, such as payload protection, tunable carrying capacity and improved biodistribution. However, due to the dysfunction of targeting moieties and payload loss during preparation, immunoliposomes have yet to be favoured in commercial manufacturing. Here we report a chemical modification-free biophysical approach for producing immunoliposomes in one step through the self-assembly of a chimeric nanobody (cNB) into liposome bilayers. cNB consists of a nanobody against human epidermal growth factor receptor 2 (HER2), a flexible peptide linker and a hydrophobic single transmembrane domain. We determined that 64% of therapeutic compounds can be encapsulated into 100-nm liposomes, and up to 2,500 cNBs can be anchored on liposomal membranes without steric hindrance under facile conditions. Subsequently, we demonstrate that drug-loaded immunoliposomes increase cytotoxicity on HER2-overexpressing cancer cell lines by 10- to 20-fold, inhibit the growth of xenograft tumours by 3.4-fold and improve survival by more than twofold.

Transport of dynamic biochemical signals in steady flow in a shallow Y-shaped microfluidic channel: effect of transverse diffusion and longitudinal dispersion.

Dynamic biochemical signal control is important in in vitro cell studies. This work analyzes the transportation of dynamic biochemical signals in steady and mixing flow in a shallow, Y-shaped microfluidic channel. The characteristics of transportation of different signals are investigated, and the combined effect of transverse diffusion and longitudinal dispersion is studied. A method is presented to control the widths of two steady flows in the mixing channel from two inlets. The transfer function and the cutoff frequency of the mixing channel as a transmission system are presented by analytically solving the governing equations for the time-dependent Taylor-Aris dispersion and molecular diffusion. The amplitude and phase spectra show that the mixing Y-shaped microfluidic channel acts as a low-pass filter due to the longitudinal dispersion. With transverse molecular diffusion, the magnitudes of the output dynamic signal are reduced compared to those without transverse molecular diffusion. The inverse problem of signal transportation for signal control is also solved and analyzed.

The influence of polarized membrane ion carriers and extracellular electrical/pH gradients on cell ionic homeostasis and locomotion.

Anisotropic environmental signals or polarized membrane ion/solute carriers can generate spatially-varying intracellular gradients, leading to polarized cell dynamics. For example, directional migration of neutrophils, galvanotaxis of glioblastoma, and water flux in kidney cells, all result from the polarized distribution of membrane ion carriers and other intracellular components. The underlying physical mechanisms behind how polarized ion carriers interact with environmental signals are not well studied. Here, we use a physiologically-relevant, physics-based mathematical model to reveal how ion carriers generate intracellular ionic and voltage gradients. The model is able to discern the contribution of individual ion carriers to the intracellular pH gradient, electric potential, and water flux. We discover that an extracellular pH gradient leads to an intracellular pH gradient via chloride-bicarbonate exchangers, whereas an extracellular electric field leads to an intracellular electric potential gradient via passive potassium channels. In addition, the mechanical-biochemical coupling can modulate actin distribution and flow, and create biphasic dependence of the cell speed on water flux. Moreover, we find that F-actin interaction with NHE alone can generate cell movement, even when other ion carriers are not polarized. Taken together, the model shows the importance of cell ion dynamics in modulating cell migration and cytoskeletal dynamics.

On the role of myosin-induced actin depolymerization during cell migration.

Mammalian cell migration in open spaces requires F-actin polymerization and myosin contraction. While many studies have focused on myosin's coupling to focal adhesion and stress fibers, the indirect effect of myosin contraction on cell migration through actin depolymerization is not well studied. In this work, we quantified how cell velocity and effective power output are influenced by the rate of actin depolymerization, which is affected by myosin contraction. In addition, we derived scaling laws to provide physical insights into cell migration. Model analysis shows that the cell migration velocity displays a biphasic dependence on the rate of actin depolymerization and myosin contraction. Our model further predicts that the effective cell energy output depends not only on the cell velocity but also on myosin contractility. The work has implications on in vivo processes such as immune response and cancer metastasis, where cells overcome barriers imposed by the physical environment.

Cytoskeletal activation of NHE1 regulates cell volume and DNA methylation.

The regulation of mammalian cell volume is crucial for maintaining key cellular processes. Cells can rapidly respond to osmotic and hydrostatic pressure imbalances during environmental challenges, generating fluxes of water and ions that alter volume within minutes. While the role of ion pump and leak in cell volume regulation has been well-established, the role of the actomyosin cytoskeleton and its substantial interplay with ion transporters are still unclear. In this work, we discover a system of cell volume regulation controlled by cytoskeletal activation of ion transporters. Under hypotonic shock, NIH-3T3 and MCF-10A display a 20% secondary volume increase (SVI) following the initial regulatory volume decrease. We show that SVI is initiated by Ca 2+ influx through stretch activated channel Piezo1 and subsequent actomyosin remodeling. Rather than contracting cells, actomyosin triggers cell swelling by activating Na + -H + exchanger 1 (NHE1) through their co-binding partner ezrin. Cytoskeletal activation of NHE1 can be similarly triggered by mechanical stretch and attenuated by soft substrates. This mechanism is absent in certain cancer cell lines such as HT1080 and MDA-MB-231, where volume regulation is dominated by intrinsic response of ion transporters. Moreover, cytoskeletal activation of NHE1 during SVI induces nuclear deformation, leading to DNA demethylation and a significant, immediate transcriptomic response in 3T3 cells, a phenomenon that is absent in HT1080 cells. Overall, our findings reveal the central role of Ca 2+ and actomyosin-mediated mechanosensation in the regulation of ion transport, cell volume, DNA methylation, and transcriptomics.

Direction of wave propagation in the cochlea for internally excited basilar membrane.

Otoacoustic emissions are an indicator of a normally functioning cochlea and as such are a useful tool for non-invasive diagnosis as well as for understanding cochlear function. While these emitted waves are hypothesized to arise from active processes and exit through the cochlear fluids, neither the precise mechanism by which these emissions are generated nor the transmission pathway is completely known. With regard to the acoustic pathway, two competing hypotheses exist to explain the dominant mode of emission. One hypothesis, the backward-traveling wave hypothesis, posits that the emitted wave propagates as a coupled fluid-structure wave while the alternate hypothesis implicates a fast, compressional wave in the fluid as the main mechanism of energy transfer. In this paper, we study the acoustic pathway for transmission of energy from the inside of the cochlea to the outside through a physiologically-based theoretical model. Using a well-defined, compact source of internal excitation, we predict that the emission is dominated by a backward traveling fluid-structure wave. However, in an active model of the cochlea, a forward traveling wave basal to the location of the force is possible in a limited region around the best place. Finally, the model does predict the dominance of compressional waves under a different excitation, such as an apical excitation.

Effective Force Generation During Mammalian Cell Migration Under Different Molecular and Physical Mechanisms.

We have developed much understanding of actin-driven cell migration and the forces that propel cell motility. However, fewer studies focused on estimating the effective forces generated by migrating cells. Since cells in vivo are exposed to complex physical environments with various barriers, understanding the forces generated by cells will provide insights into how cells manage to navigate challenging environments. In this work, we use theoretical models to discuss actin-driven and water-driven cell migration and the effect of cell shapes on force generation. The results show that the effective force generated by actin-driven cell migration is proportional to the rate of actin polymerization and the strength of focal adhesion; the energy source comes from the actin polymerization against the actin network pressure. The effective force generated by water-driven cell migration is proportional to the rate of active solute flux and the coefficient of external hydraulic resistance; the energy sources come from active solute pumping against the solute concentration gradient. The model further predicts that the actin network distribution is mechanosensitive and the presence of globular actin helps to establish a biphasic cell velocity in the strength of focal adhesion. The cell velocity and effective force generation also depend on the cell shape through the intracellular actin flow field.

Extracellular Hydraulic Resistance Enhances Cell Migration.

Cells migrating in vivo encounter microenvironments with varying physical properties. One such physical variable is the fluid viscosity surrounding the cell. Increased viscosity is expected to increase the hydraulic resistance experienced by the cell and decrease cell speed. The authors demonstrate that contrary to this expected result, cells migrate faster in high viscosity media on 2-dimensional substrates. Both actin dynamics and water dynamics driven by ion channel activity are examined. Results show that cells increase in area in high viscosity and actomyosin dynamics remain similar. Inhibiting ion channel fluxes in high viscosity media results in a large reduction in cell speed, suggesting that water flux contributes to the observed speed increase. Moreover, inhibiting actin-dependent vesicular trafficking that transports ion channels to the cell boundary changes ion channel spatial positioning and reduces cell speed in high viscosity media. Cells also display altered Ca2+ activity in high viscosity media, and when cytoplasmic Ca2+ is sequestered, cell speed reduction and altered ion channel positioning are observed. Taken together, it is found that the cytoplasmic actin-phase and water-phase are coupled to drive cell migration in high viscosity media, in agreement with physical modeling that also predicts the observed cell speedup in high viscosity environments.