Matrix confinement modulates 3D spheroid sorting and burst-like collective migration.

While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we tune matrix confinement, mimicking changes in the stiffness and confinement of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. High levels of confinement lead to cell sorting while reducing matrix confinement triggers the collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon. Burst-like migration does not occur for spheroids that have not undergone sorting, regardless of the degree of matrix confinement. Using computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. The findings support a model where matrix confinement modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites. STATEMENT OF SIGNIFICANCE: The mechanical properties of the tumor microenvironment significantly influence cancer cell migration within the primary tumor, yet how these properties affect intercellular interactions in heterogeneous tumors is not well understood. By utilizing calcium and calcium chelators, we dynamically alter collagen-alginate hydrogel stiffness and investigate tumor cell behavior within co-culture spheroids in response to varying degrees of matrix confinement. High confinement is found to trigger cell sorting while reducing confinement for sorted spheroids facilitates collective cell invasion. Notably, without prior sorting, spheroids do not exhibit burst-like migration, regardless of confinement levels. This work establishes that matrix confinement and intercellular adhesion regulate 3D spheroid dynamics, offering insights into cellular organization and migration within the primary tumor.

Matrix confinement modulates 3D spheroid sorting and burst-like collective migration.

While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we tune matrix confinement, mimicking changes in the stiffness and confinement of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. High levels of confinement lead to cell sorting while reducing matrix confinement triggers the collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon. Burst-like migration does not occur for spheroids that have not undergone sorting, regardless of the degree of matrix confinement. Using computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. The findings support a model where matrix confinement modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites.

Compressive stress drives adhesion-dependent unjamming transitions in breast cancer cell migration.

Cellular unjamming is the collective fluidization of cell motion and has been linked to many biological processes, including development, wound repair, and tumor growth. In tumor growth, the uncontrolled proliferation of cancer cells in a confined space generates mechanical compressive stress. However, because multiple cellular and molecular mechanisms may be operating simultaneously, the role of compressive stress in unjamming transitions during cancer progression remains unknown. Here, we investigate which mechanism dominates in a dense, mechanically stressed monolayer. We find that long-term mechanical compression triggers cell arrest in benign epithelial cells and enhances cancer cell migration in transitions correlated with cell shape, leading us to examine the contributions of cell-cell adhesion and substrate traction in unjamming transitions. We show that cadherin-mediated cell-cell adhesion regulates differential cellular responses to compressive stress and is an important driver of unjamming in stressed monolayers. Importantly, compressive stress does not induce the epithelial-mesenchymal transition in unjammed cells. Furthermore, traction force microscopy reveals the attenuation of traction stresses in compressed cells within the bulk monolayer regardless of cell type and motility. As traction within the bulk monolayer decreases with compressive pressure, cancer cells at the leading edge of the cell layer exhibit sustained traction under compression. Together, strengthened intercellular adhesion and attenuation of traction forces within the bulk cell sheet under compression lead to fluidization of the cell layer and may impact collective cell motion in tumor development and breast cancer progression.

Compression enhances invasive phenotype and matrix degradation of breast Cancer cells via Piezo1 activation.

BACKGROUND: Uncontrolled growth in solid breast cancer generates mechanical compression that may drive the cancer cells into a more invasive phenotype, but little is known about how such compression affects the key events and corresponding regulatory mechanisms associated with invasion of breast cancer cells including cellular behaviors and matrix degradation. RESULTS: Here we show that compression enhanced invasion and matrix degradation of breast cancer cells. We also identified Piezo1 as the putative mechanosensitive cellular component that transmitted compression to not only enhance the invasive phenotype, but also induce calcium influx and downstream Src signaling. Furthermore, we demonstrated that Piezo1 was mainly localized in caveolae, and both Piezo1 expression and compression-enhanced invasive phenotype of the breast cancer cells were reduced when caveolar integrity was compromised by either knocking down caveolin1 expression or depleting cholesterol content. CONCLUSIONS: Taken together, our data indicate that mechanical compression activates Piezo1 channels to mediate enhanced breast cancer cell invasion, which involves both cellular events and matrix degradation. This may be a critical mechanotransduction pathway during breast cancer metastasis, and thus potentially a novel therapeutic target for the disease.

Facile formation of giant elastin-like polypeptide vesicles as synthetic cells.

We demonstrate the facile and robust generation of giant peptide vesicles by using an emulsion transfer method. These robust vesicles can sustain chemical and physical stresses. The peptide vesicles can host cell-free expression reactions by encapsulating essential ingredients. We show the incorporation of another cell-free expressed elastin-like polypeptide into the existing membrane of the peptide vesicles.

ß-arrestin mediates communication between plasma membrane and intracellular GPCRs to regulate signaling.

It has become increasingly apparent that G protein-coupled receptor (GPCR) localization is a master regulator of cell signaling. However, the molecular mechanisms involved in this process are not well understood. To date, observations of intracellular GPCR activation can be organized into two categories: a dependence on OCT3 cationic channel-permeable ligands or the necessity of endocytic trafficking. Using CXC chemokine receptor 4 (CXCR4) as a model, we identified a third mechanism of intracellular GPCR signaling. We show that independent of membrane permeable ligands and endocytosis, upon stimulation, plasma membrane and internal pools of CXCR4 are post-translationally modified and collectively regulate EGR1 transcription. We found that ß-arrestin-1 (arrestin 2) is necessary to mediate communication between plasma membrane and internal pools of CXCR4. Notably, these observations may explain that while CXCR4 overexpression is highly correlated with cancer metastasis and mortality, plasma membrane localization is not. Together these data support a model where a small initial pool of plasma membrane-localized GPCRs are capable of activating internal receptor-dependent signaling events.

In search of a novel chassis material for synthetic cells: emergence of synthetic peptide compartment.

Giant lipid vesicles have been used extensively as a synthetic cell model to recapitulate various life-like processes, including in vitro protein synthesis, DNA replication, and cytoskeleton organization. Cell-sized lipid vesicles are mechanically fragile in nature and prone to rupture due to osmotic stress, which limits their usability. Recently, peptide vesicles have been introduced as a synthetic cell model that would potentially overcome the aforementioned limitations. Peptide vesicles are robust, reasonably more stable than lipid vesicles and can withstand harsh conditions including pH, thermal, and osmotic variations. This mini-review summarizes the current state-of-the-art in the design, engineering, and realization of peptide-based chassis materials, including both experimental and computational work. We present an outlook for simulation-aided and data-driven design and experimental realization of engineered and multifunctional synthetic cells.

Shock wave impact on the viability of MDA-MB-231 cells.

Shock waves are gaining interests in biological and medical applications. In this work, we investigated the mechanical characteristics of shock waves that affect cell viability. In vitro testing was conducted using the metastatic breast epithelial cell line MDA-MB-231. Shock waves were generated using a high-power pulse laser. Two different coating materials and different laser energy levels were used to vary the peak pressure, decay time, and the strength of subsequent peaks of the shock waves. Within the testing capability of the current study, it is shown that shock waves with a higher impulse led to lower cell viability, a higher detached cell ratio, and a higher cell death ratio, while shock waves with the same peak pressure could lead to different levels of cell damage. The results also showed that the detached cells had a higher cell death ratio compared to the attached cells. Moreover, a critical shock impulse of 5 Pa·s was found to cause the cell death ratio of the detached cells to exceed 50%. This work has demonstrated that, within the testing range shown here, the impulse, rather than the peak pressure, is the governing shock wave parameter for the damage of MDA-MB-231 breast cancer cells. The result suggests that a lower-pressure shock wave with a longer duration, or multiple sequential low amplitude shock waves can be applied over a duration shorter than the fundamental response period of the cells to achieve the same impact as shock waves with a high peak pressure but a short duration. The finding that cell viability is better correlated with shock impulse rather than peak pressure has potential significant implications on how shock waves should be tailored for cancer treatments, enhanced drug delivery, and diagnostic techniques to maximize efficacy while minimizing potential side effects.

The Machado-Joseph disease-associated form of ataxin-3 impacts dynamics of clathrin-coated pits.

Expansion above a certain threshold in the polyglutamine (polyQ) tract of ataxin-3 is the main cause of neurodegeneration in Machado-Joseph disease. Ataxin-3 contains an N-terminal catalytic domain, called Josephin domain, and a highly aggregation-prone C-terminal domain containing the polyQ tract. Recent work has shown that protein aggregation inhibits clathrin-mediated endocytosis (CME). However, the effects of polyQ expansion in ataxin-3 on CME have not been investigated. We hypothesize that the expansion of the polyQ tract in ataxin-3 could impact CME. Here, we report that both the wild-type and the expanded ataxin-3 reduce transferrin internalization and expanded ataxin-3 impacts dynamics of clathrin-coated pits (CCPs) by reducing CCP nucleation and increasing short-lived abortive CCPs. Since endocytosis plays a central role in regulating receptor uptake and cargo release, our work highlights a potential mechanism linking protein aggregation to cellular dysregulation.

A Novel Synthetic Toehold Switch for MicroRNA Detection in Mammalian Cells.

MicroRNAs (miRNA or miR) are short noncoding RNA of about 21-23 nucleotides that play critical roles in multiple aspects of biological processes by mediating translational repression through targeting messenger RNA (mRNA). Conventional methods for miRNA detection, including RT-PCR and Northern blot, are limited due to the requirement of cell disruption. Here, we developed a novel synthetic toehold switch, inspired by the toehold switches developed for bacterial systems, to detect endogenous and exogenously expressed miRNAs in mammalian cells, including HEK 293, HeLa, and MDA-MB-231 cells. Transforming growth factor ß-induced miR-155 expression in MDA-MB-231 cells could be detected by the synthetic toehold switch. The experimental results showed the dynamic range of current design of toehold switch is about two. Furthermore, we tested multiplex detection of miR-155 and miR-21 in HEK 293 cells by using miR-155 and miR-21 toehold switches. These toehold switches provide a modest level of orthogonality and could be optimized to achieve a better dynamic range. Our experimental results demonstrate the capability of miRNA toehold switch for detecting and visualizing miRNA expression in mammalian cells, which may potentially lead to new therapeutic or diagnostic applications.

Loss of PTEN promotes formation of signaling-capable clathrin-coated pits.

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs) display highly heterogeneous dynamics on the plasma membrane where they can take from 20 s to over 1 min to form cytosolic coated vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], which is dephosphorylated by phosphatase and tensin homolog (PTEN), is a potent tumorigenic signaling lipid. By using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we found that EGF-bound EGFR and PTEN are enriched in a distinct subset of short-lived CCPs that correspond with clathrin-dependent EGF-induced signaling. We demonstrated that PTEN plays a role in the regulation of CCP dynamics. Furthermore, increased PI(3,4,5)P3 resulted in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Altogether, our findings provide evidence for the existence of short-lived 'signaling-capable' CCPs.

The effect of mechanosensitive channel MscL expression in cancer cells on 3D confined migration.

Metastatic cancer cells migrate through constricted spaces and experience significant compressive stress, but mechanisms enabling migration in confined geometries remain unclear. Cancer cell migration within confined 3-dimensional (3D) microfluidic channels has been shown to be distinct from 2D cell migration. However, whether 3D confined migration can be manipulated by mechanosensory components has not been examined in detail. In this work, we exogenously introduced a mechanosensitive channel of large conductance (MscL) into metastatic breast cancer cells MDA-MB-231. We discovered that inducing expression of a gain-of-function G22S mutant of MscL in MDA-MB-231 cells significantly reduced spontaneous lung metastasis without affecting the growth of orthotopic tumor implants. To further investigate the effects of G22S MscL on cell migration, we designed a microfluidic device with channels of various cross-sections ranging from a 2D planar environment to narrow 3D constrictions. Both MscL G22S and control breast cancer cells migrated progressively slower in more constricted environments. Migration of cells expressing MscL G22S did not differ from control cells, even though MscL was activated in cells in constricted channels of 3 µm width. Interestingly, we found MscL expressing cells to be more frequently "stuck" at the entrance of the 3 µm channels and failed to migrate into the microchannel. Our work demonstrates the possibility of engineering mechanotransduction for controlling confined cell migration.

Development of an advanced microfluidic micropipette aspiration device for single cell mechanics studies.

Various micro-engineered tools or platforms have been developed recently for cell mechanics studies based on acoustic, magnetic, and optical actuations. Compared with other techniques for single cell manipulations, microfluidics has the advantages with simple working principles and device implementations. In this work, we develop a multi-layer microfluidic pipette aspiration device integrated with pneumatically actuated microfluidic control valves. This configuration enables decoupling of cell trapping and aspiration, and hence causes less mechanical perturbation on trapped single cells before aspiration. A high trapping efficiency is achieved by the microfluidic channel design based on fluid resistance model and deterministic microfluidics. Compared to conventional micropipette aspiration, the suction pressure applied on the aspirating cells is highly stable due to the viscous nature of low Reynolds number flow. As a proof-of-concept of this novel microfluidic technology, we built a microfluidic pipette aspiration device with 2 × 13 trapping arrays and used this device to measure the stiffness of a human breast cancer cell line, MDA-MB-231, through the observation of cell deformations during aspiration. As a comparison, we studied the effect of Taxol, a FDA-approved anticancer drug on single cancer cell stiffness. We found that cancer cells treated with Taxol were less deformable with a higher Young's modulus. The multi-layer microfluidic pipette aspiration device is a scalable technology for single cell mechanophenotyping studies and drug discovery applications.

Engineering artificial cells by combining HeLa-based cell-free expression and ultrathin double emulsion template.

Generation of artificial cells provides the bridge needed to cover the gap between studying the complexity of biological processes in whole cells and studying these same processes in an in vitro reconstituted system. Artificial cells are defined as the encapsulation of biologically active material in a biological or synthetic membrane. Here, we describe a robust and general method to produce artificial cells for the purpose of mimicking one or more behaviors of a cell. A microfluidic double emulsion system is used to encapsulate a mammalian cell-free expression system that is able to express membrane proteins into the bilayer or soluble proteins inside the vesicles. The development of a robust platform that allows the assembly of artificial cells is valuable in understanding subcellular functions and emergent behaviors in a more cell-like environment as well as for creating novel signaling pathways to achieve specific cellular behaviors.

A microfluidic pipette array for mechanophenotyping of cancer cells and mechanical gating of mechanosensitive channels.

Micropipette aspiration measures the mechanical properties of single cells. A traditional micropipette aspiration system requires a bulky infrastructure and has a low throughput and limited potential for automation. We have developed a simple microfluidic device which is able to trap and apply pressure to single cells in designated aspiration arrays. By changing the volume flow rate using a syringe pump, we can accurately exert a pressure difference across the trapped cells for pipette aspiration. By examining cell deformation and protrusion length into the pipette under an optical microscope, several important cell mechanical properties, such as the cortical tension and the Young's modulus, can be measured quantitatively using automated image analysis. Using the microfluidic pipette array, the stiffness of breast cancer cells and healthy breast epithelial cells was measured and compared. Finally, we applied our device to examine the gating threshold of the mechanosensitive channel MscL expressed in mammalian cells. Together, the development of a microfluidic pipette array could enable rapid mechanophenotyping of individual cells and for mechanotransduction studies.

Cofactoring and dimerization of proteinase-activated receptors.

Proteinase-activated receptors (PARs) are G protein-coupled receptors that transmit cellular responses to extracellular proteases and have important functions in vascular physiology, development, inflammation, and cancer progression. The established paradigm for PAR activation involves proteolytic cleavage of the extracellular N terminus, which reveals a new N terminus that functions as a tethered ligand by binding intramolecularly to the receptor to trigger transmembrane signaling. Most cells express more than one PAR, which can influence the mode of PAR activation and signaling. Clear examples include murine PAR3 cofactoring of PAR4 and transactivation of PAR2 by PAR1. Thrombin binds to and cleaves murine PAR3, which facilitates PAR4 cleavage and activation. This process is essential for thrombin signaling and platelet activation, since murine PAR3 cannot signal alone. Although PAR1 and PAR4 are both competent to signal, PAR1 is able to act as a cofactor for PAR4, facilitating more rapid cleavage and activation by thrombin. PAR1 can also facilitate PAR2 activation through a different mechanism. Cleavage of the PAR1 N terminus by thrombin generates a tethered ligand domain that can bind intermolecularly to PAR2 to activate signaling. Thus, PARs can regulate each other's activity by localizing thrombin when in complex with PAR3 and PAR4 or by cleaved PAR1, providing its tethered ligand domain for PAR2 activation. The ability of PARs to cofactor or transactivate other PARs would necessitate that the two receptors be in close proximity, likely in the form of a heterodimer. Here, we discuss the cofactoring and dimerization of PARs and the functional consequences on signaling.

Local clustering of transferrin receptors promotes clathrin-coated pit initiation.

Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor-ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.

Multivalent display and receptor-mediated endocytosis of transferrin on virus-like particles.

The structurally regular and stable self-assembled capsids derived from viruses can be used as scaffolds for the display of multiple copies of cell- and tissue-targeting molecules and therapeutic agents in a convenient and well-defined manner. The human iron-transfer protein transferrin, a high affinity ligand for receptors upregulated in a variety of cancers, has been arrayed on the exterior surface of the protein capsid of bacteriophage Qbeta. Selective oxidation of the sialic acid residues on the glycan chains of transferrin was followed by introduction of a terminal alkyne functionality through an oxime linkage. Attachment of the protein to azide-functionalized Qbeta capsid particles in an orientation allowing access to the receptor binding site was accomplished by the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. Transferrin conjugation to Qbeta particles allowed specific recognition by transferrin receptors and cellular internalization through clathrin-mediated endocytosis, as determined by fluorescence microscopy on cells expressing GFP-labeled clathrin light chains. By testing Qbeta particles bearing different numbers of transferrin molecules, it was demonstrated that cellular uptake was proportional to ligand density, but that internalization was inhibited by equivalent concentrations of free transferrin. These results suggest that cell targeting with transferrin can be improved by local concentration (avidity) effects.

Global and local regulation of clathrin-coated pit dynamics detected on patterned substrates.

Live-cell imaging of individual clathrin-coated pit (CCP) dynamics has revealed a broad variation in their internalization kinetics, but the functional significance and mechanistic underpinnings of this heterogeneity remain unknown. One contributing factor may be the spatial variations in the underlying actin cortex. To test this, we cultured cells on fibronectin (Fn) micropatterned substrates to vary the cortical actin mechanics in a defined manner. Under these conditions, stress fibers became organized to bridge adhesive islands, creating spatial heterogeneity in the cortical actin architecture. CCP lifetimes within the Fn-coated islands were selectively prolonged. This differential effect was not due to adherence to Fn-coated surfaces, and was not observed in cells grown on patterned surfaces that did not induce organized stress fiber assembly. Pharmacological agents that lower cortical tension selectively lowered CCP lifetimes within Fn islands, thus abolishing the spatial heterogeneity in the CCP dynamics. Although we cannot rule out the possibility that other factors might locally affect CCP dynamics at Fn islands, our data suggest that localized modulation in cortical tension may spatially regulate clathrin-mediated endocytosis.