Oscillatory ERK Signaling and Morphology Determine Heterogeneity of Breast Cancer Cell Chemotaxis via MEK-ERK and p38-MAPK Signaling Pathways.

Chemotaxis, regulated by oscillatory signals, drives critical processes in cancer metastasis. Crucial chemoattractant molecules in breast cancer, CXCL12 and EGF, drive the activation of ERK and Akt. Regulated by feedback and crosstalk mechanisms, oscillatory signals in ERK and Akt control resultant changes in cell morphology and chemotaxis. While commonly studied at the population scale, metastasis arises from small numbers of cells that successfully disseminate, underscoring the need to analyze processes that cancer cells use to connect oscillatory signaling to chemotaxis at single-cell resolution. Furthermore, little is known about how to successfully target fast-migrating cells to block metastasis. We investigated to what extent oscillatory networks in single cells associate with heterogeneous chemotactic responses and how targeted inhibitors block signaling processes in chemotaxis. We integrated live, single-cell imaging with time-dependent data processing to discover oscillatory signal processes defining heterogeneous chemotactic responses. We identified that short ERK and Akt waves, regulated by MEK-ERK and p38-MAPK signaling pathways, determine the heterogeneous random migration of cancer cells. By comparison, long ERK waves and the morphological changes regulated by MEK-ERK signaling, determine heterogeneous directed motion. This study indicates that treatments against chemotaxis in consider must interrupt oscillatory signaling.

Ultrasound-Induced Mechanical Compaction in Acoustically Responsive Scaffolds Promotes Spatiotemporally Modulated Signaling in Triple Negative Breast Cancer.

Cancer cells continually sense and respond to mechanical cues from the extracellular matrix (ECM). Interaction with the ECM can alter intracellular signaling cascades, leading to changes in processes that promote cancer cell growth, migration, and survival. The present study used a recently developed composite hydrogel composed of a fibrin matrix and phase-shift emulsion, termed an acoustically responsive scaffold (ARS), to investigate effects of local mechanical properties on breast cancer cell signaling. Treatment of ARSs with focused ultrasound drives acoustic droplet vaporization (ADV) in a spatiotemporally controlled manner, inducing local compaction and stiffening of the fibrin matrix adjacent to the matrix-bubble interface. Combining ARSs and live single cell imaging of triple-negative breast cancer cells, it is discovered that both basal and growth-factor stimulated activities of protein kinase B (also known as Akt) and extracellular signal-regulated kinase (ERK), two major kinases driving cancer progression, negatively correlate with increasing distance from the ADV-induced bubble both in vitro and in a mouse model. Together, these data demonstrate that local changes in ECM compaction regulate Akt and ERK signaling in breast cancer and support further applications of the novel ARS technology to analyze spatial and temporal effects of ECM mechanics on cell signaling and cancer biology.

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.

Physiologic biomechanics enhance reproducible contractile development in a stem cell derived cardiac muscle platform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) allow investigations in a human cardiac model system, but disorganized mechanics and immaturity of hPSC-CMs on standard two-dimensional surfaces have been hurdles. Here, we developed a platform of micron-scale cardiac muscle bundles to control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips on two-dimensional elastomer substrates with far greater throughput than single cell methods. By defining geometry and workload in this reductionist platform, we show that myofibrillar alignment and auxotonic contractions at physiologic workload drive maturation of contractile function, calcium handling, and electrophysiology. Using transcriptomics, reporter hPSC-CMs, and quantitative immunofluorescence, these cardiac muscle bundles can be used to parse orthogonal cues in early development, including contractile force, calcium load, and metabolic signals. Additionally, the resultant organized biomechanics facilitates automated extraction of contractile kinetics from brightfield microscopy imaging, increasing the accessibility, reproducibility, and throughput of pharmacologic testing and cardiomyopathy disease modeling.

Human induced pluripotent stem cell-derived lung organoids in an ex vivo model of the congenital diaphragmatic hernia fetal lung.

Three-dimensional lung organoids (LOs) derived from pluripotent stem cells have the potential to enhance our understanding of disease mechanisms and to enable novel therapeutic approaches in neonates with pulmonary disorders. We established a reproducible ex vivo model of lung development using transgene-free human induced pluripotent stem cells generated from fetuses and infants with Bochdalek congenital diaphragmatic hernia (CDH), a polygenic disorder associated with fetal lung compression and pulmonary hypoplasia at birth. Molecular and cellular comparisons of CDH LOs revealed impaired generation of NKX2.1+ progenitors, type II alveolar epithelial cells, and PDGFRα+ myofibroblasts. We then subjected these LOs to disease relevant mechanical cues through ex vivo compression and observed significant changes in genes associated with pulmonary progenitors, alveolar epithelial cells, and mesenchymal fibroblasts. Collectively, these data suggest both primary cell-intrinsic and secondary mechanical causes of CDH lung hypoplasia and support the use of this stem cell-based approach for disease modeling in CDH.

Evaluation of Continuous Lactate Monitoring Systems within a Heparinized In Vivo Porcine Model Intravenously and Subcutaneously.

We present an animal model used to evaluate the in vivo performance of electrochemical amperometric continuous lactate sensors compared to blood gas instruments. Electrochemical lactate sensors were fabricated, placed into 5 Fr central venous catheters (CVCs), and paired with wireless potentiostat devices. Following in vivo evaluation and calibration, sensors were placed within the jugular and femoral veins of a porcine subject as a preliminary assessment of in vivo measurement accuracy. The mobile electronic circuit potentiostat devices supplied the operational voltage for the sensors, measured the resultant steady-state current, and recorded the sensor response values in internal memory storages. An in vivo time trace of implanted intravenous (IV) sensors demonstrated lactate values that correlated well with the discrete measurements of blood samples on a benchtop point-of-care sensor-based instrument. Currents measured continuously from the implanted lactate sensors over 10 h were converted into lactate concentration values through use of a two-point in vivo calibration. Study shows that intravenously implanted sensors had more accurate readings, faster peak-reaching rates, and shorter peak-detection times compared to subcutaneously placed sensors. IV implanted and subcutaneously placed sensors closer to the upper body (in this case neck) showed faster response rates and more accurate measurements compared to those implanted in the lower portion of the porcine model. This study represents an important milestone not only towards continuous lactate monitoring for early diagnosis and intervention in neonatal patients with congenital heart disease undergoing cardiopulmonary bypass surgeries, but also in the intervention of critical ill patients in the Intensive Care Units or during complex surgical procedures.

Advanced Microfluidic Device Designed for Cyclic Compression of Single Adherent Cells.

Cells in our body experience different types of stress including compression, tension, and shear. It has been shown that some cells experience permanent plastic deformation after a mechanical tensile load was removed. However, it was unclear whether cells are plastically deformed after repetitive compressive loading and unloading. There have been few tools available to exert cyclic compression at the single cell level. To address technical challenges found in a previous microfluidic compression device, we developed a new single-cell microfluidic compression device that combines an elastomeric membrane block geometry to ensure a flat contact surface and microcontact printing to confine cell spreading within cell trapping chambers. The design of the block geometry inside the compression chamber was optimized by using computational simulations. Additionally, we have implemented step-wise pneumatically controlled cell trapping to allow more compression chambers to be incorporated while minimizing mechanical perturbation on trapped cells. Using breast epithelial MCF10A cells stably expressing a fluorescent actin marker, we successfully demonstrated the new device design by separately trapping single cells in different chambers, confining cell spreading on microcontact printed islands, and applying cyclic planar compression onto single cells. We found that there is no permanent deformation after a 0.5 Hz cyclic compressive load for 6 min was removed. Overall, the development of the single-cell compression microfluidic device opens up new opportunities in mechanobiology and cell mechanics studies.

Fetal lung transcriptome patterns in an ex vivo compression model of diaphragmatic hernia.

BACKGROUND: The purpose of this study was to employ a novel ex vivo lung model of congenital diaphragmatic hernia (CDH) to determine how a mechanical compression affects early pulmonary development. METHODS: Day-15 whole fetal rat lungs (n = 6-12/group) from nitrofen-exposed and normal (vehicle only) dams were explanted and cultured ex vivo in compression microdevices (0.2 or 0.4 kPa) for 16 h to mimic physiologic compression forces that occur in CDH in vivo. Lungs were evaluated with significance set at P < 0.05. RESULTS: Nitrofen-exposed lungs were hypoplastic and expressed lower levels of surfactant protein C at baseline. Although compression alone did not alter the α-smooth muscle actin (ACTA2) expression in normal lungs, nitrofen-exposed lungs had significantly increased ACTA2 transcripts (0.2 kPa: 2.04 ± 0.15; 0.4 kPa: 2.22 ± 0.11; both P < 0.001). Nitrofen-exposed lungs also showed further reductions in surfactant protein C expression at 0.2 and 0.4 kPa (0.53 ± 0.04, P < 0.01; 0.69 ± 0.23, P < 0.001; respectively). Whereas normal lungs exposed to 0.2 and 0.4 kPa showed significant increases in periostin (POSTN), a mechanical stress-response molecule (1.79 ± 0.10 and 2.12 ± 0.39, respectively; both P < 0.001), nitrofen-exposed lungs had a significant decrease in POSTN expression (0.4 kPa: 0.67 ± 0.15, P < 0.001), which was confirmed by immunohistochemistry. CONCLUSIONS: Collectively, these pilot data in a model of CDH lung hypoplasia suggest a primary aberration in response to mechanical stress within the nitrofen lung, characterized by an upregulation of ACTA2 and a downregulation in SPFTC and POSTN. This ex vivo compression system may serve as a novel research platform to better understand the mechanobiology and complex regulation of matricellular dynamics during CDH fetal lung development.

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.

Protein aggregation with poly(vinyl) alcohol surfactant reduces double emulsion-encapsulated mammalian cell-free expression.

Development of artificial cell models requires encapsulation of biomolecules within membrane-bound compartments. There have been limited studies of using mammalian cell-free expression (CFE) system as the 'cytosol' of artificial cells. We exploit glass capillary droplet microfluidics for the encapsulation of mammalian CFE within double emulsion templated vesicles. The complexity of the physicochemical properties of HeLa cell-free lysate poses a challenge compared with encapsulating simple buffer solutions. In particular, we discovered the formation of aggregates in double emulsion templated vesicles encapsulating mammalian HeLa CFE, but not with bacterial CFE. The aggregates did not arise from insolubility of the proteins made from CFE nor due to the interaction of mammalian CFE with the organic solvents in the middle phase of the double emulsions. We found that aggregation is dependent on the concentration of poly(vinyl) alcohol (PVA) surfactant, a critical double emulsion-stabilizing surfactant, and the lysate concentration in mammalian CFE. Despite vesicle instability and reduced protein expression, we demonstrate protein expression by encapsulating mammalian CFE system. Using mass spectrometry and Western blot, we identified and verified that actin is one of the proteins inside the mammalian CFE that aggregated with PVA surfactant. Our work establishes a baseline description of mammalian CFE system encapsulated in double emulsion templated vesicles as a platform for building artificial cells.

Mechanically activated artificial cell by using microfluidics.

All living organisms sense mechanical forces. Engineering mechanosensitive artificial cell through bottom-up in vitro reconstitution offers a way to understand how mixtures of macromolecules assemble and organize into a complex system that responds to forces. We use stable double emulsion droplets (aqueous/oil/aqueous) to prototype mechanosensitive artificial cells. In order to demonstrate mechanosensation in artificial cells, we develop a novel microfluidic device that is capable of trapping double emulsions into designated chambers, followed by compression and aspiration in a parallel manner. The microfluidic device is fabricated using multilayer soft lithography technology, and consists of a control layer and a deformable flow channel. Deflections of the PDMS membrane above the main microfluidic flow channels and trapping chamber array are independently regulated pneumatically by two sets of integrated microfluidic valves. We successfully compress and aspirate the double emulsions, which result in transient increase and permanent decrease in oil thickness, respectively. Finally, we demonstrate the influx of calcium ions as a response of our mechanically activated artificial cell through thinning of oil. The development of a microfluidic device to mechanically activate artificial cells creates new opportunities in force-activated synthetic biology.

Cell-free compartmentalized protein synthesis inside double emulsion templated liposomes with in vitro synthesized and assembled ribosomes.

A cell-free expression platform for making bacterial ribosomes encapsulated within giant liposomes was capable of synthesizing sfGFP. The liposomes were prepared using a double emulsion template, and compartmentalized in vitro protein synthesis was analysed using spinning disk confocal microscopy. Two different liposome phospholipid formulations were investigated to characterize their effects on the compartmentalized reaction kinetics. This study was performed as a necessary step towards the synthesis of minimal cells.

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.

Interferometric time-stretch microscopy for ultrafast quantitative cellular and tissue imaging at 1 µm.

Quantitative phase imaging (QPI) has been proven to be a powerful tool for label-free characterization of biological specimens. However, the imaging speed, largely limited by the image sensor technology, impedes its utility in applications where high-throughput screening and efficient big-data analysis are mandated. We here demonstrate interferometric time-stretch (iTS) microscopy for delivering ultrafast quantitative phase cellular and tissue imaging at an imaging line-scan rate >20 MHz­orders-of-magnitude faster than conventional QPI. Enabling an efficient time-stretch operation in the 1-µm wavelength window, we present an iTS microscope system for practical ultrafast QPI of fixed cells and tissue sections, as well as ultrafast flowing cells (at a flow speed of up to 8 m∕s). To the best of our knowledge, this is the first time that time-stretch imaging could reveal quantitative morphological information of cells and tissues with nanometer precision. As many parameters can be further extracted from the phase and can serve as the intrinsic biomarkers for disease diagnosis, iTS microscopy could find its niche in high-throughput and high-content cellular assays (e.g., imaging flow cytometry) as well as tissue refractometric imaging (e.g., whole-slide imaging for digital pathology).

Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow.

Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity--a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry--permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay.