The tumor microenvironment plays a critical role in drug resistance, with extracellular matrix mechanics, cell-cell crosstalk, and transport barriers contributing to poor therapeutic outcomes. Traditional two-dimensional (2D) cultures fail to capture these features, and drug efficacy in 2D often does not translate to three-dimensional (3D) models orin vivotumors. Here, we present a 3D tumor model integrated with a high-throughput biomechanical sensor array that enables simultaneous measurement of cellular forces and matrix remodeling. The platform, fabricated using a scalable and cost-effective micro-milling approach, supports the parallel generation of multiple tumor constructs within a single dish. To demonstrate feasibility, we formedin vitrotumors using patient-derived pancreatic ductal adenocarcinoma organoids, cancer cells, and stromal fibroblasts. The sensors were then applied to characterize the evolving biophysical properties of these tumors (tissue force and stiffness) and to evaluate responses to chemotherapy drug, Gemcitabine, and the investigational agent, all-trans retinoic acid. Drug responses in 3D tumors were compared with those in 2D cultures. By combining biochemical and biomechanical readouts, this 3D platform provides a more physiologically relevant tumor model and a powerful tool for preclinical drug testing and personalized medicine.

Physical exercise promotes brain health in part through muscle-derived factors that enter the brain, but how peripheral signals are translated into neural responses remain unclear. Here, we identify astrocyte contraction as a previously unrecognized physiological response to exercise signals that may contribute to adult hippocampal neurogenesis. In vivo, voluntary running rapidly induced nuclear localization of the mechanically sensitive transcriptional regulator Yes-associated protein (YAP), and increased non-muscle myosin II phosphorylation in hilar astrocytes in mice, consistent with acute contraction. Using an in vitro platform with ultrasensitive force sensors, we found that factors released by contracting skeletal muscles activated astrocytes which in turn increased contraction that was necessary and sufficient for their proliferation and expansion. Activated astrocytes subsequently released soluble factors that modulated neuronal network tension and promoted immature neuron abundance. These findings identify astrocyte contractility as a physiological transducer of exercise-derived muscle signals and establish cellular force generation as a potential mechanism regulating neuroplasticity.

Colorectal cancer metastasis is governed by a variety of chemical and mechanical signaling that are largely influenced by cancer-associated fibroblasts (CAFs) in the tumor microenvironment. Here, we deconvolute the chemical from mechanical signaling in the case of the colon cancer cell line HCT-116 and CAFs. We examined three chemoattractants (CXCL12, TGF-β, and activin A) which allegedly are secreted by CAFs and induce HCT-116 cell migration. None of the chemoattractants tested resulted in enhanced migration of HCT-116 in a 2D transwell assay, at low cell density. Similarly, CAF-conditioned media also did not lead to enhanced HCT-116 migration, while CAFs co-cultured in the transwell assay did lead to increased HCT-116 migration. This result suggests that either high cell densities are required for chemotaxis, and/or a reciprocal two-way signaling network between CAFs and HCT-116 is necessary to induce chemotaxis. Surprisingly, we find that HCT-116 cells exhibit enhanced migration along the axis of mechanical stress in a 3D collagen matrix, at very high cell densities. This migration is independent of whether the strain is induced mechanically or by CAFs. By comparing purely mechanical and purely chemical migration to a 3D co-culture of CAFs and HCT-116 containing both chemical and mechanical cues, it is concluded that HCT-116 migration is dominated by mechanical signaling, while chemical cues are less influential.

Cell-matrix interactions, mediated by cellular force and matrix remodeling, result in a dynamic reciprocity that drives numerous biological processes and disease progression. Currently, there is no available method for direct quantification cell traction force and matrix remodeling in 3D matrices as a function of time. To address this long-standing need, we recently developed a high-resolution microfabricated sensor 1 that measures cell force, tissue-stiffness and can apply mechanical stimulation to the tissue. Here the tissue self-assembles and self-integrates with the sensor. With primary fibroblasts, cancer cells and neurons, we demonstrated the feasibility of the sensor by measuring single/multiple cell force with a resolution of 1 nN, and tissue stiffness 1 due to matrix remodeling by the cells. The sensor can be translated into a high-throughput system for clinical assays such as patient-specific drug and phenotypic screening. In this paper, we present the detailed protocol for manufacturing the sensors, preparing experimental setup, developing assays with different tissues, and for imaging and analyzing the data.

The field of biohybrid robotics focuses on using biological actuators to study the emergent properties of tissues and the locomotion of living organisms. On the basis of models of swimming at small size scales, we designed and fabricated a muscle-powered, flagellate swimmer. We investigate the design of a compliant mechanism based on nonlinear mechanics and its mechanical integration with a muscle ring and motor neurons. We find that within a range of anchor stiffnesses around 1 micronewton per micrometer, the homeostatic tension in muscle is insensitive to stiffness, offering greater design flexibility. The proximity of motor neurons results in a fourfold improvement in muscle contractility. Improved contractility and nonlinear design allow for a peak swimming speed about two orders of magnitude higher than previous biohybrid flagellate swimmers, reaching 0.58 body lengths per minute (86.8 micrometers per second), by a mechanism involving inertia that we verify through flow field imaging. This swimmer opens the door for a class of intermediate–Reynolds number swimmers.

Integration of biological actuators and sensors with soft, synthetic scaffolds has allowed for the development of a class of microscale, emergent, and motile biohybrid robots, including small scale swimmers and walkers. Swimmers generated thrust from time irreversible dynamics of their flagella-like thin compliant tails. However, these swimmers were limited in their wider application by slow speed (0.7 micrometers per second, 0.014 body lengths/min, with Re ~ 0.001). Low Reynolds number (Re) hydrodynamic theory, appropriate for locomotion with negligible inertial effects, predicts that swimming speed can be increased by orders of magnitude by increasing angular actuation of the tails of these swimmers. This study investigates a novel design for a fast swimmer achieving speeds up to 86.8 micrometers per second, 0.58 body lengths/minute. Here, living muscle tissue applies force on the swimmer scaffold upon electrical actuation. The compliant mechanism transduces the contraction to twisting of the tails by up to 7°, resulting in high speeds. Muscle maturity was modulated through coculture with motor neurons and mechanical coupling to the compliant swimmer scaffold. Although the design was motivated by low Reynolds number theory, the swimmer achieves high speed benefiting from inertial effects with Re ~ 0.1. Our study provides a simple design for generating high thrust using bio-actuators at small scale and a design for introducing neurons into biohybrid systems with 3D muscle tissues. ### Competing Interest Statement The authors have declared no competing interest.

Understanding the molecular and physical complexity of the tissue microenvironment (TiME) in the context of its spatiotemporal organization has remained an enduring challenge. Recent advances in engineering and data science are now promising the ability to study the structure, functions, and dynamics of the TiME in unprecedented detail; however, many advances still occur in silos that rarely integrate information to study the TiME in its full detail. This review provides an integrative overview of the engineering principles underlying chemical, optical, electrical, mechanical, and computational science to probe, sense, model, and fabricate the TiME. In individual sections, we first summarize the underlying principles, capabilities, and scope of emerging technologies, the breakthrough discoveries enabled by each technology and recent, promising innovations. We provide perspectives on the potential of these advances in answering critical questions about the TiME and its role in various disease and developmental processes. Finally, we present an integrative view that appreciates the major scientific and educational aspects in the study of the TiME.

Despite considerable advances to improve colorectal cancer (CRC) survival over the last decade, therapeutic challenges remain due to the rapid metastatic dissemination of primary tumors and screening limitations. Meanwhile, the rise of CRC in younger adults (Early-onset CRC), commonly diagnosed with a metastatic form of the disease, shows the pressing need to develop more effective targeted therapies to decrease the high mortality rates associated with metastatic disease. Hyperactivation of the Rictor-mTORC2-AKT signaling pathway drives key metastatic players in diverse malignant tumors, including early- and late-onset colorectal cancer. Selective mTORC2 inhibitors are becoming a potential treatment strategy for CRC due to the therapeutic limitations of mTORC1 inhibitors. Veratridine (VTD), a lipid-soluble alkaloid extracted from Liliaceae plants, can transcriptionally increase UBXN2A, which induces 26S proteasomal degradation of the Rictor protein, a key member in the mTORC2 complex. Destabilization of Rictor protein by VTD decreases Akt phosphorylation on Ser473, which is responsible for metastatic signaling downstream of the mTORC2 pathway in diverse malignant tumors. VTD decreases the population of metastatic colon cancer stem cells and functions as an angiogenesis inhibitor. VTD effectively reduces the spheroid growth rate and restricts cell migration. Live cell migration and invasion assays alongside biomechanical-force-based experiments revealed that VTD suppresses colon cancer cell invasiveness and the ensuing risk of tumor metastasis. A CRC mouse model that mimics the natural stages of human sporadic CRC revealed that VTD treatment significantly decreases tumor growth in a UBXN2A-dependent manner. This study showed a novel mechanistic connection between a ubiquitin-like protein and mTORC2-dependent migration and invasion in CRC tumors. This study revealed the therapeutic benefit of selective inhibition of Rictor in CRC, particularly in tumors with a hyperactive Rictor-mTORC2 signaling pathway. Finally, this study opened a new platform for repurposing VTD, a supplemental anti-hypertension molecule, into an effective targeted therapy in CRC tumors.

Cells cultured on stiff 2D substrates exert high intracellular force, resulting in mechanical deformation of their nuclei. This nuclear deformation (ND) plays a crucial role in the transport of Yes Associated Protein (YAP) from the cytoplasm to the nucleus. However, cells in vivo are in soft 3D environment with potentially much lower intracellular forces. Whether and how cells may deform their nuclei in 3D for YAP localization remains unclear. Here, by culturing human colon cancer associated fibroblasts (CAFs) on 2D, 2.5D, and 3D substrates, we differentiated the effects of stiffness, force, and ND on YAP localization. We found that nuclear translocation of YAP depends on the degree of ND irrespective of dimensionality, stiffness and total force. ND induced by the perinuclear force, not the total force, and nuclear membrane curvature correlate strongly with YAP activation. Immunostained slices of human tumors further supported the association between ND and YAP nuclear localization, suggesting ND as a potential biomarker for YAP activation in tumors. Additionally, we conducted quantitative analysis of the force dynamics of CAFs on 2D substrates to construct a stochastic model of YAP kinetics. This model revealed that the probability of YAP nuclear translocation, as well as the residence time in the nucleus follow a power law. This study provides valuable insights into the regulatory mechanisms governing YAP dynamics and highlights the significance of threshold activation in YAP localization.

Neurons in the brain communicate with each other at their synapses. It has long been understood that this communication occurs through biochemical processes. Here, we reveal that mechanical tension in neurons is essential for communication. Using in vitro rat hippocampal neurons, we find that 1) neurons become tout/tensed after forming synapses resulting in a contractile neural network, and 2) without this contractility, neurons fail to fire. To measure time evolution of network contractility in 3D (not 2D) extracellular matrix, we developed an ultrasensitive force sensor with 1 nN resolution. We employed Multi-Electrode Array and iGluSnFR, a glutamate sensor, to quantify neuronal firing at the network and at the single synapse scale, respectively. When neuron contractility is relaxed, both techniques show significantly reduced firing. Firing resumes when contractility is restored. This finding highlights the essential contribution of neural contractility in fundamental brain functions and has implications for our understanding of neural physiology.

Most solid tumors become stiff with progression of cancer. Cancer Associated Fibroblasts (CAFs), most abundant stromal cells in the tumor microenvironment (TME), are known to mediate such stiffening. While the biochemical crosstalk between CAFs and cancer cells have been widely investigated, it is not clear if and how CAFs in stiffer TME promote metastatic progression. To gather insights into the process, we controlled the mechanical stiffness of the substrates and collected gene expression data with human colorectal CAFs. We cultured human primary CAFs on 2D polyacrylamide hydrogels with increasing elastic modulus (E) of 1, 10 and 40 kPa, and performed genome-wide transcriptome analyses in these cells to identify expression levels of ~16000 genes. The high-quality RNAseq results can be an excellent data-source for bioinformatic analysis for identifying novel pathways and biomarkers in cancer development and metastatic progression. With thorough analysis and accurate interpretation, this data may help researchers understand the role of mechanical stiffness of the TME in CAF-cancer cell crosstalk.

Intravital microscopy reveals that the transmission of solid stresses within tumours depends on tumour size and on the tumour microenvironment.

Cells in functional tissues execute various collective activities to achieve diverse ordered processes including wound healing, organogenesis, and tumor formation. How a group of individually operating cells initiate such complex collective processes is still not clear. Here, we report that cells in 3D extracellular matrix (ECM) initiate collective behavior by forming cell-ECM network when the cells are within a critical distance from each other. We employed compaction of free-floating (FF) 3D collagen gels with embedded fibroblasts as a model system to study collective behavior and found a sharp transition in the amount of compaction as a function of cell-cell distance, reminiscent of phase transition in materials. Within the critical distance, cells remodel the ECM irreversibly, and form dense collagen bridges between each other resulting in the formation of a network. Beyond the critical distance, cells exhibit Brownian dynamics and only deform the matrix reversibly in a transient manner with no memory of history, thus maintaining the disorder. Network formation seems to be a necessary and sufficient condition to trigger collective behavior and a disorder-to order transition. STATEMENT OF SIGNIFICANCE: Macroscopic compaction of in vitro collagen gels is mediated by collective mechanical interaction of cells. Previous studies on cell-induced ECM compaction suggest the existence of a critical cell density and phase transition associated with this phenomenon. Cell-mediated mechanical remodeling and global compaction of ECM has mostly been studied at steady state. Our study reveals a link between a transition in cell dynamics and material microstructure as cells collectively compact collagen gels. It underscores the significance of temporal evolution of these cell-ECM systems in understanding the mechanism of such collective action and provides insights on the process from a mechanistic viewpoint. These insights can be valuable in understanding dynamic pathological processes such as, cancer progression and wound healing, as well as engineering biomaterials and regenerative tissue mimics.

Microelectromechanical systems (MEMS) are essential components in many electronic technologies for consumer and industrial applications. Such devices are typically made using materials selected to support long operational lifetimes, but MEMS designed to physically disintegrate or to dissolve after a targeted period could provide a route to reduce electronic waste and could enable applications that require a finite operating timeframe, such as temporary medical implants. Here we report ecoresorbable and bioresorbable MEMS that are based on fully water-soluble material platforms and can either naturally resorb into the environment to eliminate solid waste or in the body to avoid a need for surgical extraction. We illustrate the biocompatibility of the approach with mechanobiology, histology and haematology studies of the implanted devices and their dissolution end products. We also demonstrate bioresorbable encapsulating materials and deployment strategies in small animal models to reduce device damage, confine mobile fragments and provide robust adhesion with adjacent tissues.

Quantitative assessment of soft tissue elasticity is crucial to a broad range of applications, such as biomechanical modeling, physiological monitoring, and tissue diseases diagnosing. However, the modulus measurement of soft tissues, particularly in vivo, has proved challenging since the instrument has to reach the site of soft tissue and be able to measure in a very short time. Here, we present a simple method to measure the elastic modulus of soft tissues on site by exploiting buckling of a long slender bar to quantify the applied force and a spherical indentation to extract the elastic modulus. The method is realized by developing a portable pen-sized instrument (EPen: Elastic modulus pen). The measurement accuracies are verified by independent modulus measures using commercial nanoindenter. Quantitative measurements of the elastic modulus of mouse pancreas, healthy and cancerous, surgically exposed but attached to the body further confirm the potential clinical utility of the EPen.

Cells in vivo generate mechanical traction on the surrounding 3D extracellular matrix (ECM) and neighboring cells. Such traction and biochemical cues may remodel the matrix, e.g., increase stiffness, which, in turn, influences cell functions and forces. This dynamic reciprocity mediates development and tumorigenesis. Currently, there is no method available to directly quantify single-cell forces and matrix remodeling in 3D. Here, we introduce a method to fulfill this long-standing need. We developed a high-resolution microfabricated sensor that hosts a 3D cell-ECM tissue formed by self-assembly. This sensor measures cell forces and tissue stiffness and can apply mechanical stimulation to the tissue. We measured single and multicellular force dynamics of fibroblasts (3T3), human colon (FET) and lung (A549) cancer cells, and cancer-associated fibroblasts (CAF05) with 1-nN resolution. Single cells show notable force fluctuations in 3D. FET/CAF coculture system, mimicking cancer tumor microenvironment, increased tissue stiffness by three times within 24 hours.

Fluorescent microscopy employs monochromatic light which can affect the cells being observed. We reported earlier that fibroblasts relax their contractile force in response to green light of typical intensity. Here we show that such effects are independent of extracellular matrix and type of cell. In addition, we establish a threshold light that invokes minimal effect on cells. We cultured fibroblasts on soft 2D elastic hydrogels embedded with fluorescent beads to trace substrate deformation. The beads move towards cell center when cells contract, but they move away when cells relax. We use relaxation/contraction ratio, λ r, as a measure of cell response to light. The cells were exposed to green (wavelength, λ = 545-580 nm) and red ( λ = 635-650 nm) light with a range of intensities. We find red light with intensity less than ~ 57 W/m2 results in λ r = 1, i.e., cells maintain force homeostasis. Higher intensities and smaller wavelengths result in widespread force-relaxation in cells with λ r > 1. We suggest the use of λ > 650 nm light with low intensity ( I ≤ 57 W/m2) for time-lapse imaging of cells and tissues in order to avoid light-induced artifacts in experimental observations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Respiratory infections may spread through droplets and aerosols released by infected individuals coughing, sneezing, or speaking. In the case of Coronavirus Disease 2019 (COVID-19), spread can occur from symptomatic, pre-symptomatic, and asymptomatic persons. Given the limited supply of professional face masks and respirators, the U.S. Centers for Disease Control and Prevention (CDC) has recommended home-made cloth face coverings for use by the general public in areas of significant community-based transmission. There is, however, little information on the effectiveness of cloth face coverings in reducing droplet dissemination. Here, we ascertained the performance of 11 household fabrics at blocking high-velocity droplets, using a commercial medical mask as a benchmark. We also assessed their breathability (air permeability), texture, fiber composition, and water absorption properties. We found that droplet blocking efficiency anti-correlates with breathability; less breathable fabrics being more effective in blocking. However, materials with high breathability are desirable for comfort and to reduce airflow through gaps between the mask and face. Our measurements indicate that 2 or 3 layers of highly permeable fabric, such as T-shirt cloth, may block droplets with an efficacy similar to that of medical masks, while still maintaining comparable breathability. Overall, our study suggests that cloth face coverings, especially with multiple layers, may help reduce droplet transmission of respiratory infections. Furthermore, face coverings made from biodegradable fabrics such as cotton allow washing and reusing, and can help reduce the adverse environmental effects of widespread use of commercial disposable and non-biodegradable facemasks.

Coronavirus Disease 2019 (COVID-19) may spread through respiratory droplets released by infected individuals during coughing, sneezing, or speaking. Given the limited supply of professional respirators and face masks, the U.S. Centers for Disease Control and Prevention (CDC) has recommended home-made cloth face coverings for use by the general public. While there have been several studies on aerosol filtration performance of household fabrics, their effectiveness at blocking larger droplets has not been investigated. Here, we ascertained the performance of 11 common household fabrics at blocking large, high-velocity droplets, using a commercial medical mask as a benchmark. We also assessed the breathability (air permeability), texture, fiber composition, and water absorption properties of the fabrics. We found that most fabrics have substantial blocking efficiency (median values >70%). In particular, two layers of highly permeable fabric, such as T-shirt cloth, blocks droplets with an efficiency (>94%) similar to that of medical masks, while being approximately twice as breathable. The first layer allows about 17% of the droplet volume to transmit, but it significantly reduces their velocity. This allows the second layer to trap the transmitted droplets resulting in high blocking efficacy. Overall, our study suggests that cloth face coverings, especially with multiple layers, may help reduce droplet transmission of respiratory infections. Furthermore, face coverings made from materials such as cotton fabrics allow washing and reusing, and can help reduce the adverse environmental effects of widespread use of commercial disposable and non-biodegradable facemasks.