Inspiring a convergent engineering approach to measure and model the tissue microenvironment.

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.

Nuclear deformation regulates YAP dynamics in cancer associated fibroblasts.

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. STATEMENT OF SIGNIFICANCE: Yes Associated Protein (YAP), a transcription cofactor, has been identified as one of the drivers of cancer progression. High tumor stiffness is attributed to driving YAP to the nucleus, wherein it activates pro-metastatic genes. Here we show, using cancer associated fibroblasts, that YAP translocation to the nucleus depends on the degree of nuclear deformation, irrespective of stiffness. We also identified that perinuclear force induced membrane curvature correlates strongly with YAP nuclear transport. A novel stochastic model of YAP kinetics unveiled a power law relationship between the activation threshold and persistence time of YAP in the nucleus. Overall, this study provides novel insights into the regulatory mechanisms governing YAP dynamics and the probability of activation that is of immense clinical significance.

Synapses without tension fail to fire in an in vitro network of hippocampal neurons.

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.

Mechanosensitive changes in the expression of genes in colorectal cancer-associated fibroblasts.

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.

Disorder to order transition in cell-ECM systems mediated by cell-cell collective interactions.

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.

A novel method for sensor-based quantification of single/multicellular force dynamics and stiffening in 3D matrices.

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.

A portable pen-sized instrumentation to measure stiffness of soft tissues in vivo.

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.

Performance of fabrics for home-made masks against the spread of COVID-19 through droplets: A quantitative mechanistic study.

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.

Biophysics of Tumor Microenvironment and Cancer Metastasis - A Mini Review.

The role of tumor microenvironment in cancer progression is gaining significant attention. It is realized that cancer cells and the corresponding stroma co-evolve with time. Cancer cells recruit and transform the stromal cells, which in turn remodel the extra cellular matrix of the stroma. This complex interaction between the stroma and the cancer cells results in a dynamic feed-forward/feed-back loop with biochemical and biophysical cues that assist metastatic transition of the cancer cells. Although biochemistry has long been studied for the understanding of cancer progression, biophysical signaling is emerging as a critical paradigm determining cancer metastasis. In this mini review, we discuss the role of one of the biophysical cues, mostly the mechanical stiffness of tumor microenvironment, in cancer progression and its clinical implications.