The role of cell-matrix adhesion and cell migration in breast tumor growth and progression.

During breast cancer progression, there is typically increased collagen deposition resulting in elevated extracellular matrix rigidity. This results in changes to cell-matrix adhesion and cell migration, impacting processes such as the epithelial-mesenchymal transition (EMT) and metastasis. We aim to investigate the roles of cell-matrix adhesion and cell migration on breast tumor growth and progression by studying the impacts of different types of extracellular matrices and their rigidities. We embedded MCF7 spheroids within three-dimensional (3D) collagen matrices and agarose matrices. MCF7 cells adhere to collagen but not agarose. Contrasting the results between these two matrices allows us to infer the role of cell-matrix adhesion. We found that MCF7 spheroids exhibited the fastest growth rate when embedded in a collagen matrix with a rigidity of 5.1 kPa (0.5 mg/mL collagen), whereas, for the agarose matrix, the rigidity for the fastest growth rate is 15 kPa (1.0% agarose) instead. This discrepancy is attributable to the presence of cell adhesion molecules in the collagen matrix, which initiates collagen matrix remodeling and facilitates cell migration from the tumor through the EMT. As breast tumors do not adhere to agarose matrices, it is suitable to simulate the cell-cell interactions during the early stage of breast tumor growth. We conducted further analysis to characterize the stresses exerted by the expanding spheroid on the agarose matrix. We identified two distinct MCF7 cell populations, namely, those that are non-dividing and those that are dividing, which exerted low and high expansion stresses on the agarose matrix, respectively. We confirmed this using Western blot which showed the upregulation of proliferating cell nuclear antigen, a proliferation marker, in spheroids grown in the 1.0% agarose (≈13 kPa). By treating the embedded MCF7 spheroids with an inhibitor or activator of myosin contractility, we showed that the optimum spheroids' growth can be increased or decreased, respectively. This finding suggests that tumor growth in the early stage, where cell-cell interaction is more prominent, is determined by actomyosin tension, which alters cell rounding pressure during cell division. However, when breast tumors begin generating collagen into the surrounding matrix, collagen remodeling triggers EMT to promote cell migration and invasion, ultimately leading to metastasis.

Zyxin regulates embryonic stem cell fate by modulating mechanical and biochemical signaling interface.

Biochemical signaling and mechano-transduction are both critical in regulating stem cell fate. How crosstalk between mechanical and biochemical cues influences embryonic development, however, is not extensively investigated. Using a comparative study of focal adhesion constituents between mouse embryonic stem cell (mESC) and their differentiated counterparts, we find while zyxin is lowly expressed in mESCs, its levels increase dramatically during early differentiation. Interestingly, overexpression of zyxin in mESCs suppresses Oct4 and Nanog. Using an integrative biochemical and biophysical approach, we demonstrate involvement of zyxin in regulating pluripotency through actin stress fibres and focal adhesions which are known to modulate cellular traction stress and facilitate substrate rigidity-sensing. YAP signaling is identified as an important biochemical effector of zyxin-induced mechanotransduction. These results provide insights into the role of zyxin in the integration of mechanical and biochemical cues for the regulation of embryonic stem cell fate.

Integration of a microfluidic multicellular coculture array with machine learning analysis to predict adverse cutaneous drug reactions.

Adverse cutaneous reactions are potentially life-threatening skin side effects caused by drugs administered into the human body. The availability of a human-specific in vitro platform that can prospectively screen drugs and predict this risk is therefore of great importance to drug safety. However, since adverse cutaneous drug reactions are mediated by at least 2 distinct mechanisms, both involving systemic interactions between liver, immune and dermal tissues, existing in vitro skin models have not been able to comprehensively recapitulate these complex, multi-cellular interactions to predict the skin-sensitization potential of drugs. Here, we report a novel in vitro drug screening platform, which comprises a microfluidic multicellular coculture array (MCA) to model different mechanisms-of-action using a collection of simplistic cellular assays. The resultant readouts are then integrated with a machine-learning algorithm to predict the skin sensitizing potential of systemic drugs. The MCA consists of 4 cell culture compartments connected by diffusion microchannels to enable crosstalk between hepatocytes that generate drug metabolites, antigen-presenting cells (APCs) that detect the immunogenicity of the drug metabolites, and keratinocytes and dermal fibroblasts, which collectively determine drug metabolite-induced FasL-mediated apoptosis. A single drug screen using the MCA can simultaneously generate 5 readouts, which are integrated using support vector machine (SVM) and principal component analysis (PCA) to classify and visualize the drugs as skin sensitizers or non-skin sensitizers. The predictive performance of the MCA and SVM classification algorithm is then validated through a pilot screen of 11 drugs labelled by the US Food and Drug Administration (FDA), including 7 skin-sensitizing and 4 non-skin sensitizing drugs, using stratified 4-fold cross-validation (CV) on SVM. The predictive performance of our in vitro model achieves an average of 87.5% accuracy (correct prediction rate), 75% specificity (prediction rate of true negative drugs), and 100% sensitivity (prediction rate of true positive drugs). We then employ the MCA and the SVM training algorithm to prospectively identify the skin-sensitizing likelihood and mechanism-of-action for obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist which has undergone clinical trials for non-alcoholic steatohepatitis (NASH) with well-documented cutaneous side effects.

Zyxin Is Involved in Fibroblast Rigidity Sensing and Durotaxis.

Focal adhesions (FAs) are specialized structures that enable cells to sense their extracellular matrix rigidity and transmit these signals to the interior of the cells, bringing about actin cytoskeleton reorganization, FA maturation, and cell migration. It is known that cells migrate towards regions of higher substrate rigidity, a phenomenon known as durotaxis. However, the underlying molecular mechanism of durotaxis and how different proteins in the FA are involved remain unclear. Zyxin is a component of the FA that has been implicated in connecting the actin cytoskeleton to the FA. We have found that knocking down zyxin impaired NIH3T3 fibroblast's ability to sense and respond to changes in extracellular matrix in terms of their FA sizes, cell traction stress magnitudes and F-actin organization. Cell migration speed of zyxin knockdown fibroblasts was also independent of the underlying substrate rigidity, unlike wild type fibroblasts which migrated fastest at an intermediate substrate rigidity of 14 kPa. Wild type fibroblasts exhibited durotaxis by migrating toward regions of increasing substrate rigidity on polyacrylamide gels with substrate rigidity gradient, while zyxin knockdown fibroblasts did not exhibit durotaxis. Therefore, we propose zyxin as an essential protein that is required for rigidity sensing and durotaxis through modulating FA sizes, cell traction stress and F-actin organization.

Hepatic Bioactivation of Skin-Sensitizing Drugs to Immunogenic Reactive Metabolites.

The clinical use of some drugs, such as carbamazepine, phenytoin, and allopurinol, is often associated with adverse cutaneous reactions. The bioactivation of drugs into immunologically reactive metabolites by the liver is postulated to be the first step in initiating a downstream cascade of pathological immune responses. Current mechanistic understanding and the ability to predict such adverse drug cutaneous responses have been partly limited by the lack of appropriate cutaneous drug bioactivation experimental models. Although in vitro human liver models have been extensively investigated for predicting hepatotoxicity and drug-drug interactions, their ability to model the generation of antigenic reactive drug metabolites that are capable of eliciting immunological reactions is not well understood. Here, we employed a human progenitor cell (HepaRG)-derived hepatocyte model and established highly sensitive liquid chromatography-mass spectrometry analytical assays to generate and quantify different reactive metabolite species of three paradigm skin sensitizers, namely, carbamazepine, phenytoin, and allopurinol. We found that the generation of reactive drug metabolites by the HepaRG-hepatocytes was sensitive to the medium composition. In addition, a functional assay based on the activation of U937 myeloid cells into the antigen-presenting cell (APC) phenotype was established to evaluate the immunogenicity potential of the reactive drug metabolites produced by HepaRG-derived hepatocytes. We showed that the reactive drug metabolites of known skin sensitizers could significantly upregulate IL8, IL1ß, and CD86 expressions in U937 cells compared to the metabolites from a nonskin sensitizer (i.e., acetaminophen). Thus, the extent of APC activation by HepaRG-hepatocytes conditioned medium containing reactive drug metabolites can potentially be used to predict their skin sensitization potential.

Self-aligning Tetris-Like (TILE) modular microfluidic platform for mimicking multi-organ interactions.

Multi-organ perfusion systems offer the unique opportunity to mimic different physiological systemic interactions. However, existing multi-organ culture platforms have limited flexibility in specifying the culture conditions, device architectures, and fluidic connectivity simultaneously. Here, we report a modular microfluidic platform that addresses this limitation by enabling easy conversion of existing microfluidic devices into tissue and fluid control modules with self-aligning magnetic interconnects. This enables a 'stick-n-play' approach to assemble planar perfusion circuits that are amenable to both bioimaging-based and analytical measurements. A myriad of tissue culture and flow control TILE modules were successfully constructed with backward compatibility. Finally, we demonstrate applications in constructing recirculating multi-organ systems to emulate liver-mediated bioactivation of nutraceuticals and prodrugs to modulate their therapeutic efficacies in the context of atherosclerosis and cancer. This platform greatly facilitates the integration of existing organs-on-chip models to provide an intuitive and flexible way for users to configure different multi-organ perfusion systems.

A liver-immune coculture array for predicting systemic drug-induced skin sensitization.

Drug-induced skin sensitization is prevalent worldwide and can trigger life-threatening health conditions, such as Stevens Johnson Syndrome. However, existing in vitro skin models cannot adequately predict the skin sensitization effects of drugs administered into the systemic circulation because dermal inflammation and injury are preceded by conversion of parent drugs into antigenic reactive metabolites in the liver and subsequent activation of the immune system. Here, we demonstrate that recapitulation of these early tandem cellular processes in a compartmentalized liver-immune coculture array is sufficient to predict the skin sensitization potential of systemic drugs. Human progenitor cell (HepaRG)-derived hepatocyte spheroids and U937 myeloid cells, a representative antigen presenting cell (APC), can maintain their respective functions in 2 concentric micro-chambers, which are connected by a diffusion microchannel network. Paradigm drugs that are reported to cause severe cutaneous drug reactions (i.e. carbamazepine, phenytoin and allopurinol) can be metabolized into their reactive metabolites, which diffuse efficiently into the adjoining immune compartment within a 48 hour period. By measuring the extent of U937 activation as indicated by IL8, IL1ß and CD86 upregulation upon drug administration, we show that the liver-immune coculture array more consistently and reliably distinguish all 3-paradigm skin sensitizing drugs from a non-skin sensitizer than conventional bulk Transwell coculture. Given its miniaturized format, design simplicity and prediction capability, this novel in vitro system can be readily scaled into a screenable platform to identify the skin sensitization potential of systemically-administered drugs.

A pump-free microfluidic 3D perfusion platform for the efficient differentiation of human hepatocyte-like cells.

The practical application of microfluidic liver models for in vitro drug testing is partly hampered by their reliance on human primary hepatocytes, which are limited in number and have batch-to-batch variation. Human stem cell-derived hepatocytes offer an attractive alternative cell source, although their 3D differentiation and maturation in a microfluidic platform have not yet been demonstrated. We develop a pump-free microfluidic 3D perfusion platform to achieve long-term and efficient differentiation of human liver progenitor cells into hepatocyte-like cells (HLCs). The device contains a micropillar array to immobilize cells three-dimensionally in a central cell culture compartment flanked by two side perfusion channels. Constant pump-free medium perfusion is accomplished by controlling the differential heights of horizontally orientated inlet and outlet media reservoirs. Computational fluid dynamic simulation is used to estimate the hydrostatic pressure heads required to achieve different perfusion flow rates, which are experimentally validated by micro-particle image velocimetry, as well as viability and functional assessments in a primary rat hepatocyte model. We perform on-chip differentiation of HepaRG, a human bipotent progenitor cell, and discover that 3D microperfusion greatly enhances the hepatocyte differentiation efficiency over static 2D and 3D cultures. However, HepaRG progenitor cells are highly sensitive to the time-point at which microperfusion is applied. Isolated HepaRG cells that are primed as static 3D spheroids before being subjected to microperfusion yield a significantly higher proportion of HLCs (92%) than direct microperfusion of isolated HepaRG cells (62%). This platform potentially offers a simple and efficient means to develop highly functional microfluidic liver models incorporating human stem cell-derived HLCs. Biotechnol. Bioeng. 2017;114: 2360-2370. © 2017 Wiley Periodicals, Inc.