Over 90% of all cancer-related deaths are consequences of metastatic tumors. Epithelial–Mesenchymal Transition (EMT) plays a critical role in the early stages of dissemination of carcinoma leading to metastatic tumors.  Current therapeutic regimens, however, have been ineffective in the cure of metastatic cancer, thus an urgent need exists to revisit existing protocols and to improve the efficacy of newly developed therapeutics. Strategies based on preventing EMT could potentially contribute to improving the outcome of advanced stage cancers. To achieve this goal new assays are needed to identify targeted drugs capable of interfering with EMT or to revert the mesenchymal-like phenotype of carcinoma to an epithelial-like state. Current assays are limited to examining the dispersion of carcinoma cells in isolation in conventional 2-dimensional (2D) microwell systems, an approach that fails to account for the 3-dimensional (3D) environment of the tumor or the essential interactions that occur with other nearby cell types in the tumor microenvironment.  Here we present a microfluidic system that integrates tumor cell spheroids in a 3D hydrogel scaffold, in close co-culture with an endothelial monolayer. Drug candidates inhibiting receptor activation or signal transduction pathways implicated in EMT have been tested using dispersion of A549 lung adenocarcinoma cell spheroids as a metric of effectiveness. We observe significant differences in response to drugs between 2D and 3D, and between monoculture and co-culture.
Two different cancer cell lines, A549 (lung) and T24 (bladder), were tested. Dose-response assays of Src inhibitor AZD-0530 were obtained for relative dispersion as a metric of epithelial-mesenchymal transition (EMT). Although dispersion of A549 cells could be inhibited, none of the doses of AZD-0530 inhibited T24 dispersion. Hepatocyte growth factor (HGF) signaling was further identified that plays a role in T24 dispersion. This 3D microfluidic co-culture platform provides an in vivo-like surrogate for anti-invasive and anti-metastatic drug screening. It will be particularly useful for defining combination therapy for aggressive tumors such as invasive bladder carcinoma.

The use of microfluidic devices to study angiogenesis, the sprouting of new blood vessels from pre-existing vessels, in 3D has been widely adopted for the creation of in vitro assays that are closer to in vivo conditions. Our recent work develops microfluidic devices to study synergistic effects of two different angiogenic compounds, prolyl hydroxylase inhibitors (PHis) and sphingosine 1-phosphate (S1P) in the presence of fibroblasts. PHis are compounds that prevent the degradation of hypoxia inducible factor-1α (HIF-1α) in vivo thus upregulating various angiogenic factors including VEGF as a response to the induced pseudo-hypoxic condition under normoxia. S1P, the other hand, is a lysophospholipid that mediates multiple biological events including angiogenesis through specific cell surface G-protein coupled receptors. The synergistic effects of PHis and S1P are studied in an in vitro microfludic platform where endothelial cells are co-cultured with alginate bead encapsulated-fibroblasts and subjected to the stimulation of ciclopirox olamine (CPX, a type of PHis) and S1P. We find that the interactions between endothelial cells and fibroblasts are essential in maintaining immature sprouts and converting them into functional vessels with lumens and these effects are most prominent when CPX works synergistically with S1P.

We demonstrated that a cross gradient of mutually stimulating factors was generated as each cell type responded differently to the compounds, especially to the PHi. EC showed increased secretion of placental growth factor, endothelin-1, epidermal growth factor and interleukin-8 while fibroblasts showed upregulated secretion of hepatocyte growth factor, insulin-like growth factor-binding protein 2, urokinase plasminogen activator and most importantly, vascular endothelial growth factor (VEGF). We also showed that S1P induced migration was S1P receptor 1 (S1P1) dependent and more importantly the expression of S1P1 on EC could be modulated by both PHi and S1P. The downregulation of either VEGF or S1P1 showed attenuated capillary sprouting and also change of sprouting morphologies. The proangiogenic monocyte chemotactic protein (MCP)-1 was mainly secreted by fibroblasts, but only in the presence of EC-conditioned medium (CM). Antibody interference with MCP-1 also reduced the sprouting response. The EC-fibroblasts interactions could be replaced by neither fibroblasts CM nor EC-cancer cells crosstalk. These observations not only confirm the importance of EC-fibroblasts crosstalk in angiogenesis but also suggest that the combination of PHi and S1P enhances this synergy and thus might be of therapeutic value for tissue repair and regeneration.

We developed a computational model to predict oxygen levels in microfluidic plastic devices during cell culture. This model is based on experimental evaluation of oxygen levels. Conditions are determined that provide adequate oxygen supply to two cell types, hepatocytes (HEP) and endothelial cells (EC), either by diffusion through the plastic device, or by supplying a low flow rate of medium. PDMS devices where fabricated with soft lithography techniques. Alternatively, the COC (cyclic olefin copolymer) and PMP (poly-methyl pentene) chips were injection molded. Oxygen-sensitive foils (Visisens, Germany) were bonded to the bottom of the devices. All oxygen measurements were evaluated using the Visisens software after calibration against two reference solutions.

3D tissue-like cultures are now widely used as in vitro models of in vivo cellular functions such as embryonic development, wound healing, and malignancy. Compared with a 2D monolayer culture, a 3D model better mimics the in vivo tissue like cellular features in morphology, cell–cell interaction, signal transduction, and mechanical stimulation, thus providing a physiologically and pathologically relevant environment. Multicellular 3D aggregates have proven useful as a model for directing specifi c cell lineages using embryonic stem cell-formed embryoid bodies, and studying collective cell migration in cancer.

Microwell technology has revolutionized many aspects of in vitro cellular studies from 2D traditional cultures to 3D in vivo-like functional assays. However, existing lithography-based approaches are often costly and time consuming. This study presents a rapid, low-cost prototyping method of CO2 laser ablation of a conventional untreated culture dish to create concave microwells used for generating multicellular aggregates, which can be readily available for general laboratories. Polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), and polystyrene (PS) microwells are investigated, and each produces distinctive microwell features. Among these three
materials, PS cell culture dishes produce the optimal surface smoothness and roundness. A549 lung cancer cells are grown to form cancer aggregates of controllable size from ≈40 to ≈80 μ m in PS microwells. Functional assays of spheroids are performed to study migration on 2D substrates and in 3D hydrogel conditions as a step towards recapitulating the dissemination of cancer cells. Preclinical anti-cancer drug screening is investigated and reveals considerable differences between 2D and 3D conditions, indicating the importance of assay type as well as the utility of the present approach.

Cells in the human body migrate to perform numerous biological functions, including development and wound healing. For years, cell migration has been studied with two-dimensional (2D) on-the-gel assays using 2D imaging and cell tracking techniques. When cells migrate in three-dimensional (3D) environments, they exhibit strikingly different behaviors. In 3D, cells migrate by remodeling and cleaving the surrounding gel whereas in 2D, they simply move across a free surface. However, their migration behavior and interactions with the surrounding gel are still poorly understood. To help understand 3D migration and cell-gel interactions, we aim to develop a new automated image processing methodology which will provide useful insights into how a vascular structure is formed as a collection of migratory cells. We present a new approach to incorporating information from heterogeneous images of migrating cells in 3D gel. We study 3D angiogenic sprouting, where cells burrow into the gel matrix, communicate with other cells and create vascular networks. We combine time-lapse fluorescent images of stained cell nuclei and transmitted light images of the background gel to track cell trajectories. The nuclei images are sampled less frequently due to photo toxicity. Hence, 3D cell tracking can be performed more reliably when 2D sprout profiles, extracted from gel matrix images, are effectively incorporated. We employ a Bayesian filtering approach to optimally combine the two heterogeneous images with different sampling rates. We construct stochastic models to predict cell locations and sprout profiles and condition the likelihood of nuclei location by the sprout profile. The conditional distribution is non-Gaussian and the cell dynamics is non-linear. To jointly update cell and sprout estimates, we use a Rao–Blackwell particle filter. Simulation and experimental results show accurate tracking of multiple cells along with sprout formation, demonstrating synergistic effects of incorporating the two types of images.

Overview of the tracking system. Multiple sprouts, are treated as separate groups of cells, lumen and filopodia. We predict these parameters using motion models. The segmented nuclei, lumen and filopodia candidates are associated firstly with a particular sprout, and then with existing tracks in its joint estimate. Matched candidates are updated to an existing track. New sprouts may be created for dissociated sprout profiles. New nuclei, lumen and filopodia tracks in a sprout may be augmented to a joint estimate. Tracks and sprouts may also be terminated.

The mechanical properties of the materials surrounding cells influence the behaviour of cells in differentiation, proliferation, and apoptosis. We have developed a new way to change one mechanical property of the extracellular matrix (ECM), namely the stiffness, via embedded magnetic beads. These beads are conjugated in an ECM via streptavidin-coated beads that bind to biotinylated collagen fibres, then applying an external magnetic field to exert forces on the beads in order to manipulate the local strain and then the effective stiffness of the ECM. The change in the micro and macro Young’s elastic modulus of the ECM sample, when switching on or off the magnetic field, was determined by atomic force microscopy-enabled indentation (developed in Thrust 2) and uniaxial compression, respectively. The experimental results demonstrated that, by embedding magnetic beads in an ECM through bio-conjugation between the streptavidin-coated beads and the collagen fibers, the stiffness of the ECM can be actively manipulated by the application of an external magnetic field. A separate study was done by allowing endothelial cells (ECs) to migrate though the magnetic bead embedded ECM ensemble with magnetic field and without magnetic field. A VEGF gradient was also added to promote the sprouting of ECs. The results from this work have demonstrated the possibility of creating desired stiffness gradients in an ECM in vitro to influence endothelial cell behaviour. Electromagnetic needles (EMN) were designed and fabricated to be installed in the existing confocal microscope for the manipulations of magnetics beads during angiogenesis. These EMNs are designed such that their magnetic fields are focused on a smaller region to allow single magnetic bead manipulations.

An integrative cell migration model incorporating focal adhesion (FA) dynamics, cytoskeleton and nucleus remodeling and actin motor activity is developed for predicting cell migration behaviors on 3-dimensional curved surfaces, such as cylindrical lumens in the 3-D extracellular matrix (ECM). The work is motivated by 3-D microfluidic migration experiments suggesting that the migration speed and direction may vary depending on the cross sectional shape of the lumen along which the cell migrates. In this paper, the mechanical structure of the cell is modeled as double elastic membranes of cell and nucleus. The two elastic membranes are connected by stress fibers, which are extended from focal adhesions on the cell surface to the nuclear membrane. The cell deforms and gains traction as transmembrane integrins distributed over the outer cell membrane bind to ligands on the ECM, form focal adhesions, and activate stress fibers. Probabilities at which integrin ligand–receptor bonds are formed as well as ruptures are affected by the surface geometry, resulting in diverse migration behaviors that depend on the curvature of the surface. Monte Carlo simulations of the integrative model reveal that (a) the cell migration speed is dependent on the cross sectional area of the lumen with a maximum speed at a particular diameter or width, (b) as the lumen diameter increases, the cell tends to spread and migrate around the circumference of the lumen, while it moves in the longitudinal direction as the lumen diameter narrows, (c) once the cell moves in one direction, it tends to stay migrating in the same direction despite the stochastic nature of migration. The relationship between the cell migration speed and the lumen width agrees with microfluidic experimental data for cancer cell migration.

An integrative cell migration model incorporating focal adhesion (FA) dynamics, cytoskeleton and nucleus remodeling, actin motor activity, and lamellipodia protrusion is developed for predicting cell spreading and migration behaviors. This work is motivated by two experimental works: (1) cell migration on 2-D substrates under various fibronectin concentrations and (2) cell spreading on 2-D micropatterned geometries. These works suggest (1) cell migration speed takes a maximum at a particular ligand density (,1140 molecules/mm2) and (2) that strong traction forces at the corners of the patterns may exist due to combined effects exerted by actin stress fibers (SFs). The integrative model of this paper successfully reproduced these experimental results and indicates the mechanism of cell migration and spreading. In this paper, the mechanical structure of the cell is modeled as having two elastic membranes: an outer cell membrane and an inner nuclear membrane. The two elastic membranes are connected by SFs, which are extended from focal adhesions on the cortical surface to the nuclear membrane. In addition, the model also includes ventral SFs bridging two focal adhesions on the cell surface. The cell deforms and gains traction as transmembrane integrins distributed over the outer cell membrane bond to ligands on the ECM surface, activate SFs, and form focal adhesions. The relationship between the cell migration speed and fibronectin concentration agrees with existing experimental data for Chinese hamster ovary (CHO) cell migrations on fibronectin coated surfaces. In addition, the integrated model is validated by showing persistent high stress concentrations at sharp geometrically patterned edges. This model will be used as a predictive model to assist in design and data processing of upcoming microfluidic cell migration assays.

• https://www.youtube.com/watch?v=Ft3ZVlDwhvU
• https://www.youtube.com/watch?v=PO641vz2oKg

Dynamic model of cell migration. A) Integrated cell migration model consisting of the cytoskeleton, the nucleus, N integrin nodes on the surface of cytoskeleton, N nuclear nodes on the surface of nucleus, and two types of actin SFs which connect the integrin node to the nuclear node and between integrin nodes; a top view of the model showing triangular mesh network of double membranes of cytoskeleton and nucleus. B) the free body diagram of the i-th integrin node in the circle marked in A) where five external forces are acting. Note that, while shown in 2-D, the force balance exists in 3-D.		3-D integrated cell migration model A) schematic representation of cell migration model on the planar substrate, showing deformable cell and nuclear membranes, focal adhesions, and actin SFs, B) a magnified view in A) showing the structure of focal adhesion including the attachment of the end of SFs through an integrin node to the underlying extracellular matrix, illustrating a stochastic ligand-receptor bonding process at the focal adhesion site, and showing the structure of actin SFs. Note that, A) and B) represent top and side views, respectively.

Delta-like 4 (Dll4), a membrane-bound Notch ligand, plays a fundamental role in vascular development and angiogenesis. Dll4 is highly expressed in capillary endothelial tip cells and is involved in suppressing neighboring stalk cells to become tip cells during angiogenesis. Dll4-Notch signaling is mediated either by direct cell-cell contact or by Dll4-containing exosomes from a distance. However, whether Dll4-containing exosomes influence tip cells of existing capillaries is unknown. Using a 3D microfluidic device and time-lapse confocal microscopy, we show here for the first time that Dll4-containing exosomes causes tip cells to lose their filopodia and trigger capillary sprout retraction in collagen matrix. We demonstrate that Dll4 exosomes can freely travel through 3D collagen matrix and transfer Dll4 protein to distant tip cells.
Upon reaching endothelial sprout, it causes filopodia and tip cell retraction. Continuous application of Dll4 exosomes from a distance lead to significant reduction of sprout formation. This effect correlates with Notch signaling activation upon Dll4-containing exosome interaction with recipient endothelial cells. Furthermore, we show that Dll4-containing exosomes increase endothelial cell motility while suppressing their proliferation. These data revealed novel functions of Dll4 in angiogenesis through exosomes.