Elisabeth Wong

Collective and individual migration following the epithelial–mesenchymal transition

During cancer progression, malignant cells in the tumour invade surrounding tissues. This transformation of adherent cells to a motile phenotype has been associated with the epithelial mesenchymal transition (EMT). Here, we show that EMT-activated cells migrate through micropillar arrays as a collectively advancing front that scatters individual cells. Individual cells with few neighbours dispersed with fast, straight trajectories, whereas cells that encountered many neighbours migrated collectively with epithelial biomarkers. We modelled these emergent dynamics using a physical analogy to solidification phase transitions in binary mixtures, and validated it using drug perturbations, which revealed that individually migrating cells exhibit diminished chemosensitivity. Our measurements also indicate a degree of phenotypic plasticity as cells interconvert between individual and collective migration. The study of multicellular behaviours with single-cell resolution should enable further quantitative insights into heterogeneous tumour invasion.

Microfluidic platform for the quantitative analysis of leukocyte migration signatures

Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyze four qualitative migration patterns: chemo-attraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence, and fraction of cells responding. We find that established chemokines C5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T-lymphocytes migrate away than towards SDF-1 and their directional migration patterns are not persistent. Overall, our platform characterizes migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators.

Microfluidic chambers for monitoring leukocyte trafficking and humanized nano-proresolving medicines interactions

Leukocyte trafficking plays a critical role in determining the progress and resolution of inflammation. Although significant progress has been made in understanding the role of leukocyte activation in inflammation, dissecting the interactions between different leukocyte subpopulations during trafficking is hampered by the complexity of in vivo conditions and the lack of detail of current in vitro assays. To measure the effects of the interactions between neutrophils and monocytes migrating in response to various chemoattractants, at single-cell resolution, we developed a microfluidic platform that replicates critical features of focal inflammation sites. We integrated an elastase assay into the focal chemotactic chambers (FCCs) of our device that enabled us to distinguish between phlogistic and nonphlogistic cell recruitment. We found that lipoxin A4 and resolvin D1, in solution or incorporated into nano–proresolving medicines, reduced neutrophil and monocyte trafficking toward leukotriene B4. Lipoxin A4 also reduced the elastase release from homogenous and heterogenous mixtures of neutrophils and monocytes. Surprisingly, the effect of resolvin D1 on heterogenous mixtures was antisynergistic, resulting in a transient spike in elastase activity, which was quickly terminated, and the degraded elastin removed by the leukocytes inside the FCCs. Therefore, the microfluidic assay provides a robust platform for measuring the effect of leukocyte interactions during trafficking and for characterizing the effects of inflammation mediators.

Microfluidic Chemotaxis Platform for Differentiating the Roles of Soluble and Bound Amyloid-β on Microglial Accumulation

Progressive microglial accumulation at amyloid-β (Aβ) plaques is a well-established signature of the pathology of Alzheimers disease, but how and why microglia accumulate in the vicinity of Aβ plaques is unknown. To understand the distinct roles of Aβ on microglial accumulation, we quantified microglial responses to week-long lasting gradients of soluble Aβ and patterns of surface-bound Aβ in microfluidic chemotaxis platforms. We found that human microglia chemotaxis in gradients of soluble Aβ42 was most effective at two distinct concentrations of 23 pg.mL−1 and 23 ng.mL−1 Aβ42 in monomers and oligomers. We uncovered that while the chemotaxis at higher Aβ concentrations was exclusively due to Aβ gradients, chemotaxis at lower concentrations was enhanced by Aβ-induced microglial production of MCP-1. Microglial migration was inhibited by surface-bound Aβ42 in oligomers and fibrils above 45 pg.mm−2. Better understanding of microglial migration can provide insights into the pathophysiology of senile plaques in AD.

Microscale arrays for the profiling of start and stop signals coordinating human-neutrophil swarming

Neutrophil swarms protect healthy tissues by sealing off sites of infection. In the absence of swarming, microbial invasion of surrounding tissues can result in severe infections. Recent observations in animal models have shown that swarming requires rapid neutrophil responses and well-choreographed neutrophil migration patterns. However, in animal models physical access to the molecular signals coordinating neutrophil activities during swarming is limited. Here, we report the development and validation of large microscale arrays of zymosan-particle clusters for the study of human neutrophils during swarming ex vivo. We characterized the synchronized swarming of human neutrophils under the guidance of neutrophil-released chemokines, and measured the mediators released at different phases of human-neutrophil swarming against targets simulating infections. We found that the network of mediators coordinating human-neutrophil swarming includes start and stop signals, proteolytic enzymes and enzyme inhibitors, as well as modulators of activation of other immune and non-immune cells. We also show that the swarming behavior of neutrophils from patients following major trauma is deficient and gives rise to smaller swarms than those of neutrophils from healthy individuals.

A Worldwide Competition to Compare the Speed and Chemotactic Accuracy of Neutrophil-Like Cells

Chemotaxis is the ability to migrate towards the source of chemical gradients. It underlies the ability of neutrophils and other immune cells to hone in on their targets and defend against invading pathogens. Given the importance of neutrophil migration to health and disease, it is crucial to understand the basic mechanisms controlling chemotaxis so that strategies can be developed to modulate cell migration in clinical settings. Because of the complexity of human genetics, Dictyostelium and HL60 cells have long served as models system for studying chemotaxis. Since many of our current insights into chemotaxis have been gained from these two model systems, we decided to compare them side by side in a set of winner-take-all races, the Dicty World Races. These worldwide competitions challenge researchers to genetically engineer and pharmacologically enhance the model systems to compete in microfluidic racecourses. These races bring together technological innovations in genetic engineering and precision measurement of cell motility. Fourteen teams participated in the inaugural Dicty World Race 2014 and contributed cell lines, which they tuned for enhanced speed and chemotactic accuracy. The race enabled large-scale analyses of chemotaxis in complex environments and revealed an intriguing balance of speed and accuracy of the model cell lines. The successes of the first race validated the concept of using fun-spirited competition to gain insights into the complex mechanisms controlling chemotaxis, while the challenges of the first race will guide further technological development and planning of future events.

On-demand, competing gradient arrays for neutrophil chemotaxis

Neutrophils are the most abundant type of white blood cells in the circulation, protecting the body against pathogens and responding early to inflammation. Although we understand how neutrophils respond to individual stimuli, we know less about how they prioritize between competing signals or respond to combinational signals. This situation is due in part to the lack of adequate experimental systems to provide signals in controlled spatial and temporal fashion. To address these limitations, we designed a platform for generating on-demand, competing chemical gradients and for monitoring neutrophil migration. On this platform, we implemented forty-eight assays generating independent gradients and employed synchronized valves to control the timing of these gradients. We observed faster activation of neutrophils in response to fMLP than to LTB4 and unveiled for the first time a potentiating effect for fMLP during migration towards LTB4. Our observations, enabled by the new tools, challenge the current paradigm of inhibitory competition between distinct chemoattractant gradients and suggest that human neutrophils are capable of complex integration of chemical signals in their environment.