A novel jamming phase diagram links tumor invasion to non-equilibrium phase separation.

It is well established that the early malignant tumor invades surrounding extracellular matrix (ECM) in a manner that depends upon material properties of constituent cells, surrounding ECM, and their interactions. Recent studies have established the capacity of the invading tumor spheroids to evolve into coexistent solid-like, fluid-like, and gas-like phases. Using breast cancer cell lines invading into engineered ECM, here we show that the spheroid interior develops spatial and temporal heterogeneities in material phase which, depending upon cell type and matrix density, ultimately result in a variety of phase separation patterns at the invasive front. Using a computational approach, we further show that these patterns are captured by a novel jamming phase diagram. We suggest that non-equilibrium phase separation based upon jamming and unjamming transitions may provide a unifying physical picture to describe cellular migratory dynamics within, and invasion from, a tumor.

In primary airway epithelial cells, the unjamming transition is distinct from the epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) and the unjamming transition (UJT) each comprises a gateway to cellular migration, plasticity and remodeling, but the extent to which these core programs are distinct, overlapping, or identical has remained undefined. Here, we triggered partial EMT (pEMT) or UJT in differentiated primary human bronchial epithelial cells. After triggering UJT, cell-cell junctions, apico-basal polarity, and barrier function remain intact, cells elongate and align into cooperative migratory packs, and mesenchymal markers of EMT remain unapparent. After triggering pEMT these and other metrics of UJT versus pEMT diverge. A computational model attributes effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, those tissues that are subject to development, injury, or disease become endowed with rich mechanisms for cellular migration, plasticity, self-repair, and regeneration.

Epithelial layer unjamming shifts energy metabolism toward glycolysis.

In development of an embryo, healing of a wound, or progression of a carcinoma, a requisite event is collective epithelial cellular migration. For example, cells at the advancing front of a wound edge tend to migrate collectively, elongate substantially, and exert tractions more forcefully compared with cells many ranks behind. With regards to energy metabolism, striking spatial gradients have recently been reported in the wounded epithelium, as well as in the tumor, but within the wounded cell layer little is known about the link between mechanical events and underlying energy metabolism. Using the advancing confluent monolayer of MDCKII cells as a model system, here we report at single cell resolution the evolving spatiotemporal fields of cell migration speeds, cell shapes, and traction forces measured simultaneously with fields of multiple indices of cellular energy metabolism. Compared with the epithelial layer that is unwounded, which is non-migratory, solid-like and jammed, the leading edge of the advancing cell layer is shown to become progressively more migratory, fluid-like, and unjammed. In doing so the cytoplasmic redox ratio becomes progressively smaller, the NADH lifetime becomes progressively shorter, and the mitochondrial membrane potential and glucose uptake become progressively larger. These observations indicate that a metabolic shift toward glycolysis accompanies collective cellular migration but show, further, that this shift occurs throughout the cell layer, even in regions where associated changes in cell shapes, traction forces, and migration velocities have yet to penetrate. In characterizing the wound healing process these morphological, mechanical, and metabolic observations, taken on a cell-by-cell basis, comprise the most comprehensive set of biophysical data yet reported. Together, these data suggest the novel hypothesis that the unjammed phase evolved to accommodate fluid-like migratory dynamics during episodes of tissue wound healing, development, and plasticity, but is more energetically expensive compared with the jammed phase, which evolved to maintain a solid-like non-migratory state that is more energetically economical.

Irradiation Induces Epithelial Cell Unjamming.

The healthy and mature epithelial layer is ordinarily quiescent, non-migratory, solid-like, and jammed. However, in a variety of circumstances the layer transitions to a phase that is dynamic, migratory, fluid-like and unjammed. This has been demonstrated in the developing embryo, the developing avian airway, the epithelial layer reconstituted in vitro from asthmatic donors, wounding, and exposure to mechanical stress. Here we examine the extent to which ionizing radiation might similarly provoke epithelial layer unjamming. We exposed primary human bronchial epithelial (HBE) cells maintained in air-liquid interface (ALI) to sub-therapeutic doses (1 Gy) of ionizing radiation (IR). We first assessed: (1) DNA damage by measuring p-H2AX, (2) the integrity of the epithelial layer by measuring transepithelial electrical resistance (TEER), and (3) the extent of epithelial cell differentiation by detecting markers of differentiated airway epithelial cells. As expected, IR exposure induced DNA damage but, surprisingly, disrupted neither normal differentiation nor the integrity of the epithelial cell layer. We then measured cell shape and cellular migration to determine the extent of the unjamming transition (UJT). IR caused cell shape elongation and increased cellular motility, both of which are hallmarks of the UJT as previously confirmed. To understand the mechanism of IR-induced UJT, we inhibited TGF-ß receptor activity, and found that migratory responses were attenuated. Together, these observations show that IR can provoke epithelial layer unjamming in a TGF-ß receptor-dependent manner.

Unjamming and collective migration in MCF10A breast cancer cell lines.

Each cell comprising an intact, healthy, confluent epithelial layer ordinarily remains sedentary, firmly adherent to and caged by its neighbors, and thus defines an elemental constituent of a solid-like cellular collective [1,2]. After malignant transformation, however, the cellular collective can become fluid-like and migratory, as evidenced by collective motions that arise in characteristic swirls, strands, ducts, sheets, or clusters [3,4]. To transition from a solid-like to a fluid-like phase and thereafter to migrate collectively, it has been recently argued that cells comprising the disordered but confluent epithelial collective can undergo changes of cell shape so as to overcome geometric constraints attributable to the newly discovered phenomenon of cell jamming and the associated unjamming transition (UJT) [1,2,5-9]. Relevance of the jamming concept to carcinoma cells lines of graded degrees of invasive potential has never been investigated, however. Using classical in vitro cultures of six breast cancer model systems, here we investigate structural and dynamical signatures of cell jamming, and the relationship between them [1,2,10,11]. In order of roughly increasing invasive potential as previously reported, model systems examined included MCF10A, MCF10A.Vector; MCF10A.14-3-3ζ; MCF10.ErbB2, MCF10AT; and MCF10CA1a [12-15]. Migratory speed depended on the particular cell line. Unsurprisingly, for example, the MCF10CA1a cell line exhibited much faster migratory speed relative to the others. But unexpectedly, across different cell lines higher speeds were associated with enhanced size of cooperative cell packs in a manner reminiscent of a peloton [9]. Nevertheless, within each of the cell lines evaluated, cell shape and shape variability from cell-to-cell conformed with predicted structural signatures of cell layer unjamming [1]. Moreover, both structure and migratory dynamics were compatible with previous theoretical descriptions of the cell jamming mechanism [2,10,11,16,17]. As such, these findings demonstrate the richness of the cell jamming mechanism, which is now seen to apply across these cancer cell lines but remains poorly understood.

The tumor suppressor p53 can promote collective cellular migration.

Loss of function of the tumor suppressor p53 is known to increase the rate of migration of cells transiting the narrow pores of the traditional Boyden chamber assay. Here by contrast we investigate how p53 impacts the rate of cellular migration within a 2D confluent cell layer and a 3D collagen-embedded multicellular spheroid. We use two human carcinoma cell lines, the bladder carcinoma EJ and the colorectal carcinoma HCT116. In the confluent 2-D cell layer, for both EJ and HCT cells the migratory speeds and effective diffusion coefficients for the p53 null cells were significantly smaller than in p53-expressing cells. Compared to p53 expressers, p53-null cells exhibited more organized cortical actin rings together with reduced front-rear cell polarity. Furthermore, loss of p53 caused cells to exert smaller traction forces upon their substrates, and reduced formation of cryptic lamellipodia. In the 3D multicellular spheroid, loss of p53 consistently reduced collective cellular migration into surrounding collagen matrix. As regards the role of p53 in cellular migration, extrapolation from the Boyden chamber assay to other cellular microenvironments is seen to be fraught even in terms of the sign of the effect. Together, these paradoxical results show that the effects of p53 on cellular migration are context-dependent.

Geometric constraints during epithelial jamming.

As an injury heals, an embryo develops, or a carcinoma spreads, epithelial cells systematically change their shape. In each of these processes cell shape is studied extensively whereas variability of shape from cell-to-cell is regarded most often as biological noise. But where do cell shape and its variability come from? Here we report that cell shape and shape variability are mutually constrained through a relationship that is purely geometrical. That relationship is shown to govern processes as diverse as maturation of the pseudostratified bronchial epithelial layer cultured from non-asthmatic or asthmatic donors, and formation of the ventral furrow in the Drosophila embryo. Across these and other epithelial systems, shape variability collapses to a family of distributions that is common to all. That distribution, in turn, is accounted for by a mechanistic theory of cell-cell interaction showing that cell shape becomes progressively less elongated and less variable as the layer becomes progressively more jammed. These findings suggest a connection between jamming and geometry that spans living organisms and inert jammed systems, and thus transcends system details. Although molecular events are needed for any complete theory of cell shape and cell packing, observations point to the hypothesis that jamming behavior at larger scales of organization sets overriding geometrical constraints.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (10(3)-10(6)). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics.

Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (P(rec) = 3.3 × 10(-4) ± 2 × 10(-5) ) for a 1205 nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.

Eccentric rheometry for viscoelastic characterization of small, soft, anisotropic, and irregularly shaped biopolymer gels and tissue biopsies.

Quantification of the physical properties of tissue biopsies and cell-remodeled hydrogels is critical for understanding tissue development and pathophysiological tissue remodeling. However, due to the low modulus, small size, irregular shape, and anisotropy of samples from these materials, accurate viscoelastic characterization using standard rheometric methods is problematic. The goal of this work is to utilize image analysis to extend rotational rheometry to these samples. In this method, the sample is offset to increase the torque generated; a custom clear glass geometry, right angle prism, and camera are used to determine the exact shape and location of the sample relative to the axis of rotation for calculation of the sample shear modulus, G'. Values of G' for standard polydimethylsiloxane gels tested in centered and eccentric configurations were not statistically different (respectively 137 ± 37 kPa and 126 ± 8 kPa, p = 0.58), indicating accuracy of the method. Additionally, G' values from circular and irregularly shaped collagen gels yielded equivalent results (31 ± 1.8 Pa and 31 ± 5.1 Pa, p = 0.29). A blood clot and a lipid plaque sample recovered from human patients (G' ~ 4 kPa) were successfully tested with this method demonstrating applicability to clinical diagnostics.

A combination of peppermint oil and caraway oil attenuates the post-inflammatory visceral hyperalgesia in a rat model.

OBJECTIVE: Visceral hyperalgesia plays a pivotal role in manifestation of symptoms in patients with functional gastrointestinal disorders. In clinical studies combined treatment of peppermint- and caraway oil significantly reduced symptoms. Thus, the aim of this study was to characterize the effects of peppermint- and caraway oil, individually and in combination, on visceral nociception in a rat model of post-inflammatory visceral hyperalgesia. MATERIAL AND METHODS: On day 28, male Lewis rats (n=80) were randomized to treatment with a rectal administration of trinitrobenzene sulphonic acid (TNBS)/ethanol or physiological saline solution. To quantify the visceromotor response to a standardized colorectal distension, bipolar electrodes were implanted into the external oblique musculature, just superior to the inguinal ligament for electromyographic recordings on day 3. On day 0, baseline measurement was performed. Thereafter, oral treatment with peppermint- or caraway oil or combination treatment was started and continued for 14 consecutive days. After 7 and 14 days of treatment a colorectal distension was performed. Colonic tissue samples were obtained on days 0, 7 and 14 to assess histological alterations due to the different treatment groups and the influence of different compounds. RESULTS: After a single instillation of TNBS/ethanol persistent elevation of the visceromotor response at all different time-points was observed, although colonic mucosa was completely normal. After 14 days of combined treatment with peppermint- and caraway oil, a reduced visceromotor response of up to 50% compared to placebo was detected in TNBS/ethanol pretreated animals. In contrast, neither peppermint- nor caraway oil had a significant effect on post-inflammatory visceral hyperalgesia. In saline-treated controls there was no significant difference in the visceromotor response. CONCLUSIONS: These data show that combined treatment with peppermint- and caraway oil modulates post-inflammatory visceral hyperalgesia synergistically. The exact mechanisms have to be further investigated.