A subtle relationship between substrate stiffness and collective migration of cell clusters.

The physical cues from the extracellular environment mediates cell signaling spatially and temporally. Cells respond to physical cues from their environment in a non-monotonic fashion. Despite our understanding of the role of substrate rigidity on single cell migration, how cells respond collectively to increasing extracellular matrix stiffness is not well established. Here we patterned multicellular epithelial Madin-Darby canine kidney (MDCK) islands on polyacrylamide gels of varying stiffness and studied their expansion. Our findings show that the MDCK islands expanded faster with increasing stiffness only up to an optimum stiffness, over which the expansion plateaued. We then focused on the expansion of the front of the assemblies and the formation of leader cells. We observed cell front destabilization only above substrate stiffness of a few kPa. The extension of multicellular finger-like structures at the edges of the colonies for intermediate and high stiffnesses from 6 to 60 kPa responded to higher substrate stiffness by increasing focal adhesion areas and actin cable assembly. Additionally, the number of leader cells at the finger-like protrusions increased with stiffness in correlation with an increase of the area of these multicellular protrusions. Consequently, the force profile along the epithelial fingers in the parallel and transverse directions of migration showed an unexpected relationship leading to a global force decrease with the increase of stiffness. Taken together, our findings show that epithelial cell colonies respond to substrate stiffness but in a non-trivial manner that may be of importance to understand morphogenesis and collective cell invasion during tumour progression.

Activated leukocyte cell adhesion molecule (ALCAM) is a marker of recurrence and promotes cell migration, invasion, and metastasis in early-stage endometrioid endometrial cancer.

Endometrial cancer is the most common gynaecological cancer in western countries, being the most common subtype of endometrioid tumours. Most patients are diagnosed at an early stage and present an excellent prognosis. However, a number of those continue to suffer recurrence, without means of identification by risk classification systems. Thus, finding a reliable marker to predict recurrence becomes an important unmet clinical issue. ALCAM is a cell-cell adhesion molecule and member of the immunoglobulin superfamily that has been associated with the genesis of many cancers. Here, we first determined the value of ALCAM as a marker of recurrence in endometrioid endometrial cancer by conducting a retrospective multicentre study of 174 primary tumours. In early-stage patients (N = 134), recurrence-free survival was poorer in patients with ALCAM-positive compared to ALCAM-negative tumours (HR 4.237; 95% CI 1.01-17.76). This difference was more significant in patients with early-stage moderately-poorly differentiated tumours (HR 9.259; 95% CI 2.12-53.47). In multivariate analysis, ALCAM positivity was an independent prognostic factor in early-stage disease (HR 6.027; 95% CI 1.41-25.74). Then we demonstrated in vitro a role for ALCAM in cell migration and invasion by using a loss-of-function model in two endometrial cancer cell lines. ALCAM depletion resulted in a reduced primary tumour size and reduced metastatic local spread in an orthotopic murine model. Gene expression analysis of ALCAM-depleted cell lines pointed to motility, invasiveness, cellular assembly, and organization as the most deregulated functions. Finally, we assessed some of the downstream effector genes that are involved in ALCAM-mediated cell migration; specifically FLNB, TXNRD1, and LAMC2 were validated at the mRNA and protein level. In conclusion, our results highlight the potential of ALCAM as a recurrent biomarker in early-stage endometrioid endometrial cancer and point to ALCAM as an important molecule in endometrial cancer dissemination by regulating cell migration, invasion, and metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Reentrant wetting transition in the spreading of cellular aggregates.

We study spreading on soft substrates of cellular aggregates using CT26 cells that produce an extracellular matrix (ECM). Compared to our previous work on the spreading of S180 cellular aggregates, which did not secrete ECMs, we found that the spreading velocity of the precursor film is also maximal for intermediate rigidities, but new striking features show up. First, we observed a cascade of liquid-gas-liquid (L/G/L) transitions of the precursor film as the substrate rigidity is decreased. We attribute the L/G transition to a decrease of cell/cell adhesion resulting from the weakening of the cell/substrate adhesion. We attribute the reentrant liquid phase (G/L) observed on soft substrates to the slow spreading of the aggregates on ultra-soft substrates, which gives time to the cells to secrete more ECM proteins and stick together. Second, a nematic order appears in the cohesive (liquid) states of the precursor film, attributed to the gradient of cell's velocities.

How cells flow in the spreading of cellular aggregates.

Like liquid droplets, cellular aggregates, also called "living droplets," spread onto adhesive surfaces. When deposited onto fibronectin-coated glass or polyacrylamide gels, they adhere and spread by protruding a cellular monolayer (precursor film) that expands around the droplet. The dynamics of spreading results from a balance between the pulling forces exerted by the highly motile cells at the periphery of the film, and friction forces associated with two types of cellular flows: (i) permeation, corresponding to the entry of the cells from the aggregates into the film; and (ii) slippage as the film expands. We characterize these flow fields within a spreading aggregate by using fluorescent tracking of individual cells and particle imaging velocimetry of cell populations. We find that permeation is limited to a narrow ring of width ξ (approximately a few cells) at the edge of the aggregate and regulates the dynamics of spreading. Furthermore, we find that the subsequent spreading of the monolayer depends heavily on the substrate rigidity. On rigid substrates, the migration of the cells in the monolayer is similar to the flow of a viscous liquid. By contrast, as the substrate gets softer, the film under tension becomes unstable with nucleation and growth of holes, flows are irregular, and cohesion decreases. Our results demonstrate that the mechanical properties of the environment influence the balance of forces that modulate collective cell migration, and therefore have important implications for the spreading behavior of tissues in both early development and cancer.

Multicellular aggregates: a model system for tissue rheology.

Morphogenetic processes involve cell flows. The mechanical response of a tissue to active forces is linked to its effective viscosity. In order to decouple this mechanical response from the complex genetic changes occurring in a developing organism, we perform rheometry experiments on multicellular aggregates, which are good models for tissues. We observe a cell softening behavior when submitting to stresses. As our technique is very sensitive, we were able to get access to the measurement of a yield point above which a creep regime is observed obtained for strains above 12%. To explain our rheological curves we propose a model for the cytoskeleton that we represent as a dynamic network of parallel springs, which will break under stress and reattach at null strain. Such a simple model is able to reproduce most of the important behavior of cells under strain. We highlight here the importance of considering cells as complex fluids whose properties will vary with time according to the history of applied stress.

ALCAM shedding at the invasive front of the tumor is a marker of myometrial infiltration and promotes invasion in endometrioid endometrial cancer.

Endometrial cancer (EC) is the sixth deadliest cancer in women. The depth of myometrial invasion is one of the most important prognostic factors, being directly associated with tumor recurrence and mortality. In this study, ALCAM, a previously described marker of EC recurrence, was studied by immunohistochemistry at the superficial and the invasive tumor areas from 116 EC patients with different degree of myometrial invasion and related to a set of relevant epithelial and mesenchymal markers. ALCAM expression presented a heterogeneous functionality depending on its localization, it correlated with epithelial markers (E-cadherin/ß-catenin) at the superficial area, and with mesenchymal markers at the invasive front (COX-2, SNAIL, ETV5, and MMP-9). At the invasive front, ALCAM-negativity was an independent marker of myometrial invasion. This negativity, together with an increase of soluble ALCAM in uterine aspirates from patients with an invasive EC, and its positive correlation with MMP-9 levels, suggested that ALCAM shedding by MMP-9 occurs at the invasive front. In vivo and in vitro models of invasive EC were generated by ETV5-overexpression. In those, we demonstrated that ALCAM shedding was related to a more invasive pattern and that full-ALCAM recovery reverted most of the ETV5-cells mesenchymal abilities, partially through a p-ERK dependent-manner.

Fine tuning of tissues' viscosity and surface tension through contractility suggests a new role for α-catenin.

What governs tissue organization and movement? If molecular and genetic approaches are able to give some answers on these issues, more and more works are now giving a real importance to mechanics as a key component eventually triggering further signaling events. We chose embryonic cell aggregates as model systems for tissue organization and movement in order to investigate the origin of some mechanical constraints arising from cells organization. Steinberg et al. proposed a long time ago an analogy between liquids and tissues and showed that indeed tissues possess a measurable tissue surface tension and viscosity. We question here the molecular origin of these parameters and give a quantitative measurement of adhesion versus contractility in the framework of the differential interfacial tension hypothesis. Accompanying surface tension measurements by angle measurements (at vertexes of cell-cell contacts) at the cell/medium interface, we are able to extract the full parameters of this model: cortical tensions and adhesion energy. We show that a tunable surface tension and viscosity can be achieved easily through the control of cell-cell contractility compared to cell-medium one. Moreover we show that α-catenin is crucial for this regulation to occur: these molecules appear as a catalyser for the remodeling of the actin cytoskeleton underneath cell-cell contact, enabling a differential contractility between the cell-medium and cell-cell interface to take place.