Quantitatively relating brain endothelial cell–cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program

The endothelial cell–cell junctions of the blood–brain barrier (BBB) play a pivotal role in the barrier’s function. Altered cell–cell junctions can lead to barrier dysfunction and have been implicated in several diseases. Despite this, the driving forces regulating junctional protein presentation remain relatively understudied, largely due to the lack of efficient techniques to quantify their presentation at sites of cell–cell adhesion. Here, we used our novel Junction Analyzer Program (JAnaP) to quantify junction phenotype (i.e., continuous, punctate, or perpendicular) in response to various substrate compositions, cell culture times, and cAMP treatments in human brain microvascular endothelial cells (HBMECs). We then quantitatively correlated junction presentation with barrier permeability on both a “global” and “local” scale.https://doi.org/10.1186/s12987-020-0177-

Nuclear Deformation in Response to Mechanical Confinement is Cell Type Dependent

Mechanosensing of the mechanical microenvironment by cells regulates cell phenotype and function. The nucleus is critical in mechanosensing, as it transmits external forces from the cellular microenvironment to the nuclear envelope housing chromatin. This study aims to elucidate how mechanical confinement affects nuclear deformation within several cell types, and to determine the role of cytoskeletal elements in controlling nuclear deformation. Human cancer cells (MDA-MB-231), human mesenchymal stem cells (MSCs), and mouse fibroblasts (L929) were seeded within polydimethylsiloxane (PDMS) microfluidic devices containing microchannels of varying cross-sectional areas, and nuclear morphology and volume were quantified via image processing of fluorescent cell nuclei. We found that the nuclear major axis length remained fairly constant with increasing confinement in MSCs and MDA-MB-231 cells, but increased with increasing confinement in L929 cells. Nuclear volume of L929 cells and MSCs decreased in the most confining channels. However, L929 nuclei were much more isotropic in unconfined channels than MSC nuclei. When microtubule polymerization or myosin II contractility was inhibited, nuclear deformation was altered only in MSCs in wide channels. This work informs our understanding of nuclear mechanics in physiologically relevant spaces, and suggests diverging roles of the cytoskeleton in regulating nuclear deformation in different cell types

VE-Cadherin-Independent Cancer Cell Incorporation into the Vascular Endothelium Precedes Transmigration

<div><p>Metastasis is accountable for 90% of cancer deaths. During metastasis, tumor cells break away from the primary tumor, enter the blood and the lymph vessels, and use them as highways to travel to distant sites in the body to form secondary tumors. Cancer cell migration through the endothelium and into the basement membrane represents a critical step in the metastatic cascade, yet it is not well understood. This process is well characterized for immune cells that routinely transmigrate through the endothelium to sites of infection, inflammation, or injury. Previous studies with leukocytes have demonstrated that this step depends heavily on the activation status of the endothelium and subendothelial substrate stiffness. Here, we used a previously established <i>in vitro</i> model of the endothelium and live cell imaging, in order to observe cancer cell transmigration and compare this process to leukocytes. Interestingly, cancer cell transmigration includes an additional step, which we term ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs, leading to the dislocation of EC VE-cadherin away from EC junctions bordering cancer cells, and spread into the monolayer. In some cases, ECs completely detach from the matrix. Furthermore, cancer cell incorporation occurs independently of the activation status and the subendothelial substrate stiffness for breast cancer and melanoma cells, a notable difference from the process by which leukocytes transmigrate. Meanwhile, pancreatic cancer cell incorporation was dependent on the activation status of the endothelium and changed on very stiff subendothelial substrates. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation is one of the earliest steps.</p></div

Human Neutrophil Cytoskeletal Dynamics and Contractility Actively Contribute to Trans-Endothelial Migration

<div><p>Transmigration through the endothelium is a key step in the immune response. In our recent work, the mechanical properties of the subendothelial matrix and biophysical state of the endothelium have been identified as key modulators of leukocyte trans-endothelial migration. Here, we demonstrated that neutrophil contractile forces and cytoskeletal dynamics also play an active biophysical role during transmigration through endothelial cell-cell junctions. Using our previously-established model for leukocyte transmigration, we first discovered that >93% of human neutrophils preferentially exploit the paracellular mode of transmigration in our <i>in vitro</i> model, and that is independent of subendothelial matrix stiffness. We demonstrated that inhibition of actin polymerization or depolymerization completely blocks transmigration, thus establishing a critical role for neutrophil actin dynamics in transmigration. Next, inhibition of neutrophil myosin II-mediated contractile forces renders 44% of neutrophils incapable of retracting their trailing edge under the endothelium for several minutes after the majority of the neutrophil transmigrates. Meanwhile, inhibition of neutrophil contractile forces or stabilization of microtubules doubles the time to complete transmigration for the first neutrophils to cross the endothelium. Notably, the time to complete transmigration is significantly reduced for subsequent neutrophils that cross through the same path as a previous neutrophil and is less dependent on neutrophil contractile forces and microtubule dynamics. These results suggest that the first neutrophil induces a gap in endothelial cell-cell adhesions, which “opens the door” in the endothelium and facilitates transmigration of subsequent neutrophils through the same hole. Collectively, this work demonstrates that neutrophils play an active biophysical role during the transmigration step of the immune response.</p></div

Incorporation of MDA-MB-231 does not depend on whether the endothelium is activated by TNF-α.

<p>(A) Cumulative fraction of ECs or MDA-MB-231 cells (231), SW1990 (1990), and A375 cells incorporated into the endothelium as a function of time after plating. Data points represent mean ± SEM for at least 3 independent experiments (N>20 cells for each experiment). (B) Final fraction of MDA-MB-231 cells incorporated into the untreated or TNF-α-treated endothelium after 15 hours. Bars represent mean, while error bars represent SEM of at least 3 independent experiments. P>0.05 between these values indicates there is no statistical difference (n.s.). (C) Final fraction of MDA-MB-231 breast cancer cells, ECs, A375 melanoma cells, and SW1990 pancreatic cells incorporated into the endothelium after 15 hours. Bars represent mean, while error bars represent SEM of at least 3 independent experiments. (*) indicates significance (P<0.05) when compared to ECs. (D) Plot of spreading area versus time reveals differences in spreading dynamics for MDA-MB-231 cells spreading onto a fibronectin-coated coverslip (“single cells”) or into an untreated endothelium (“into monolayer”).</p

MDA-MB-231 incorporation causes detachment and rounding of some endothelial cells.

<p>Phase contrast (left) and DiIC₁₆ fluorescence (right) images of MDA-MB-231 cells plated onto an untreated HUVEC monolayer, at time points immediately after plating (top) and after 16 hours of interaction with the endothelium (bottom). Red arrows point to phase-white cells that do not emit fluorescence; these are endothelial cells that have been forced out of the monolayer and thus have detached and become rounded. Scale bar is 25 µm and applies to all images.</p

Confocal images reveal that MDA-MB-231 cells do not migrate underneath ECs during the incorporation process.

<p>(A) A representative MDA-MB-231 (green; Actin-GFP) cell infected with GFP-actin is shown spreading into a HUVEC monolayer (red; Phalloidin). Orthogonal projections are shown. (B) Schematic showing that a cancer cell (green) displaces ECs (red) by spreading between adjacent ECs during incorporation.</p