Substrate elasticity regulates the behavior of human monocyte-derived macrophages.

Macrophages play a key role in atherosclerosis, cancer, and in the response to implanted medical devices. In each of these situations, the mechanical environment of a macrophage can vary from soft to stiff. However, how stiffness affects macrophage behavior remains uncertain. Using substrates of varying stiffness, we show macrophage phenotype and function depends on substrate stiffness. Notably, the cell area increases slightly from a sphere after 18 h on substrates mimicking healthy arterial stiffness (1-5 kPa), whereas macrophages on stiffer substrates (280 kPa-70 GPa) increased in area by nearly eight-fold. Macrophage migration is random regardless of substrate stiffness. The total average track speed was 7.8 ± 0.5 µm/h, with macrophages traveling fastest on the 280-kPa substrate (12.0 ± 0.5 µm/h) and slowest on the 3-kPa substrate (5.0 ± 0.4 µm/h). In addition F-actin organization in macrophages depends on substrate stiffness. On soft substrates, F-actin is spread uniformly throughout the cytoplasm, whereas on stiff substrates F-actin is functionalized into stress fibers. The proliferation rate of macrophages was faster on stiff substrates. Cells plated on the 280-kPa gel had a significantly shorter doubling time than those plated on the softer substrate. However, the ability of macrophages to phagocytose 1-µm particles did not depend on substrate stiffness. In conclusion, the results herein show macrophages are mechanosensitive; they respond to changes in stiffness by modifying their area, migration speed, actin organization, and proliferation rate. These results are important to understanding how macrophages respond in complex mechanical environments such as an atherosclerotic plaque.

Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury.

Axons in the peripheral nervous system respond to injury by activating retrograde injury signaling (RIS) pathways, which promote local axonal protein synthesis (LPS) and neuronal regeneration. RIS is also initiated following injury of neurons in the central nervous system (CNS). However, regulation of the localization of axonal mRNA required for LPS is not well understood. We used a hippocampal explant system to probe the regulation of axonal levels of RIS-associated transcripts following axonal injury. Axonal levels of importin ß1 and RanBP1 were elevated biphasically at 1 and 24 hrs after axotomy. Transcript levels for ß-actin, a prototypic axonally synthesized protein, were similarly elevated. Our data suggest differential regulation of axonal transcripts. At 1 hr after injury, deployment of actinomycin revealed that RanBP1, but not importin ß1, requires de novo mRNA synthesis. At 24 hrs after injury, use of importazole revealed that the second wave of increased axonal mRNA levels required importin ß-mediated nuclear import. We also observed increased importin ß1 axonal protein levels at 1 and 6 hrs after injury. RanBP1 levels and vimentin levels fluctuated but were unchanged at 3 and 6 hrs after injury. This study revealed temporally complex regulation of axonal transcript levels, and it has implications for understanding neuronal response to injury in the CNS.

Endothelial cell substrate stiffness influences neutrophil transmigration via myosin light chain kinase-dependent cell contraction.

A vast amount of work has been dedicated to the effects of shear flow and cytokines on leukocyte transmigration. However, no studies have explored the effects of substrate stiffness on transmigration. Here, we investigated important aspects of endothelial cell contraction-mediated neutrophil transmigration using an in vitro model of the vascular endothelium. We modeled blood vessels of varying mechanical properties using fibronectin-coated polyacrylamide gels of varying physiologic stiffness, plated with human umbilical vein endothelial cell (HUVEC) monolayers, which were activated with tumor necrosis factor-α. Interestingly, neutrophil transmigration increased with increasing substrate stiffness below the endothelium. HUVEC intercellular adhesion molecule-1 expression, stiffness, cytoskeletal arrangement, morphology, and cell-substrate adhesion could not account for the dependence of transmigration on HUVEC substrate stiffness. We also explored the role of cell contraction and observed that large holes formed in endothelium on stiff substrates several minutes after neutrophil transmigration reached a maximum. Further, suppression of contraction through inhibition of myosin light chain kinase normalized the effects of substrate stiffness by reducing transmigration and eliminating hole formation in HUVECs on stiff substrates. These results provide strong evidence that neutrophil transmigration is regulated by myosin light chain kinase-mediated endothelial cell contraction and that this event depends on subendothelial cell matrix stiffness.

A Case for Material Stiffness as a Design Parameter in Encapsulated Islet Transplantation.

Diabetes is a disease that plagues over 463 million people globally. Approximately 40 million of these patients have type 1 diabetes mellitus (T1DM), and the global incidence is increasing by up to 5% per year. T1DM is where the body's immune system attacks the pancreas, specifically the pancreatic beta cells, with antibodies to prevent insulin production. Although current treatments such as exogenous insulin injections have been successful, exorbitant insulin costs and meticulous administration present the need for alternative long-term solutions to glucose dysregulation caused by diabetes. Encapsulated islet transplantation (EIT) is a tissue-engineered solution to diabetes. Donor islets are encapsulated in a semipermeable hydrogel, allowing the diffusion of oxygen, glucose, and insulin but preventing leukocyte infiltration and antibody access to the transplanted cells. Although successful in small animal models, EIT is still far from commercial use owing to necessary long-term systemic immunosuppressants and consistent immune rejection. Most published research has focused on tailoring the characteristics of the capsule material to promote clinical viability. However, most studies have been limited in scope to biochemical changes. Current mechanobiology studies on the effect of substrate stiffness on the function of leukocytes, especially macrophages-primary foreign body response (FBR) orchestrators, show promise in tailoring a favorable response to tissue-engineered therapies such as EIT. In this review, we explore strategies to improve the clinical viability of EIT. A brief overview of the immune system, the FBR, and current biochemical approaches will be elucidated throughout this exploration. Furthermore, an argument for using substrate stiffness as a capsule design parameter to increase EIT efficacy and clinical viability will be posed.

Micromechanical characterizations and viscoelastic modeling reveal elastic and viscoelastic heterogeneities in ovarian tissue and the significant viscoelastic contribution to the apparent elastic modulus determined by AFM indentation.

Ovarian follicles develop in a highly regulated mechanical microenvironment and disruptions to the microenvironment may cause infertility. However, the viscoelastic properties of the ovarian tissue are not well studied. Here, we characterize both the elastic and viscoelastic properties of ovarian tissue from both reproductively older and younger domestic cats using atomic force microscopy (AFM) indentation and viscoelastic models of stress relaxation. Importantly, our analyses reveal the apparent elastic modulus obtained from the conventional AFM indentation measurement is significantly higher than the intrinsic elastic modulus and insignificantly different from the equivalent elastic modulus that is the summation of the intrinsic elastic modulus and the viscoelastic contribution to modulus at time 0. Interestingly, the ovarian cortex of both reproductive age groups has a higher apparent/intrinsic modulus than that of the medulla. Furthermore, two different kinetics of stress relaxation are identified with rate constants of ∼1 s and ∼20-40 s, respectively. Moreover, the rate constant of the slow kinetics is significantly different between the cortex and medulla in the reproductively older ovaries. Finally, these mechanical heterogeneities appear to follow the heterogeneous distribution of hyaluronic acid (HA) in the ovary. These findings may be invaluable to the development of biomimetic follicle culture for treating infertility. STATEMENT OF SIGNIFICANCE: This study investigates not only elastic but also the viscoelastic heterogeneity in both reproductively younger and older ovarian tissues for the first time. Further, by combining AFM indentation measurement and viscoelastic modeling, we show the apparent elastic modulus conventionally reported in the literature for AFM indentation measurement is the summation of the intrinsic elastic modulus and a significant viscoelastic contribution to the modulus at time 0. This is an important consideration for others who use this method to quantify biomaterial properties. In addition, the possible connection between the mechanical and compositional heterogeneities is explored. These findings may be invaluable for designing biomaterials to recapitulate the mechanical environment of the ovary and possibly many other organs for biomimetic tissue engineering.

Comparative Tensile Properties and Collagen Patterns in Domestic Cat (Felis catus) and Dog (Canis lupus familiaris) Ovarian Cortical Tissues.

The importance of the ovarian extracellular environment and tissue rigidity on follicle survival and development has gained attention in recent years. Our laboratory has anecdotally observed differences in the rigidity of domestic cat and dog ovarian cortical tissues, which have been postulated to underlie the differences in in vitro culture responses between the species, wherein cat ovarian tissues display higher survival in extended incubation. Here, the tensile strengths of cat and dog ovarian cortical tissues were compared via micropipette aspiration. The underlying collagen patterns, including fiber length, thickness, alignment, curvature, branch points and end points, and overall tissue lacunary and high-density matrix (HDM) were quantified via picrosirius red staining and TWOMBLI analysis. Finally, we explored the potential of MMP (-1 and -9) and TIMP1 supplementation in modulating tissue rigidity, collagen structure, and follicle activation in vitro. No differences in stiffness were observed between cat or dog cortical tissues, or pre- versus post-pubertal status. Cat ovarian collagen was characterized by an increased number of branch points, thinner fibers, and lower HDM compared with dog ovarian collagen, and cat tissues exposed to MMP9 in vitro displayed a reduced Young's modulus. Yet, MMP exposure had a minor impact on follicle development in vitro in either species. This study contributes to our growing understanding of the interactions among the physical properties of the ovarian microenvironment, collagen patterns, and follicle development in vitro.

Hyperglycemic Conditions Enhance the Mechanosensitivity of Proinflammatory RAW264.7 Macrophages.

Macrophages are a primary contributor to the orchestration and severity of the foreign body response. As phagocytes and antigen-presenting cells, macrophages engage foreign objects, producing chemokines, degrading enzymes, and proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Encapsulated islet transplantation (EIT) is a return of function therapy in which donor insulin-secreting cells are encased in a biomaterial and implanted into a diabetic patient to regulate blood glucose levels. However, the foreign body response by macrophages to the encapsulated islet allograft may cause rejection. Recent studies have shown that substrate stiffness affects macrophage activity, which can inform EIT capsule design. However, due to the dysregulation of glucose maintenance in diabetic patients, varying from normoglycemic to hypoglycemic or hyperglycemic conditions, it is imperative to determine if glucose dysregulation affects macrophage mechanosensitivity to EIT biomaterials. This study explores the relationship between glucose metabolism and mechanosensitivity and the ultimate impact on proinflammatory macrophage function in static hyperglycemic and normoglycemic conditions. Using a 2-dimensional (2D) polyacrylamide model of 3-order magnitude in stiffness, 2, 15, and 274 kPa Young's moduli, the effect of glycemic condition on the mechanosensitive characteristics of unstimulated and proinflammatory RAW264.7 macrophage function in vitro using lipopolysaccharide (LPS) was examined. Hyperglycemic conditions were found to impact macrophage response to substrate stiffness significantly. Notably, TNF-α secretion was significantly reduced as substrate stiffness increased in LPS-stimulated hyperglycemic conditions, whereas normoglycemic macrophages held similar secretion across all stiffnesses. Stiffness-influenced differences in cytokine secretion were also induced in IL-6 secretion by hyperglycemic conditions. Hyperglycemic conditions promoted a biphasic trend in IL-6 cytokine secretion and gene expression by proinflammatory macrophages with significantly decreased production when cultured on 15 kPa compared to production on 2 and 274 kPa. Although hyperglycemic conditions drastically increased IL-10 secretion, stiffness-influenced differences were not shown when compared to the same glycemic condition. Furthermore, under LPS stimulation, lactate secretion had an inverse relationship to TNF-α secretion. However, no significant stiffness-influenced difference was demonstrated in glucose transporter 1 (GLUT1) expression, glucose uptake, or GAPDH. These findings suggest that hyperglycemic conditions enhance the mechanosensitivity of proinflammatory macrophages and should be explored further. Impact statement The work presented increases our understanding of the effect of glycemic condition on macrophage mechanosensitivity related to substrate stiffness. This has ramifications on the design of material-based therapies, such as encapsulated islet transplantation, for type 1 diabetic patients who experience glycemic dysregulation.

Endothelial cells undergo morphological, biomechanical, and dynamic changes in response to tumor necrosis factor-α.

The immune response triggers a complicated sequence of events, one of which is release of the cytokine tumor necrosis factor-α (TNF-α) from stromal cells, for example monocytes and macrophages. In this work we investigated the biophysical effects of TNF-α on endothelial cells (ECs), including changes in cell morphology, biomechanics, migration, and cytoskeletal dynamics. We found that TNF-α induces a wide distribution of cell area and aspect ratio, with these properties increasing on average during treatment. Interestingly, aspect ratio peaks after approximately 10 h of exposure to TNF-α, corresponding also to a peak in exerted traction forces. Meanwhile, ECs treated with TNF-α soften, and we associate this with significant increases in estimated cellular volume. In addition, our evaluation of migratory dynamics revealed an inverse correlation between cell aspect ratio and migration speed after TNF-α treatment, suggesting that cell shape may be an important functional regulator of EC migration during an inflammatory response. Finally, we addressed the basic mechanics of how the reorganization of F-actin filaments occurs during TNF-α treatment, and observed a dynamic shift of existing actin filaments. Together, our results suggest a functional link between EC morphology, biomechanics, migration, and cytoskeletal dynamics during an inflammatory response.

Material properties of matrix lipids determine the conformation and intermolecular reactivity of diacetylenic phosphatidylcholine in the lipid bilayer.

Photopolymerizable phospholipid DC(8,9)PC (1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) exhibits unique assembly characteristics in the lipid bilayer. Because of the presence of the diacetylene groups, DC(8,9)PC undergoes polymerization upon UV (254 nm) exposure and assumes chromogenic properties. DC(8,9)PC photopolymerization in gel-phase matrix lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monitored by UV-vis absorption spectroscopy occurred within 2 min after UV treatment, whereas no spectral shifts were observed when DC(8,9)PC was incorporated into liquid-phase matrix 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Liquid chromatography-tandem mass spectrometry analysis showed a decrease in the amount of DC(8,9)PC monomer in both DPPC and POPC environments without any change in the matrix lipids in UV-treated samples. Molecular dynamics (MD) simulations of DPPC/DC(8,9)PC and POPC/DC(8,9)PC bilayers indicate that the DC(8,9)PC molecules adjust to the thickness of the matrix lipid bilayer. Furthermore, the motions of DC(8,9)PC in the gel-phase bilayer are more restricted than in the fluid bilayer. The restricted motional flexibility of DC(8,9)PC (in the gel phase) enables the reactive diacetylenes in individual molecules to align and undergo polymerization, whereas the unrestricted motions in the fluid bilayer restrict polymerization because of the lack of appropriate alignment of the DC(8,9)PC fatty acyl chains. Fluorescence microscopy data indicates the homogeneous distribution of lipid probe 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (N-Rh-PE) in POPC/DC(8,9)PC monolayers but domain formation in DPPC/DC(8,9)PC monolayers. These results show that the DC(8,9)PC molecules cluster and assume the preferred conformation in the gel-phase matrix for the UV-triggered polymerization reaction.

Cell blebbing and membrane area homeostasis in spreading and retracting cells.

Cells remodel their plasma membrane and cytoskeleton during numerous physiological processes, including spreading and motility. Morphological changes require the cell to adjust its membrane tension on different timescales. While it is known that endo- and exocytosis regulate the cell membrane area in a timescale of 1 h, faster processes, such as abrupt cell detachment, require faster regulation of the plasma membrane tension. In this article, we demonstrate that cell blebbing plays a critical role in the global mechanical homeostasis of the cell through regulation of membrane tension. Abrupt cell detachment leads to pronounced blebbing (which slow detachment does not), and blebbing decreases with time in a dynamin-dependent fashion. Cells only start spreading after a lag period whose duration depends on the cell's blebbing activity. Our model quantitatively reproduces the monotonic decay of the blebbing activity and accounts for the lag phase in the spreading of blebbing cells.

Neutrophils display biphasic relationship between migration and substrate stiffness.

Neutrophils are one type of migrating cell in the body's innate immune system and are the first line of defense against inflammation or infection. While extensive work exists on the effect of adhesive proteins on neutrophil motility, little is known about how neutrophil motility is affected by the mechanical properties of their physical environment. This study investigated the effects of substrate stiffness on the morphology, random motility coefficient, track speed (v), spreading area, and distribution of turning angles of neutrophils during chemokinesis. Human neutrophils were plated onto polyacrylamide gels of varying stiffness, ranging from 3 to 13 kPa, and coated with the extracellular matrix protein fibronectin, and timelapse images were taken with phase contrast microscopy. Our results show a biphasic behavior between neutrophil motility and substrate stiffness, with the optimum stiffness for motility depending on the concentration of fibronectin on the surface of the gel. On 100 microg/mL fibronectin, the optimum stiffness is 4 kPa (v = 6.9 +/- 0.6 microm/min) while on 10 microg/mL fibronectin, the optimum stiffness increases to 7 kPa (v = 4.5 +/- 2.0 microm/min). This biphasic behavior most likely arises because neutrophils on soft gels are less adherent, preventing production of traction forces, while neutrophils on stiff gels adhere strongly, resulting in decreased migration. At intermediate stiffness, however, neutrophils can attain optimal motility as a function of extracellular matrix coating.

Nuclear pores and membrane holes: generic models for confined chains and entropic barriers in pore stabilization.

The lumen of the nuclear pore complex is increasingly understood to be lined by a polymer brush that entropically regulates transport in and out of the nucleus-and it seems likely that similar effects probably arise with glycocalyx-lined holes in cell membranes. Here we mimic such pore-confined brushes with self-assembled polymer membranes imbued with nano-holes. Experiment and theory help elucidate the entropic origin and stabilization of the pores, which appear to have a similar basis as steric stabilization of colloids bearing polymer brushes. Free energies of interacting brushes reveal stable minima at pore sizes smaller than the classical metastable point, with little effect of the particular pore geometry. Such entropic forces have potential implications for lock and key mechanisms of nuclear pore assembly as well as transient poration of cells and synthetic nano-pores with regulatory mechanisms for transport.

Extracellular matrix stiffness and cell contractility control RNA localization to promote cell migration.

Numerous RNAs are enriched within cellular protrusions, but the underlying mechanisms are largely unknown. We had shown that the APC (adenomatous polyposis coli) protein controls localization of some RNAs at protrusions. Here, using protrusion-isolation schemes and RNA-Seq, we find that RNAs localized in protrusions of migrating fibroblasts can be distinguished in two groups, which are differentially enriched in distinct types of protrusions, and are additionally differentially dependent on APC. APC-dependent RNAs become enriched in high-contractility protrusions and, accordingly, their localization is promoted by increasing stiffness of the extracellular matrix. Dissecting the underlying mechanism, we show that actomyosin contractility activates a RhoA-mDia1 signaling pathway that leads to formation of a detyrosinated-microtubule network, which in turn is required for localization of APC-dependent RNAs. Importantly, a competition-based approach to specifically mislocalize APC-dependent RNAs suggests that localization of the APC-dependent RNA subgroup is functionally important for cell migration.Adenomatous polyposis coli (APC) regulates the localization of some mRNAs at cellular protrusions but the underlying mechanisms and functional roles are not known. Here the authors show that APC-dependent RNAs are enriched in contractile protrusions, via detyrosinated microtubules, and enhance cell migration.

TRPV4 calcium-permeable channel is a novel regulator of oxidized LDL-induced macrophage foam cell formation.

Cardiovascular disease is the number one cause of death in United States, and atherosclerosis, a chronic inflammatory arterial disease, is the most dominant underlying pathology. Macrophages are thought to orchestrate atherosclerosis by generating lipid-laden foam cells and by secreting inflammatory mediators. Emerging data support a role for a mechanical factor, e.g., matrix stiffness, in regulation of macrophage function, vascular elasticity, and atherogenesis. However, the identity of the plasma membrane mechanosensor and the mechanisms by which pro-atherogenic signals are transduced/maintained are unknown. We have obtained evidence that TRPV4, an ion channel in the transient receptor potential vanilloid family and a known mechanosensor, is the likely mediator of oxidized low-density lipoprotein (oxLDL)-dependent macrophage foam cell formation, a critical process in atherogenesis. Specifically, we found that: i) genetic ablation of TRPV4 or pharmacologic inhibition of TRPV4 activity by a specific antagonist blocked oxLDL-induced macrophage foam cell formation, and ii) TRPV4 deficiency prevented pathophysiological range matrix stiffness or scratch-induced exacerbation of oxLDL-induced foam cell formation. Mechanistically, we found that: i) plasma membrane localization of TRPV4 was sensitized to the increasing level of matrix stiffness, ii) lack of foam cell formation in TRPV4 null cells was not due to lack of expression of CD36, a major receptor for oxLDL, and iii) TRPV4 channel activity regulated oxLDL uptake but not its binding on macrophages. Altogether, these findings identify a novel role for TRPV4 in regulating macrophage foam cell formation by modulating uptake of oxLDL. These findings suggest that therapeutic targeting of TRPV4 may provide a selective approach to the treatment of atherosclerosis.

NKCC1 Regulates Migration Ability of Glioblastoma Cells by Modulation of Actin Dynamics and Interacting with Cofilin.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. The mechanisms that confer GBM cells their invasive behavior are poorly understood. The electroneutral Na+-K+-2Cl- co-transporter 1 (NKCC1) is an important cell volume regulator that participates in cell migration. We have shown that inhibition of NKCC1 in GBM cells leads to decreased cell migration, in vitro and in vivo. We now report on the role of NKCC1 on cytoskeletal dynamics. We show that GBM cells display a significant decrease in F-actin content upon NKCC1 knockdown (NKCC1-KD). To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition, we studied NKCC1 protein interactions. We found that NKCC1 interacts with the actin-regulating protein Cofilin-1 and can regulate its membrane localization. Finally, we analyzed whether NKCC1 could regulate the activity of the small Rho-GTPases RhoA and Rac1. We observed that the active forms of RhoA and Rac1 were decreased in NKCC1-KD cells. In summary, we report that NKCC1 regulates GBM cell migration by modulating the cytoskeleton through multiple targets including F-actin regulation through Cofilin-1 and RhoGTPase activity. Due to its essential role in cell migration NKCC1 may serve as a specific therapeutic target to decrease cell invasion in patients with primary brain cancer.

Development of a 3D Printed, Bioengineered Placenta Model to Evaluate the Role of Trophoblast Migration in Preeclampsia.

Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality. Current research suggests that the impaired trophoblastic invasion of maternal spiral arteries contributes significantly to the development of PE. However, the pathobiology of PE remains poorly understood, and there is a lack of treatment options largely due to ineffective experimental models. Utilizing the capability of bioprinting and shear wave elastography, we developed a 3D, bioengineered placenta model (BPM) to study and quantify cell migration. Through BPM, we evaluated the effect of epidermal growth factor (EGF) on the migratory behavior of trophoblast and human mesenchymal stem cells. Our results demonstrate a positive correlation between cell migration rates and EGF concentration. These results indicate that a feasible ex vivo placental model can be bioprinted to examine cellular, molecular, and pharmacologic interactions. In addition, EGF clearly affects the celluar migration, a potential therapeutic agent to treat preeclampsia. We envision that our ex vivo tissue modeling approach can be readily transferred to study other normal biologic and abnormal pathologic processes such as fibroblast migration in wound healing and stem cell homing.

Domain unfolding in neurofilament sidearms: effects of phosphorylation and ATP.

Lateral projections of neurofilaments (NF) called sidearms (SA) affect axon stability and caliber. SA phosphorylation is thought to modulate inter-NF distance and interactions between NF and other subcellular organelles. SA were probed by atomic force microscopy (AFM) and dynamic light scattering (DLS) as a function of phosphorylation and ATP content. DLS shows SA are larger when phosphorylated, and AFM shows four unfoldable domains in SA regardless of phosphorylation state or the presence of ATP. However, the native phosphorylated SA requires three-fold higher force to unfold by AFM than dephosphorylated SA, suggesting a less pliant as well as larger structure when phosphorylated.

Mouse hippocampal explant culture system to study isolated axons.

BACKGROUND: Studies of neuronal regeneration require examination of axons independently of their cell bodies. Several effective strategies have been deployed to compartmentalize long axons of the peripheral nervous system (PNS). However, current strategies to compartmentalize axons of the central nervous system (CNS) may be limited by physical damage to cells during tissue dissociation or slicing, perturbation of three-dimensional tissue architecture, or insufficient axonal tissue for biological analysis. NEW METHODS: We developed a novel mouse neonate whole-hippocampus explant culture system, to probe neuronal regeneration in the central nervous system. This system enables imaging, biological, and biophysical analysis of isolated axons. RESULTS: We validated this model by isolating pure axonal populations. Additionally, cells within the explant were viable and amenable to transfection. We implemented the explant system to characterize axonal outgrowth following crush injury to the explant at the time of harvest, and also a secondary axonal transection injury 2 days post-culture. The initial crush injury delayed axonal outgrowth; however, axotomy did not alter rates of outgrowth up to 1h post-injury, with or without initial tissue crush injury. COMPARISON WITH EXISTING METHODS: Our explant system addresses shortcomings of other strategies developed to compartmentalize CNS axons. It provides a simple method to examine axonal activity and function without requiring additional equipment to slice tissue or segregate axons. CONCLUSION: Our hippocampal explant model may be used to study axonal response to injury. We have demonstrated the feasibility of probing axonal biology, biochemistry, and outgrowth free from confounding effects of neuronal cell bodies.

VE-cadherin-independent cancer cell incorporation into the vascular endothelium precedes transmigration.

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 in vitro 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.

Human neutrophil cytoskeletal dynamics and contractility actively contribute to trans-endothelial migration.

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 in vitro 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.