Confluent Endothelial Cell Layer Research Articles

Embedding Biomimetic Vascular Networks via Coaxial Sacrificial Writing into Functional Tissue.

Printing human tissues and organs replete with biomimetic vascular networks is of growing interest. While it is possible to embed perfusable channels within acellular and densely cellular matrices, they do not currently possess the biomimetic architectures found in native vessels. Here, coaxialsacrificial writing into functional tissues (co-SWIFT) is developed, an embedded bioprinting method capable of generating hierarchically branching, multilayered vascular networks within both granular hydrogel and densely cellular matrices. Coaxial printheads are designed with an extended core-shell configuration to facilitate robust core-core and shell-shell interconnections between printed branching vessels during embedded bioprinting. Using optimized core-shell ink combinations, biomimetic vessels composed of a smooth muscle cell-laden shell that surrounds perfusable lumens are coaxially printed into granular matrices composed of: 1) transparent alginate microparticles, 2) sacrificial microparticle-laden collagen, or 3) cardiac spheroids derived from human induced pluripotent stem cells. Biomimetic blood vessels that exhibit good barrier function are produced by seeding these interconnected lumens with a confluent layer of endothelial cells. Importantly, it is found that co-SWIFT cardiac tissues mature under perfusion, beat synchronously, and exhibit a cardio-effective drug response in vitro. This advance opens new avenues for the scalable biomanufacturing of vascularized organ-specific tissues for drug testing, disease modeling, and therapeutic use.

Tetramethylpyrazine-derived polyurethane for improved hemocompatibility and rapid endothelialization.

Thrombosis and intimal hyperplasia (IH) are the main factors affecting the long-term patency of small-diameter vascular grafts (SDVGs). Fabricating a confluent endothelial cell (EC) layer on surfaces with physiological elasticity to mimic vascular endothelium should be an effective strategy to prevent restenosis that is caused by thrombosis and IH. However, the vascular endothelialization process is time-consuming and always constrained by hemocompatibility of the vascular grafts, since excellent hemocompatibility could guarantee a sufficient time window for the endothelialization process. Tetramethylpyrazine (TMP)-derived polyurethane (PU) with improved hemocompatibility and accelerated endothelialization ability is synthesized by incorporating TMP moieties into PU backbones. Results show that TMP-contained PU films possess improved hemocompatibility by down-regulating platelet adhesion/activation and increasing the clotting time. Furthermore, the in vitro human umbilical vein endothelial cell (HUVEC) test demonstrates that the introduction of TMP can significantly promote HUVEC adhesion and proliferation, and thus accelerate luminal endothelialization of vascular grafts. Moreover, the TMP-containing PU films exhibit excellent biocompatibility especially for HUVECs, and their excellent, adjustable elasticity (1123%) guarantees compliance accommodation of vascular grafts. This newly synthesized TMP-containing material with multiple biological functions is expected to make up for the shortcomings of available SDVGs in clinical practice, and has significant potential in improving the long-term patency of SDVGs.

Microvasculature‐on‐a‐Post Chip That Recapitulates Prothrombotic Vascular Geometries and 3D Flow Disturbance

AbstractStenosis, characterized by partial vessel narrowing, alters blood hemodynamics and can lead to unpredictable thrombosis. Existing models struggle to accurately represent the complex vascular geometries and hemodynamics involved in such conditions. To address this challenge, a microvasculature‐on‐a‐post chip is developed to mimic partially stenotic vascular geometries and thrombogenicity, featuring isolated 3D micropost structures with variable sizes that recreate disturbed flow profiles. To emulate the diseased vessel wall, the post microfluidics are vascularized with a confluent layer of endothelial cells. Subsequently, human blood is perfused through the endothelialized post microfluidics, observing the temporal and spatial thrombotic response governed by Virchow's triad, including vessel wall injury, hemodynamic disturbance, and hypercoagulability. The innovative model offers valuable insights into stenosis‐induced thrombosis and endothelial behavior, paving the way for improved assessment of thrombotic risks associated with stenotic vessels. This advanced microfluidic platform also offers new approaches for evaluation of prothrombotic phenotypes and cardiovascular risk assessment in the future.

Endothelial-Smooth Muscle Cell Interactions in a Shear-Exposed Intimal Hyperplasia on-a-Dish Model to Evaluate Therapeutic Strategies.

Intimal hyperplasia (IH) represents a major challenge following cardiovascular interventions. While mechanisms are poorly understood, the inefficient preventive methods incentivize the search for novel therapies. A vessel‐on‐a‐dish platform is presented, consisting of direct‐contact cocultures with human primary endothelial cells (ECs) and smooth muscle cells (SMCs) exposed to both laminar pulsatile and disturbed flow on an orbital shaker. With contractile SMCs sitting below a confluent EC layer, a model that successfully replicates the architecture of a quiescent vessel wall is created. In the novel IH model, ECs are seeded on synthetic SMCs at low density, mimicking reendothelization after vascular injury. Over 3 days of coculture, ECs transition from a network conformation to confluent 2D islands, as promoted by pulsatile flow, resulting in a “defected” EC monolayer. In defected regions, SMCs incorporated plasma fibronectin into fibers, increased proliferation, and formed multilayers, similarly to IH in vivo. These phenomena are inhibited under confluent EC layers, supporting therapeutic approaches that focus on endothelial regeneration rather than inhibiting proliferation, as illustrated in a proof‐of‐concept experiment with Paclitaxel. Thus, this in vitro system offers a new tool to study EC‐SMC communication in IH pathophysiology, while providing an easy‐to‐use translational disease model platform for low‐cost and high‐content therapeutic development.

Micro/nanoscale surface engineering to enhance hemocompatibility and reduce bacterial adhesion for cardiovascular implants

Precise engineering of surface properties is essential for improving bio/hemocompatibility of medical implants. In this study, potential of electrospraying (ES) as a coating method to control surface properties was evaluated in vitro. Poly (lactic acid) was coated on 316 L stainless steel plates by ES and spin-coating methods. Surface topography and morphology were studied using confocal and scanning electron microscope (SEM), respectively. Surface hydrophobicity and mechanical properties were investigated by contact angle and nanoindentation tests. Hemocompatibility of samples evaluated using protein adsorption, hemolysis, blood coagulation, complement system and leukocyte activation experiments. Platelet response to the coatings was examined using LDH assay, SEM and ELISA tests. Cell viability studies performed on human endothelial cells using MTT assay and SEM after 1, 4 and 7 days. SEM was used to consider formation of pseudomonas aeruginosa biofilms on the coatings. The results showed that ES increased hydrophobicity (122֯ vs 90֯) and surface roughness (2 μm vs 1.2 μm) but reduced surface stiffness (200 MPa vs 10800 MPa) in comparison to spin-coated samples. ES-coatings had microbead/nanofiber morphology while spin-coated samples had micro/nanoporous structure. Importantly, an almost confluent layer of endothelial cells was observed on ES-coated samples after 4 days in contrast to spin-coated ones. Various blood experiments revealed both coatings were hemocompatible and there was no significant difference among ES and spin-coated samples. Furthermore, ES-coated samples showed significantly lower propensity for biofilm formation. In conclusion, in vitro results revealed potential of ES to engineer different coating properties including hemo/biocompatibility and biofilm resistance for cardiovascular implants applications.

Accelerated in vitro recellularization of decellularized porcine pericardium for cardiovascular grafts

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient’s cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

A pre-conditioning protocol of peripheral blood derived endothelial colony forming cells for endothelialization of tissue engineered constructs

In regenerative medicine, autologous endothelial colony forming cells (ECFCs) bear the greatest potential to be used for surface endothelialization of tissue engineered constructs, as they are easily attainable and possess a high proliferation rate. The aim of this study was to develop a standardized pre-conditioning protocol under dynamic conditions simulating the physiology of human circulation to improve the formation of a flow resistant monolayer of ECFCs and to enhance the antithrombogenicity of the endothelial cells. The main focus of the study was to consequently compare the cellular behavior under a steady laminar flow against a pulsatile flow. Mononuclear cells were isolated out of peripheral blood (PB) buffy coats and plated on uncoated tissue culture flasks in anticipation of guidelines for Advanced Therapy Medicinal Products. ECFCs were identified by typical surface markers such as CD31, CD146 and VE-Cadherin. To explore the effects of dynamic cultivation, ECFCs and human umbilical vein endothelial cells were comparatively cultured under either laminar or pulsatile (1 Hz) flow conditions with different grades of shear stress (5 dyn/cm2versus 20 dyn/cm2). High shear stress of 20 dyn/cm2 led to a significant upregulation of the antithrombotic gene marker thrombomodulin in both cell types, but only ECFCs orientated and elongated significantly after shear stress application forming a confluent endothelial cell layer. The work therefore documents a suitable protocol to pre-condition PB-derived ECFCs for sustainable endothelialization of blood contacting surfaces and provides essential knowledge for future cultivations in bioreactor systems.

Fabrication of a Perfusable 3D In Vitro Artery-Mimicking Multichannel System for Artery Disease Models.

Fabrication of a 3D in vitro model that mimics the artery takes an important role in understanding pathological cell behaviors and mechanisms of vascular diseases by proposing an advanced model that can recapitulate a native vessel condition in a controlled manner. Because a model geometry and the structure of cells are significant for the recapitulation of the hemodynamics of arterial and cell functions, it is necessary to mimic geometries and to induce the proper morphology and orientation of the cells when fabricating a model. In this study, smooth muscle cells (SMCs) and endothelial cells (ECs), which were the main elements in the arterial wall, were cocultured in a multichannel device connected with polydimethylsiloxane (PDMS) fluidic chamber modules to parallelly fabricate a pefusable 3D in vitro human artery-mimicking multichannel system. In the coculture model, a circular PDMS channel with a wrinkled-surface guided directionality and contractile morphology to SMCs, and media perfusion induced directionality to a confluent EC layer as in vivo. Protein markers of cells and synthesized extracellular matrices were demonstrated. Because multichannels were connected to a microfluidic module in a device, it was possible to easily control the microenvironmental conditions and to fabricate coculture models in parallel with a single flow system. Coculture models that can be tuned in designs such as diameter, wall shear stress, and geometry of artery disease were constructed by 3D-printed molds to recapitulate various cellular microenvironments and to model vessels effectively. Finally, the effect of wall shear stress on cells was compared using a device with four different degrees of stenosis channels and investigated in parallel.

Fabrication, characterization, and in vitro evaluation of electrospun polyurethane-gelatin-carbon nanotube scaffolds for cardiovascular tissue engineering applications.

Myocardial infarction occurs because coronary arteries insufficiency is one of the major causes of mortality worldwide. Recent studies have shown that tissue engineering of myocardial tissue to regenerate infarcted tissue or engineering of the coronary artery may help overcome this problem. In the present research, gelatin and single-walled carbon nanotube were firstly administrated to physico-chemically and biologically modulate polyurethane nanofibers. Electrospinning, as versatile and effective technique for production of functional nanoscale fiber, was applied. Incorporation of both gelatin and SWNTs reduced mean diameter of nanofibrous scaffolds from 210 to 140 nm, which influenced on initial cell behavior. Possible interaction between gelatin and SWNTs with polyurethane chains was evaluated using FTIR and DSC techniques. Regarding the incorporation of both gelatin and SWNTs, it was found that hydrophilicity of nanofibrous scaffolds dramatically improved. Scaffold degradation profile was adjusted by incorporation of gelatin. Biomimetic mechanical properties of composite scaffolds like normal blood vessel were developed and SWNTs improved the Young modulus and ultimate strength of scaffolds up to 16.47 ± 0.5 and 23.73 ± 0.5 MPa, respectively. However, addition of gelatin increased elongation at break due to its softening effect. The incorporation of the SWNTs led to significant enhancement of electrical conductivity of the scaffolds. Biological evaluation using SEM and MTT assay demonstrated that nanofibrous surface was covered by confluent and dense layer of both myocardial myoblast and endothelial cells after 7 days of culture, which is crucial for cardiovascular tissue engineering. Results verified that the fabricated scaffolds could be effective for cardiovascular tissue engineering.

Polyurethane End-Capped by Tetramethylpyrazine-Nitrone for Promoting Endothelialization Under Oxidative Stress.

Thrombus and restenosis are two main factors that cause the failure of vascular implants. Constructing a functional and confluent layer of endothelial cells (ECs) is considered an ideal method to prevent these problems. However, oxidative stress induced by the disease and implantation can damage ECs and hinder the endothelialization of implants. Thus, developing biomaterials that can protect ECs adhesion and proliferation from oxidative stress is urgently needed for the rapid endothelialization of vascular implants. In this work, a novel polyurethane (PU-TBN) is synthesized by employing tetramethylpyrazine-nitrone (TBN) as end-group to endow polymers with dual functions of antioxidant activity and promoting endothelialization. Common PU without TBN is also prepared to be control. Compared to PU, PU-TBN significantly promotes human umbilical vein endothelial cells (HUVECs) adhesion and proliferation, where cells spread well and a confluent endothelial layer is formed. PU-TBN also shows obvious free radical scavenging activity, and thus effectively attenuates oxidative stress to protect HUVECs from oxidative apoptosis. Moreover, PU-TBN exhibits enhanced antiplatelets effect, excellent biocompatibility, and similar mechanical properties to PU. These characteristics can endow PU-TBN with great potential to be used as vascular implants or coatings of other materials for rapid endothelialization under complex oxidative stress environment.

Blood‐Brain Barrier as a Novel Link between Crude Oil Exposure and Behavioral Dysfunctions?

Low levels of exposure to crude oil and/or its derivatives are associated with numerous changes in coping responses from molecular to organismal levels in vertebrates ranging from fishes to humans. Less understood are behavioral implications of exposure, with a handful of studies suggesting that crude oil leads to behavior that is “riskier” and more aggressive. Moreover, the mechanisms underlying such behavioral changes are largely unknown. Given that blood‐brain barrier (BBB) disruption is implicated in many behavioral abnormalities, a likely, although uninvestigated, link between crude oil and associated behavioral changes is blood‐brain barrier dysfunction. We hypothesize that alterations in the integrity of the BBB is correlated with oil‐induced behavioral changes. In this poster we describe our programmatic approach consisting of three phases to test this hypothesis. Our strategy, based on three experimental approaches, tests the effects of crude oil exposure in the form of High Energy Water Accommodated Fractions (HEWAFs). 1) We have quantified behavioral changes in animal models (zebrafish, betta fish, and mouse). 2) We have assessed BBB integrity by recording real‐time transendothelial electrical resistance (TEER) of in vitro “BBBs” formed by a confluent layer of brain microvascular endothelial cells (BMECs), and assessed gene expression in these cultured endothelial cells. 3) We will correlate in vivo with in vitro findings. In the first phase of our study, initial in vivo assessment using both Siamese fighting fish and zebrafish as models, indicates that fish exposed to very low levels of crude oil HEWAF show significantly increased aggressive behavior, and reduced reproductive and other behaviors, without otherwise showing morphological or growth effects. The second phase involved high‐throughput in vitro assessment using mouse BMECs as a simplified model of the BBB. These experiments indicated that acute exposure to crude oil extracts, as well as environmentally relevant concentrations ofbenzo(a)pyrene (one of the components of crude oil), resulted in significant decreases in TEER within the first 3 to 6 h of exposure. These barrier changes were accompanied by changes in expression of tight junction‐associated proteins such as significantly reduced expression in BMECs of the cell adhesion protein Cldn5, 48h after exposure to 8% HEWAF. Expression of Tjp1, a scaffolding protein that links tight junction (TJ) transmembrane proteins, was also decreased after exposure to just 2% HEWAF. The third, future phase will correlate in vivo with in vitro findings by first inducing behavioral dysfunction in animal models using crude oil HEWAF exposure, and then isolating and culturing BMECs of these animals to determine if in vivo exposure results in the same persistent changes in BBB leakiness and reduced BBB viability that was observed in the phase two in vitro studies. By applying our multi‐faceted experimental approach, we will be able to more fully elucidate the impact of crude oil on the blood‐brain barrier, and discover how this can, in turn, affect behavior in animals and man.Support or Funding InformationGulf of Mexico Research Initiative U.S. Army/Department of DefenseThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro measurements of hemodynamic forces and their effects on endothelial cell mechanics at the sub-cellular level.

This paper presents micro-particle tracking velocimetry measurements over cultured bovine aortic endothelial cell monolayers in microchannels. The objective was to quantify fluid forces and cell morphology at the sub-cellular scale for monolayers subjected to steady shear rates of 5, 10, and 20 dyn/cm2. The ultimate goal of this study was to develop an experimental methodology for in vitro detailed study of physiologically realistic healthy and diseased conditions. Cell topography, shear stress, and pressure distributions were calculated from sets of velocity fields made in planes parallel to the microchannel wall. For each experiment, measurements were made in 3 h intervals for 18 h. It was found that there is a three-dimensional change in cell morphology as a result of applied shear stress. That is, cells flatten and become more wedge shaped in the stream direction while conserving volume by spreading laterally, i.e., in the cross-stream direction. These changes in cell morphology are directly related to local variations in fluid loading, i.e., shear stress and pressure. This paper describes the first flow measurements over a confluent layer of endothelial cells that are spatially resolved at the sub-cellular scale with a simultaneous temporal resolution to quantify the response of cells to fluid loading.

A portable culture chamber for studying the effects of hydrostatic pressure on cellular monolayers

Hydrostatic pressure is one of the most fundamental and common mechanical stimuli in the body, playing a critical role in the homeostasis of all organ systems. Kidney function is affected by high blood pressure, namely hypertension, by the increased pressure acting on the glomerular capillary walls. This general effect of hypertension is diagnosed as a chronic disease, but underlying mechanistic causes are still not well understood. This paper reports a portable and adaptive device for studying the effects of hydrostatic pressure on a monolayer of cells. The fabricated device fits within a conventional incubation system and microscope. The effects of various pressures and durations were evaluated on a confluent layer of human endothelial cells. We found that a fluid pressure (i.e. hydrostatic pressure) can alter the morphology of the cells and that returning to an ambient pressure can reverse the changes in morphology. Thus, this study provides a proof-of-principle demonstration that this tool can be utilized for exploring the effects of hydrostatic pressure on mammalian cells.

A novel polyurethane modified with biomacromolecules for small-diameter vascular graft applications

There is a growing demand for small-caliber tissue-engineered vascular grafts to replace damaged vessels. Fabricated scaffolds are unable to precisely mimic the mechanical properties of native vessels, provide long-term patency and support cell adhesion and growth, in particular support endothelialization. In this study, a new biodegradable poly(ether ester urethane) urea (PEEUU) was synthesized. The synthesized polyurethane was then functionalized by introducing free amino groups through aminolysis for further surface modification by immobilization of biomacromolecules on the surface of vascular grafts. The modified surfaces were then characterized using attenuated total reflectance-Fourier transform infrared spectroscopy, water contact angle measurement and atomic force microscopy. The mechanical properties of the fabricated scaffolds were analyzed, revealing mechanical properties close to that of the natural vessels. Surface modifications led to improved cell–scaffold interactions, showing appropriate cell attachment and function on the scaffolds. A confluent layer of endothelial cells was formed on biomacromolecule-immobilized PEEUU vascular grafts. The preliminary results of this study demonstrated that the new polyurethane modified with biomacromolecules can be considered as a candidate material for vascular tissue engineering application with capability to support endothelialization of fabricated vascular grafts.

De-adhesion dynamics of melanoma cells from brain endothelial layer

Metastasis formation is a complex and not entirely understood process. The poorest prognosis and the most feared complications are associated to brain metastases. Melanoma derived brain metastases show the highest prevalence. Due to the lack of classical lymphatic drainage, in the process of brain metastases formation the haematogenous route is of primordial importance. The first and crucial step in this multistep process is the establishment of firm adhesion between the blood travelling melanoma cells and the tightly connected layer of the endothelium, which is the fundamental structure of the blood-brain barrier. This study compares the de-adhesion properties and dynamics of three melanoma cells types (WM35, A2058 and A375) to a confluent layer of brain micro-capillary endothelial cells. Cell type dependent adhesion characteristics are presented, pointing towards the existence of metastatic potential related nanomechanical aspects. Apparent mechanical properties such as elasticity, maximal adhesion force, number, size and distance of individual rupture events showed altered values pointing towards cell type dependent aspects. Our results underline the importance of mechanical details in case of intercellular interactions. Nevertheless, it suggests that in adequate circumstances elastic and adhesive characterizations might be used as biomarkers.

Cellular Shuttles: Monocytes/Macrophages Exhibit Transendothelial Transport of Nanoparticles under Physiological Flow.

A major hurdle in the development of biomedical nanoparticles (NP) is understanding how they interact with complex biological systems and navigate biological barriers to arrive at pathological targets. It is becoming increasingly evident that merely controlling particle physicochemical properties may not be sufficient to mediate particle biodistribution in dynamic environments. Thus, researchers are increasingly turning toward more complex but likewise more physiological in vitro systems to study particle--cell/particle-system interactions. An emerging paradigm is to utilize naturally migratory cells to act as so-called "Trojan horses" or cellular shuttles. We report here the use of monocytes/macrophages to transport NP across a confluent endothelial cell layer using a microfluidic in vitro model. With a custom-built flow chamber, we showed that physiological shear stress, when compared to low flow or static conditions, increased NP uptake by macrophages. We further provided a mathematical explanation for the effect of flow on NP uptake, namely that the physical exposure times of NP to cells is dictated by shear stress (i.e., flow rate) and results in increased particle uptake under flow. This study was extended to a multicellular, hydrodynamic in vitro model. Because monocytes are cells that naturally translocate across biological barriers, we utilized a monocyte/macrophage cell line as cellular NP transporters across an endothelial layer. In this exploratory study, we showed that monocyte/macrophage cells adhere to an endothelial layer and dynamically interact with the endothelial cells. The monocytes/macrophages took up NP and diapedesed across the endothelial layer with NP accumulating within the cellular uropod. These data illustrate that monocytes/macrophages may therefore act as active shuttles to deliver particles across endothelial barriers.

Abstract A38: Biophysical Regulation of Breast Cancer Metastasis

Abstract Introduction: Despite many advances in our understanding of breast cancer biology over the last decades, there are no therapies that can effectively prevent or treat metastatic cancer. During the growth of the primary tumor mass and its dissemination through the body during metastasis, cancer cells are exposed to a host of mechanical environments generated by the local matrix compliance, pressure and tension from tumor mass expansion, and fluidic shear stresses from interstitial and vascular fluid flow. These biophysical forces are emerging as powerful regulators of cancer growth, quiescence and metastasis; however, our understanding of the mechanisms of the biomechanical regulation of tumor biology remains very limited. The overall goal was to identify the role of mechanical forces in regulating key steps in tumor metastasis and progression. Results: We recently developed a mesofluidic system that allows the high throughput study of tumor cell interactions under flow. This device allows us to simulate the adhesion of cancer cells to endothelial cells under physiological flow conditions to model this step of the metastatic cascade and to apply shear forces to cells in a 96-well format. In addition, we have designed a system to apply mechanical stretch to cells in a high throughput format (576 wells simultaneously). Using these two systems, we examined the interplay between mechanical cues and the propensity of breast cancer cells to metastasize and undergo epithelial-to-mesenchymal transition (EMT). Mechanical strain increases circulating tumor cell adhesion to endothelial cells and extracellular matrix (ECM). We applied cyclic mechanical strain (5% maximal strain) to MDA-MB-231 and MCF-7 cancer cells for 24 hours and then performed adhesion assays of the cancer cells to activated and non-activated endothelial cells (ECS) and purified ECM proteins. We found that cyclic strain increased cancer cell adhesion to activated ECs in comparison to non-strained cells. In addition cancer cells exposed to mechanical strain adhered more to collagen I, laminin, and vitronectin, while they adhered less to collagen II and fibronectin in comparison to non-strained control cells. To determine which integrins were involved in the strain-induced change in adhesion, we treated the cells with a library of integrin inhibitors while applying strain for 24 hours. Cilengitide, P11, ATN-161, Bio 1211, and RGDS peptides reduced the adhesion of cancer cells back to the level of non-strained cancer cells, indicating the role of αvβ3, αvβ5, α5β1, and α4β1 integrins in the biophysical regulation of circulating tumor cell adhesion. Cyclic strain alters the ability of breast cancer cells to invade through an endothelial monolayer. We next used the high throughput system to apply multiple levels of strain to the cancer cell lines (0, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5% strain). We then trypsinized the cells and measured their invasion in a Transwell assay with a confluent layer of endothelial cells cultured on a porous membrane. We found that the mid-level strains ranging from 7.5-15% strain decreased the invasiveness of the MDA-MB-231 cells compared to the non-strained control cells. For the MCF-7 cells, mechanical strain of 5% or higher led to decreased invasion through the endothelial layer. Mechanical forces alter signaling through the TGF-β and Yap/Taz pathways as well the expression of markers of epithelial-to-mesenchymal transition (EMT). We applied cyclic strain at multiple levels (2.5-17.5% strain) to breast cancer cells for 24 hours then immunostained for various signaling proteins. In MDA-MB-231 cells, we saw a significant decrease in the intensity of Smad2/3 and nuclear phospho-Smad2/3 at mid-range strains (5-12.5% strain) as well as an increase in the nuclear localization of Yap/Taz using fluorescent immunostaining. At high levels of strain (15 and 17.5% strain), there was increased nuclear p-Smad2/3 intensity. Consistent with these findings, we found increased Smad activity in the cells using a luciferase reporter assay. An analysis of EMT markers using PCR, western blotting and immunostaining showed a reduction in mesenchymal markers including α−SMA, Slug, ZEB, fibronectin and vimentin with mid-range mechanical strain levels. Conclusion: Together, our studies demonstrate that mechanical forces can profoundly alter the propensity of cancer cells to adhere and invade during metastasis. Moreover, the response to mechanical strain is dependent on the magnitude of the strain and cancer cell type. These effects are mediated, in part, through integrin interactions and the differential regulation of the TGF-β and Hippo pathways by biophysical forces. Citation Format: Adrianne Spencer, Jason Lee, Katerina Lee, Darshil Choksi, Jerry Wang, Christopher Spruell, Aaron Baker. Biophysical Regulation of Breast Cancer Metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A38.

Direct mapping of melanoma cell - endothelial cell interactions.

The most life-threatening aspect of cancer is metastasis; cancer patient mortality is mainly due to metastasis. Among all metastases, presence of brain metastasis is one with the poorest prognosis; the median survival time can be counted in months. Therefore, prevention or decreasing their incidence would be highly desired both by patients and physicians. Metastatic cells invading the brain must breach the cerebral vasculature, primarily the blood-brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, a high-resolution force spectrograph, our aim was to explore the connections among the cell morphology, cellular mechanics, and biological function in the process of transendothelial migration of metastatic cancer cells. By immobilization of a melanoma cell to an atomic force microscope's cantilever, intercellular adhesion was directly measured at quasi-physiological conditions. Hereby, we present our latest results by using this melanoma-decorated probe. Binding characteristics to a confluent layer of brain endothelial cells was directly measured by means of single-cell force spectroscopy. Adhesion dynamics and strength were characterized, and we present data about spatial distribution of elasticity and detachment strength. These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential toward exploration of intercellular dynamic-related processes.

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface. This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell Adhesion on Surface-Functionalized Magnesium.

The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance.