Cell Transendothelial Migration Research Articles

Noncanonical EphA2 Signaling Is a Driver of Tumor-Endothelial Cell Interactions and Metastatic Dissemination in BRAF Inhibitor‒Resistant Melanoma

Acquired BRAF/MAPK/extracellular signal‒regulated kinase inhibitor resistance in melanoma results in a new transcriptional state associated with an increased risk of metastasis. In this study, we identified noncanonical ephrin receptor (Eph) EphA2 signaling as a driver of the resistance-associated metastatic state. We used mass spectrometry‒based proteomic and phenotypic assays to demonstrate that the expression of active noncanonical EphA2-S897E in melanoma cells led to a mesenchymal-to-amoeboid transition driven by Cdc42 activation. The induction of mesenchymal-to-amoeboid transition promoted melanoma cell invasion, survival under shear stress, adhesion to endothelial cells under continuous-flow conditions, increased permeability of endothelial cell monolayers, and stimulated melanoma transendothelial cell migration. Invivo, melanoma cells expressing EphA2-S897E or active Cdc42 showed superior lung retention after tail-vain injection. Analysis of BRAF inhibitor‒sensitive and ‒resistant melanoma cells demonstrated resistance to be associated with a mesenchymal-to-amoeboid transition switch, upregulation of Cdc42 activity, increased invasion, and transendothelial migration. The drug-resistant metastatic state was dependent on histone deacetylase 8 activity. Silencing of histone deacetylase 8 led to the inhibition of EphA2 and protein kinase B phosphorylation, reduced invasion, and impaired melanoma cell-endothelial cell interactions. In summary, we have demonstrated that the metastatic state associated with acquired BRAF inhibitor resistance is dependent on noncanonical EphA2 signaling, leading to increased melanoma-endothelial cell interactions and enhanced tumor dissemination.

Heparanase promotes trans-endothelial migration of hepatocarcinoma cells by inducing apoptosis of microvascular endothelial cells

To explore the effect of heparanase (HPSE) on apoptosis of microvascular endothelial cells (MVECs) and trans-endothelial migration of hepatocellular carcinoma (HCC) cells. A HCC cell line with high HPSE expression was selected by real-time quantitative PCR (qRT-PCR) and Western blotting and transefected with a lentiviral vector containing an interfering RNA sequence of HPSE. Transwell migration assay was performed to detect the trans-endothelial migration (TEM) rate of the transfected HCC cells across human umbilical vein endothelial cells (HUVECs). In a Transwell indirect co-culture system, the effect of HPSE silencing in the HCC cells was determined on apoptosis of HUVECs in vitro. A nude mouse model of HCC was used to verify the effect of HPSE on apoptosis of MVECs and liver metastasis of the tumor. HCCLM3 cell line highly expressing HPSE was selected for the experiment. Transfection of the HCC cells with the lentiviral vector for HPSE interference the HCC cells resulted in significantly lowered TEM rate as compared with the cells transfected with the control vector (P < 0.01). In the indirect co-culture system, the survival rate of HUVECs co-cultured with HCCLM3 cells with HPSE interference was significantly higher and their apoptotic index was significantly lower than those in the control group (P < 0.05). Ultrastructural observation showed no obvious apoptosis of HUVECs co-cultured with HCCLM3 cells with HPSE interference but revealed obvious apoptotic changes in the control group. In the animal experiment, the tumor formation rate in the liver was 100% (6/6) in the control group, significantly higher than that in RNAi group (33.3%, 2/6) (P < 0.05). Under optical microscope, necrosis and apoptosis of the MVECs was detected in the liver of the control mice, while the endothelial cells remained almost intact in RNAi group. HPSE promotes the metastasis of HCC cells by inducing apoptosis of MVECs.

Adipose-derived mesenchymal stromal cells promote corneal wound healing by accelerating the clearance of neutrophils in cornea

The dome-shaped cornea is a transparent, non-vascularized, and epithelialized highly organized tissue. Physical and chemical injuries may trigger corneal wound healing (CWH) response and result in neovascularization that impairs the visual function. CWH involves not only migration, proliferation, and differentiation of the cells in different layers of cornea, but also the mobilization of immune cells. We demonstrated here that human adipose-derived mesenchymal stromal cells (ADSCs) could effectively inhibit neovascularization during ethanol-induced injury in mouse cornea. Importantly, we found that while neutrophils are essential for CWH, excessive and prolonged neutrophil retention during the granulation stage contributes to neovascularization. ADSCs were found to promote the clearance of neutrophils in the cornea during the granulation stage, likely via increasing the reverse transendothelial cell migration of CXCR4high neutrophils from cornea to the lung. Our results demonstrate that ADSCs are effective in treating CWH-induced neovascularization and modulation of neutrophil clearance could be novel strategies for better vision recovery after injury.

Bulk and single-cell gene expression analyses reveal aging human choriocapillaris has pro-inflammatory phenotype

The human choroidal vasculature is subject to age-related structural and gene expression changes implicated in age-related macular degeneration (AMD). In this study, we performed both bulk and single-cell RNA sequencing on infant (n = 4 for bulk experiments, n = 2 for single-cell experiments) and adult (n = 13 for bulk experiments, n = 6 for single-cell experiments) human donors to characterize how choroidal gene expression changes with age. Differential expression analysis revealed that aged choroidal samples were enriched in genes encoding pro-inflammatory transcription factors and leukocyte transendothelial cell migration adhesion proteins. Such genes were observed to be differentially expressed specifically within choroidal endothelial cells at the single-cell level. Immunohistochemistry experiments support transcriptional findings that CD34 is elevated in infant choriocapillaris endothelial cells while ICAM-1 is enriched in adults. These results suggest several potential drivers of the pro-inflammatory vascular phenotype observed with advancing age.

Inhibition of trans-endothelial migration of HIV-1 infected myeloid cells using virus expression dependent anti-ALCAM CRISPR-Cas9

Abstract Migration of HIV-1 infected monocytes across the endothelial barrier plays an essential role in establishing and maintenance of viral reservoir in the brain and continues despite antiretroviral therapy (ART). Using the CRISPR-Cas9 gene-editing platform, we created an inducible knockout for activated leukocytes cell adhesion molecule gene (ALCAM/CD166), an HIV-1 host dependency factor shown to play a critical role in CNS entry of infected myeloid cells. First, using lentiviral delivery, we developed several ALCAM knockout clones in monocytic U937, and their latently infected with HIV-1 equivalent: U1 cells. As expected, ALCAM−/− myeloid cells showed markedly reduced adhesion to and transmigration through monolayers of endothelial cells. Next, using AAV6 delivery, we replicated these results in primary human monocytes from three different healthy donors. Finally, to limit CRISPR-Cas9 editing to HIV-1 infected cells, we placed Cas9 expression under the control of minimal HIV-1 Tat responsive LTR promoter (−80/+66). HIV-1BAL-GFP infection of AAV6/adeno-LTR-CRISPR-anti-ALCAM treated CD4+ T cells, monocytes, and monocyte-derived macrophages (MDMs) resulted in the induction of Cas9 expression and CRISPR mediated cleavage of exon 1 of ALCAM gene in Tat expression dependent manner and inhibition of their transendothelial migration. We demonstrated the feasibility of using CRISPR gene editing to disrupt a single CAM gene, i.e, ALCAM to disable adhesion and transmigration ability of HIV-1 infected myeloid cell line cells, primary monocytes, and macrophages. Moreover, we provided data proving the achievability of creating an HIV expression dependent CRISPR platform to restrict Cas9 cleavage activity only to HIV infected cells.

CX3CL1 Induces Vertebral Microvascular Barrier Dysfunction via the Src/P115-RhoGEF/ROCK Signaling Pathway.

Trans-endothelial migration (TEM) of cancer cells is a critical step in metastasis. Micro-vascular barrier disruptions of distant organs play important roles in tumor cells TEM. The spine is a preferred site for multiple cancer cell metastases. Our previous study found that vertebral spongy bone was rich in CX3CL1 and that CX3CL1 can attract fractalkine receptor-expressing tumor cells to the spine. In the present study, we determined whether CX3CL1 was involved in vertebral micro-vascular barrier disruption and promoted tumor cell TEM after circulating tumor cells were arrested in the vertebral micro-vasculature. We examined the role of CX3CL1 in the barrier function of vertebral micro-vascular endothelial cells (VMECs) and explored the molecular mechanisms of CX3CL1-induced VMEC barrier disruption. Our results demonstrated that CX3CL1 led to F-actin formation and ZO-1 disruption in VMECs and induced the vertebral micro-vascular barrier disruption. Importantly, we found that the activation of the Src/P115-RhoGEF/ROCK signaling pathway plays an important role in CX3CL1-induced VMEC stress fiber formation, ZO-1 disruption and then vertebral micro-vascular barrier hyper-permeability. Inhibiting Src/P115-RhoGEF/ROCK signaling in VMECs effectively blocked CX3CL1-induced vertebral vascular endothelial dysfunction and subsequent tumor cell TEM. The results of this study and our previous study indicate that in addition to its chemotaxis, CX3CL1 plays a critical role in regulating vertebral micro-vascular barrier function and tumor cell TEM. CX3CL1 induced VMECs stress fiber formation, ZO-1 disruption and then vascular endothelial hyperpermeability via activation of the Src/P115-RhoGEF/ROCK signaling pathway. The inhibition of the Src/P115-RhoGEF/ROCK signaling pathway in VMECs effectively blocked tumor cells TEMs in vertebral spongy bone and maybe a potential therapeutic strategy for spine metastases in the future.

The lymph node stromal laminin α5 shapes alloimmunity.

Lymph node stromal cells (LNSCs) regulate immunity through constructing lymphocyte niches. LNSC-produced laminin α5 (Lama5) regulates CD4+ T cells but the underlying mechanisms of its functions are poorly understood. Here we show that depleting Lama5 in LNSCs resulted in decreased Lama5 protein in the LN cortical ridge (CR) and around high endothelial venules (HEVs). Lama5 depletion affected LN structure with increased HEVs, upregulated chemokines, and cell adhesion molecules, and led to greater numbers of Tregs in the T cell zone. Mouse and human T cell transendothelial migration and T cell entry into LNs were suppressed by Lama5 through the receptors α6 integrin and α-dystroglycan. During immune responses and allograft transplantation, depleting Lama5 promoted antigen-specific CD4+ T cell entry into the CR through HEVs, suppressed T cell activation, and altered T cell differentiation to suppressive regulatory phenotypes. Enhanced allograft acceptance resulted from depleting Lama5 or blockade of T cell Lama5 receptors. Lama5 and Lama4/Lama5 ratios in allografts were associated with the rejection severity. Overall, our results demonstrated that stromal Lama5 regulated immune responses through altering LN structures and T cell behaviors. This study delineated a stromal Lama5-T cell receptor axis that can be targeted for immune tolerance modulation.

Local microvascular leakage promotes trafficking of activated neutrophils to remote organs.

Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability–enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.

A biomechanical model for the transendothelial migration of cancer cells

We propose a biomechanical model for the extravasation of a tumor cell (TC) through the endothelium of a blood vessel. Based on prior in vitro observations, we assume that the TC extends a protrusion between adjacent endothelial cells (ECs) that adheres to the basement membrane via focal adhesions (FAs). As the protrusion grows in size and branches out, the actomyosin contraction along the stress fibers (SFs) inside the protrusion pulls the relatively rigid nucleus through the endothelial opening. We model the chemo-mechanics of the SFs and the FAs by following the kinetics of the active myosin motors and high-affinity integrins, subject to mechanical feedback. This is incorporated into a finite-element simulation of the extravasation process, with the contractile force pulling the nucleus of the TC against elastic resistance of the ECs. To account for the interaction between the TC nucleus and the endothelium, we consider two scenarios: solid–solid contact and lubrication by cytosol. The former gives a lower bound for the required contractile force to realize transmigration, while the latter provides a more realistic representation of the process. Using physiologically reasonable parameters, our model shows that the SF and FA ensemble can produce a contractile force on the order of 70 nN, which is sufficient to deform the ECs and enable transmigration. Furthermore, we use an atomic force microscope to measure the resistant force on a human bladder cancer cell that is pushed through an endothelium cultured in vitro. The magnitude of the required force turns out to be in the range of 70–100 nN, comparable to the model predictions.

Regulatory T cells specifically suppress conventional CD8\u03b1\u03b2 T cells in intestinal tumors of APCMin/+ mice

The presence of activated T cells in colorectal cancer tissues is a strong predictor of patient survival. Our previous studies have shown that regulatory T cells (Treg) are able to reduce T cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo in the murine APCMin/+ model for microsatellite stable intestinal tumors. In this study, we investigated the effect of Treg depletion on the density and effector functions of different TCRαβ+ and TCRγδ+ T cell populations in intestinal tumors. We used the APCMin/+\\DEREG mouse model, which harbor a diphtheria toxin receptor under the control of the FOXP3 promoter, to deplete Treg in tumor bearing mice. We found that the density of conventional TCRαβ+CD8αβ+ T cells was significantly increased in Treg-depleted tumors in comparison with Treg-proficient tumors. Furthermore, TCRαβ+CD8αβ+ T cells showed increased proliferation and activation as well as increased Granzyme B and IFN-γ production in Treg-depleted tumors. In sharp contrast, the densities and effector functions of TCRαβ+CD8αα+ T cells and TCRγδ+ T cells remained unchanged by Treg depletion. We also documented a distinct population of IL-17A+TNF+ TCRγδ+CD8− T cells in tumors, which were not affected by Treg depletion. We conclude that Treg depletion affects only conventional TCRαβ+CD8αβ+ T cells in intestinal tumors, while unconventional T cells and T cells in unaffected tissue are not altered. Immunotherapies aimed at depleting Treg from tumors may thus be a viable option for reinvigoration of conventional cytotoxic T cells with a Th1 cytokine profile.

Cerebrovascular inflammation promotes the formation of brain metastases.

Brain metastases are the most prevalent intracranial malignancy. Patient outcome is poor and treatment options are limited. Hence, new avenues must be explored to identify potential therapeutic targets. Inflammation is a known critical component of cancer progression. Intratumoral inflammation drives progression and leads to the release of circulating tumor cells (CTCs). Inflammation at distant sites promotes adhesion of CTCs to the activated endothelium and then initiates the formation of metastases. These interactions mostly involve cell adhesion molecules expressed by activated endothelial cells. For example, the vascular cell adhesion molecule-1 (VCAM-1) is known to promote transendothelial migration of cancer cells in different organs. However, it is unclear whether a similar mechanism occurs within the specialized environment of the brain. Our objective was therefore to use molecular imaging to assess the potential role of VCAM-1 in promoting the entry of CTCs into the brain. First, magnetic resonance imaging (MRI) and histological analyses revealed that cerebrovascular inflammation induced by intracranial injection of lipopolysaccharide significantly increased the expression of VCAM-1 in the Balb/c mouse brain. Next, intracardiac injection of 4T1 mammary carcinoma cancer cells in animals with cerebrovascular inflammation yielded a higher brain metastasis burden than in the control animals. Finally, blocking VCAM-1 prior to 4T1 cells injection prevented this increased metastatic burden. Here, we demonstrated that by contributing to CTCs adhesion to the activated cerebrovascular endothelium, VCAM-1 improves the capacity of CTCs to form metastatic foci in the brain.

Abstract P3-01-29: Impact of heparan sulphate binding domain of chemokine CCL21 to migration of breast cancer cells

Abstract Lymph node metastasis constitutes a key event in breast cancer progression. Chemokines are small proteins, which can promote metastatic spread by inducing cancer cell migration and invasion. Chemokine function is dependant upon their binding to both cell surface heparan sulphate (HS) molecules and to their specific receptor. Our group has demonstrated a significant increase in chemokine receptor CCR7 expression in cancerous breast epithelia compared to healthy controls. This study is designed to test the hypothesis that a non-HS binding forms of chemokine CCL21 can disrupt the normal response to CCL21, therefore reducing the metastasis of CCR7-expressing cancer cells. Truncated CCL21 chemokine (Δ98-134 c-terminal basic extension), was synthesised to investigate a possible linkage between chemokine binding capacity and cell activation. Wild type (WT) and mutant-CCL21 were tested for their ability to stimulate a dose-dependent increase in intracellular-free calcium in peripheral blood mononuclear cell (PBMC) and breast cancer epithelial cells MDA-MB-231. Mutant-CCL21 at concentrations 5 and 10nM showed potential to mobilise Ca2+ at levels similar to that produced by WT-CCl21. A series of experiments was performed to determine how deletion of the HS-binding site altered the ability of CCL21 to stimulate chemotaxis within a concentration gradient generated by free solute diffusion. PBMC stimulated to migrate by wild-type CCL21 was not significantly different from that stimulated by mutant (P> 0.05). Similar results were observed in assays using MDA-MB-231 cells. A further series of experiments was performed to compare the potential of WT and mutant-CCL21 to stimulate the migration of cells across endothelium. In contrast to results for trans-filter migration, it was found that the non HS-binding mutant stimulated no increased in transendothelial cell migration above the background at each of the tested concentrations, 10, 30 and 50 nM respectively (P>0.05). However, WT-CCL21 stimulated significant increased PBMC migration at each of the tested concentration (all P <0.001). Furthermore, the effect of heparin on chemotactic properties of WT and mutant- CCL21 was examined. Interestingly, heparin (250 µg/ml) completely inhibit the chemotaxis mediated by WT-CCL21 (5nM) (P < 0.001), whereas it did not inhibit the chemotaxis at concentrations 100, 250 & 500 µg/ml in response to mutant CCL21 (5nM) (P > 0.05). Similar assay will be performed using MDA-MB-231 cells. Work is ongoing to characterise the biophysical properties of mutant-CCL21 and determine its potential role for a therapeutic blockade of the migration of breast cancer cells in-vivo. Our primarily data showed that mutant CCL21 in xenograft brain tumor models showed substantial inhibition of tumour growth. Our results indicate that truncated CCL21 chemokine might be a potential preventive biofactor for human breast cancer metastasis by targeting chemokine receptor genes. Citation Format: Mohammed Imad Malki. Impact of heparan sulphate binding domain of chemokine CCL21 to migration of breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-29.

Abstract PD8-10: Regulation of breast tumor metastasis by the dynamic interaction between the TMEM doorway macrophage, tumor and endothelial cells

Abstract Tumor cell intravasation is an essential step in the metastatic cascade, but its exact mechanism is not completely understood. We have previously shown that the direct physical association of a tumor cell over-expressing Mena, a Tie2hi/Vegfhi macrophage and an endothelial cell, creates a micro-anatomic doorway called “tumor microenvironment of metastasis” (TMEM). TMEM are responsible for cancer cell intravasation and dissemination to distant sites. The density of TMEM doorways is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that TMEM doorways create increased localized vascular permeability which cancer cells utilize to intravasate, the precise molecular mechanisms relating TMEM-doorway function and intravasation has not been elucidated. Active TMEM doorways are found in pre-invasive and invasive ductal breast carcinoma as well as in metastatic foci in lymph nodes and lungs, indicating that TMEM-mediated cancer cell dissemination occurs not only at the primary tumor site but also at metastatic sites, which may perpetuate metastatic dissemination even after removal of the primary tumor. Thus it is essential to understand the exact molecular mechanism of TMEM-doorway function so that specific targeted therapies can be developed to intercept systemic cancer cell dissemination. We outline here the exact molecular mechanism of TMEM-doorway functions. TMEM doorway endothelial cell-secreted Ang2 (a Tie2 ligand) stimulates VEGF expression and production by the Tie2hi TMEM macrophage. Subsequently, the TMEM doorway tumor cell- secreted CSF1 stimulates local secretion of VEGF from the Tie2hi TMEM macrophages, leading to dissociation of endothelial adherens and tight junctions near TMEM and cancer cell intravasation. In addition, we show that acute blockage of CSF1R and Tie2-Ang2 signaling by inhibitors and blocking antibodies both in vitro and in mammary tumors leads to decreased macrophage VEGF production and secretion, decreased tumor cell trans-endothelial migration, and decreased TMEM-dependent vascular permeability, tumor cell dissemination and circulating tumor cells. This is the first description of the molecular mechanisms regulating TMEM doorway function and thus represents a major step in defining new biomarkers and targets for the treatment of metastatic tumors. Citation Format: Chinmay Surve, Allison S. Harney, Mary Chen, Yarong Wang, Xianjun Ye, Yu Lin, Ved Sharma, Richard Stanley, Maja H. Oktay, John S. Condeelis. Regulation of breast tumor metastasis by the dynamic interaction between the TMEM doorway macrophage, tumor and endothelial cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-10.

The role of opioids in cancer progression.

The role of opioids in cancer progression.

Engineered fluidic systems to understand lymphatic cancer metastasis.

The majority of all cancers metastasize initially through the lymphatic system. Despite this, the mechanisms of lymphogenous metastasis remain poorly understood and understudied compared to hematogenous metastasis. Over the past few decades, microfluidic devices have been used to model pathophysiological processes and drug interactions in numerous contexts. These devices carry many advantages over traditional 2D in vitro systems, allowing for better replication of in vivo microenvironments. This review highlights prominent fluidic devices used to model the stages of cancer metastasis via the lymphatic system, specifically within lymphangiogenesis, vessel permeability, tumor cell chemotaxis, transendothelial migration, lymphatic circulation, and micrometastases within the lymph nodes. In addition, we present perspectives for the future roles that microfluidics might play within these settings and beyond.

Zoledronic Acid Inhibits the RhoA-mediated Amoeboid Motility of Prostate Cancer Cells.

The bisphosphonate Zoledronic acid (ZA) is a potent osteoclast inhibitor currently used in the clinic to reduce osteoporosis and cancer-induced osteolysis. Moreover, ZA exerts an anti-tumor effect in several tumors. Despite this evidence, the relevance of ZA in prostate cancer (PCa) is not completely understood. To investigate the effect of ZA administration on the invasive properties of PC3 cells, which are characterised by RhoA-dependent amoeboid motility. The effect of ZA administration on the in vitro invasive properties of PC3 cells was evaluated by cell migration in 3D collagen matrices, immunofluorescence and Boyden assays or transendothelial migration. Lung retention and colonization assays were performed to assess the efficacy of ZA administration in vivo. PC3 cells are characterised by RhoA-dependent amoeboid motility. We now report a clear inhibition of in vitro PC3 cell invasion and RhoA activity upon ZA treatment. Moreover, to confirm a specific role of ZA in the inhibition of amoeboid motility of PC3 cells, we demonstrate that ZA interferes only partially with PC3 cells showing a mesenchymal phenotype due to both treatment with conditioned medium of cancer associated fibroblasts or to the acquisition of chemoresistance. Furthermore, we demonstrate that ZA impairs adhesion to endothelial cells and the trans-endothelial cell migration, two essential properties characterising amoeboid motility and PC3 metastatic dissemination. In vivo experiments prove the ability of ZA to inhibit the metastatic process of PC3 cells as shown by the decrease in lung colonization. This study demonstrates that ZA inhibits Rho-dependent amoeboid motility of PC3 cells, thus suggesting ZA as a potential therapy to impede the metastatic dissemination of PC3 cells.

Exosomal Thrombospondin-1 Disrupts the Integrity of Endothelial Intercellular Junctions to Facilitate Breast Cancer Cell Metastasis.

Transendothelial migration of malignant cells plays an essential role in tumor progression and metastasis. The present study revealed that treating human umbilical vein endothelial cells (HUVECs) with exosomes derived from metastatic breast cancer cells increased the number of cancer cells migrating through the endothelial cell layer and impaired the tube formation of HUVECs. Furthermore, the expression of intercellular junction proteins, including vascular endothelial cadherin (VE-cadherin) and zona occluden-1 (ZO-1), was reduced significantly in HUVECs treated with carcinoma-derived exosomes. Proteomic analyses revealed that thrombospondin-1 (TSP1) was highly expressed in breast cancer cell MDA-MB-231-derived exosomes. Treating HUVECs with TSP1-enriched exosomes similarly promoted the transendothelial migration of malignant cells and decreased the expression of intercellular junction proteins. TSP1-down regulation abolished the effects of exosomes on HUVECs. The migration of breast cancer cells was markedly increased in a zebrafish in vivo model injected with TSP1-overexpressing breast cancer cells. Taken together, these results suggest that carcinoma-derived exosomal TSP1 facilitated the transendothelial migration of breast cancer cells via disrupting the intercellular integrity of endothelial cells.

The contribution of platelets to intravascular arrest, extravasation, and outgrowth of disseminated tumor cells.

Platelets are primarily known for their contribution to hemostasis and subsequent wound healing. In addition to these functions, platelets play a role in the process of metastasis. Since the first study that suggested a metastasis-promoting function for platelets was published in 1968, various mechanisms have been proposed to explain how platelets contribute to the metastatic process. These include roles in the intravascular arrest of tumor cells, in tumor cell transendothelial migration, in the degradation of basement membrane barriers, in migration and invasion at the metastatic site, and in the proliferation of disseminated tumor cells. Nevertheless, conflicting observations about the role of platelets in these processes have also been reported. Here, we review the in vivo evidence that supports a role for platelets in metastasis formation, propose several scenarios for the contribution of platelets to tumor cell arrest and transendothelial migration, and discuss the evidence that platelets contribute to metastatic invasion and outgrowth.

(−)-Epicatechin metabolites promote vascular health through epigenetic reprogramming of endothelial-immune cell signaling and reversing systemic low-grade inflammation

Ingestion of (−)-epicatechin flavanols reverses endothelial dysfunction by increasing flow mediated dilation and by reducing vascular inflammation and oxidative stress, monocyte-endothelial cell adhesion and transendothelial monocyte migration in vitro and in vivo. This involves multiple changes in gene expression and epigenetic DNA methylation by poorly understood mechanisms. By in silico docking and molecular modeling we demonstrate favorable binding of different glucuronidated, sulfated or methylated (−)-epicatechin metabolites to different DNA methyltransferases (DNMT1/DNMT3A). In favor of this model, genome-wide DNA methylation profiling of endothelial cells treated with TNF and different (−)-epicatechin metabolites revealed specific DNA methylation changes in gene networks controlling cell adhesion-extravasation endothelial hyperpermeability as well as gamma-aminobutyric acid, renin-angiotensin and nitric oxide hypertension pathways. Remarkably, blood epigenetic profiles of an 8 weeks intervention with monomeric and oligomeric flavanols (MOF) including (−)-epicatechin in male smokers revealed individual epigenetic gene changes targeting similar pathways as the in vitro exposure experiments in endothelial cells. Furthermore, epigenetic changes following MOF diet intervention oppose atherosclerosis associated epigenetic changes. In line with biological data, the individual epigenetic response to a MOF diet is associated with different vascular health parameters (glutathione peroxidase 1 and endothelin-1 expression, acetylcholine-mediated microvascular response), in part involving systemic shifts in blood immune cell types which reduce the neutrophil–lymphocyte ratio (NLR). Altogether, our study suggests that different (−)-epicatechin metabolites promote vascular health in part via epigenetic reprogramming of endothelial-immune cell signaling and reversing systemic low-grade inflammation.

Ionizing radiation increases the endothelial permeability and the transendothelial migration of tumor cells through ADAM10-activation and subsequent degradation of VE-cadherin

BackgroundWe analyzed the changes in permeability of endothelial cell layers after photon irradiation, with a focus on the metalloproteases ADAM10 and ADAM17, and on VE-cadherin, components crucial for the integrity of endothelial intercellular junctions, and their roles in the transmigration of cancer cells through endothelial cell monolayers.MethodsPrimary HUVEC were irradiated with 2 or 4 Gy photons at a dose rate of 5 Gy/min. The permeability of an irradiated endothelial monolayer for macromolecules and tumor cells was analyzed in the presence or absence of the ADAM10/17 inhibitors GI254023X and GW280264X. Expression of ADAM10, ADAM17 and VE-Cadherin in endothelial cells was quantified by immunoblotting and qRT. VE-Cadherin was additionally analyzed by immunofluorescence microscopy and ELISA.ResultsIonizing radiation increased the permeability of endothelial monolayers and the transendothelial migration of tumor cells. This was effectively blocked by a selective inhibition (GI254023X) of ADAM10. Irradiation increased both, the expression and activity of ADAM10, which led to increased degradation of VE-cadherin, but also led to higher rates of VE-cadherin internalization. Increased degradation of VE-cadherin was also observed when endothelial monolayers were exposed to tumor-cell conditioned medium, similar to when exposed to recombinant VEGF.ConclusionsOur results suggest a mechanism of irradiation-induced increased permeability and transendothelial migration of tumor cells based on the activation of ADAM10 and the subsequent change of endothelial permeability through the degradation and internalization of VE-cadherin.