Tumor Cell Transmigration Research Articles

Normalization of Snai1-mediated vessel dysfunction increases drug response in cancer

Blood vessels in tumors are often dysfunctional. This impairs the delivery of therapeutic agents to and distribution among the cancer cells. Subsequently, treatment efficacy is reduced, and dose escalation can increase adverse effects on non-malignant tissues. The dysfunctional vessel phenotypes are attributed to aberrant pro-angiogenic signaling, and anti-angiogenic agents can ameliorate traits of vessel dysfunctionality. However, they simultaneously reduce vessel density and thereby impede drug delivery and distribution. Exploring possibilities to improve vessel functionality without compromising vessel density in the tumor microenvironment, we evaluated transcription factors (TFs) involved in epithelial-mesenchymal transition (EMT) as potential targets. Based on similarities between EMT and angiogenic activation of endothelial cells, we hypothesized that these TFs, Snai1 in particular, might serve as key regulators of vessel dysfunctionality. In vitro, experiments demonstrated that Snai1 (similarly Slug and Twist1) regulates endothelial permeability, permissiveness for tumor cell transmigration, and tip/stalk cell formation. Endothelial-specific, heterozygous knock-down of Snai1 in mice improved vascular quality in implanted tumors. This resulted in better oxygenation and reduced metastasis. Notably, the tumors in Snai1KD mice responded significantly better to chemotherapeutics as drugs were transported into the tumors at strongly increased rates and more homogeneously distributed. Thus, we demonstrate that restoring vessel homeostasis without affecting vessel density is feasible in malignant tumors. Combining such vessel re-engineering with anti-cancer drugs allows for strategic treatment approaches that reduce treatment toxicity on non-malignant tissues.

S-Nitrosylation in endothelial cells contributes to tumor cell adhesion and extravasation during breast cancer metastasis

BackgroundNitric oxide is produced by different nitric oxide synthases isoforms. NO activates two signaling pathways, one dependent on soluble guanylate cyclase and protein kinase G, and other where NO post-translationally modifies proteins through S-nitrosylation, which is the modification induced by NO in free-thiol cysteines in proteins to form S-nitrosothiols. High levels of NO have been detected in blood of breast cancer patients and increased NOS activity has been detected in invasive breast tumors compared to benign or normal breast tissue, suggesting a positive correlation between NO biosynthesis, degree of malignancy and metastasis. During metastasis, the endothelium plays a key role allowing the adhesion of tumor cells, which is the first step in the extravasation process leading to metastasis. This step shares similarities with leukocyte adhesion to the endothelium, and it is plausible that it may also share some regulatory elements. The vascular cell adhesion molecule-1 (VCAM-1) expressed on the endothelial cell surface promotes interactions between the endothelium and tumor cells, as well as leukocytes. Data show that breast tumor cells adhere to areas in the vasculature where NO production is increased, however, the mechanisms involved are unknown.ResultsWe report that the stimulation of endothelial cells with interleukin-8, and conditioned medium from breast tumor cells activates the S-nitrosylation pathway in the endothelium to induce leukocyte adhesion and tumor cell extravasation by a mechanism that involves an increased VCAM-1 cell surface expression in endothelial cells. We identified VCAM-1 as an S-nitrosylation target during this process. The inhibition of NO signaling and S-nitrosylation blocked the transmigration of tumor cells through endothelial monolayers. Using an in vivo model, the number of lung metastases was inhibited in the presence of the S-nitrosylation inhibitor N-acetylcysteine (NAC), which was correlated with lower levels of S-nitrosylated VCAM-1 in the metastases.ConclusionsS-Nitrosylation in the endothelium activates pathways that enhance VCAM-1 surface localization to promote binding of leukocytes and extravasation of tumor cells leading to metastasis. NAC is positioned as an important tool that might be tested as a co-therapy against breast cancer metastasis.

Involvement of platelet-derived VWF in metastatic growth of melanoma in the brain.

BackgroundThe prognosis of patients with brain metastases (BM) is poor despite advances in our understanding of the underlying pathophysiology. The high incidence of thrombotic complications defines tumor progression and the high mortality rate. We, therefore, postulated that von Willebrand factor (VWF) promotes BM via its ability to induce platelet aggregation and thrombosis.MethodsWe measured the abundance of VWF in the blood and intravascular platelet aggregates of patients with BM, and determined the specific contribution of endothelial and platelet-derived VWF using in vitro models and microfluidics. The relevance for the brain metastatic cascade in vivo was demonstrated in ret transgenic mice, which spontaneously develop BM, and by the intracardiac injection of melanoma cells.ResultsHigher levels of plasma VWF in patients with BM were associated with enhanced intraluminal VWF fiber formation and platelet aggregation in the metastatic tissue and peritumoral regions. Platelet activation triggered the formation of VWF multimers, promoting platelet aggregation and activation, in turn enhancing tumor invasiveness. The absence of VWF in platelets, or the blocking of platelet activation, abolished platelet aggregation, and reduced tumor cell transmigration. Anticoagulation and platelet inhibition consistently reduced the number of BM in preclinical animal models.ConclusionsOur data indicate that platelet-derived VWF is involved in cerebral clot formation and in metastatic growth of melanoma in the brain. Targeting platelet activation with low-molecular-weight heparins represents a promising therapeutic approach to prevent melanoma BM.

HOXB9 enhances the ability of lung cancer cells to penetrate the blood-brain barrier.

Even after multimodal therapy, the prognosis is dismal for patients with brain metastases from non-small cell lung cancer (NSCLC). Although the blood-brain barrier (BBB) limits tumor cell penetration into the brain parenchyma, some nevertheless colonize brain tissue through mechanisms that are not fully clear. Here we show that homeobox B9 (HOXB9), which is commonly overexpressed in NSCLC, promotes epithelial-to-mesenchymal transition (EMT) and tumor migration and invasion. Animal experiments showed that HOXB9 expression correlates positively with the brain metastatic potential of human NSCLC cells, while brain metastatic cells derived through in vivo selection showed greater HOXB9 expression than their cells of origin. Comparable results were obtained after immunohistochemical analysis of clinical primary NSCLC and matched brain metastasis samples obtained after surgery. Using an in vitro BBB model, knockdown and overexpression experiments showed that HOXB9-dependent expression of MMP9 in NSCLC cells leads to reduced expression of junctional proteins in cultured human vascular endothelial cells and enhanced transmigration of tumor cells. These data indicate that HOXB9 enables NSCLC cells to break away from the primary tumor by inducing EMT, and promotes brain metastasis by driving MMP9 production and degradation of intercellular adhesion proteins in endothelial cells comprising the BBB.

Circulating Tumor Cells Exhibit Metastatic Tropism and Reveal Brain Metastasis Drivers.

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood-brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis. SIGNIFICANCE: Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo-cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain.This article is highlighted in the In This Issue feature, p. 1.

Liver sinusoidal endothelial cell ICAM-1 mediated tumor/endothelial crosstalk drives the development of liver metastasis by initiating inflammatory and angiogenic responses

The prometastatic stroma generated through tumor cells/host cells interaction is critical for metastatic growth. To elucidate the role of ICAM-1 on the crosstalk between tumor and primary liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), implicated in tumor adhesion and angiogenesis, we performed in vitro cocultures and an in vivo model of liver metastasis of colorectal cancer (CRC). ICAM-1 blockade in the LSECs decreased the adhesion and transmigration of tumor cells through an LSEC in vitro and vivo. Cocultures of C26 cells and LSECs contained higher amounts of IL-1β, IL-6, PGE-2, TNF-α and ICAM-1 than monocultures. C26 cells incubated with sICAM-1 secreted higher amounts of PGE-2, IL-6, VEGF, and MMPs, while enhanced the migration of LSECs and HSCs. HSCs cultures activated by media from C26 cells pretreated with sICAM-1 contained the largest amounts of VEGF and MMPs. C26 cell activation with sICAM-1 enhanced their metastasizing potential in vivo, while tumor LFA-1 blockade reduced tumor burden and LSECs and HSC-derived myofibroblasts recruitment. In vivo ICAM-1 silencing produced similar results. These findings uncover LSEC ICAM-1 as a mediator of the CRC metastatic cascade in the liver and identifies it as target for the inhibition of liver colonization and metastatic progression.

The vacuolar-type ATPase inhibitor archazolid increases tumor cell adhesion to endothelial cells by accumulating extracellular collagen.

The vacuolar-type H+-ATPase (v-ATPase) is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells (ECs), in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.

Endothelial Rab7 GTPase mediates tumor growth and metastasis in lysosomal acid lipase–deficient mice

Tumors depend on their microenvironment for sustained growth, invasion, and metastasis. In this environment, endothelial cells (ECs) are an important stromal cell type interacting with malignant cells to facilitate tumor angiogenesis and cancer cell extravasation. Of note, lysosomal acid lipase (LAL) deficiency facilitates melanoma growth and metastasis. ECs from LAL-deficient (lal-/-) mice possess enhanced proliferation, migration, and permeability of inflammatory cells by activating the mammalian target of rapamycin (mTOR) pathway. Here we report that lal-/- ECs facilitated in vivo tumor angiogenesis, growth, and metastasis, largely by stimulating tumor cell proliferation, migration, adhesion, and transendothelial migration via increased expression of IL-6 and monocyte chemoattractant protein 1 (MCP-1). This prompted us to look for lysosomal proteins that are involved in lal-/- EC dysfunctions. We found that lal-/- ECs displayed increased expression of Rab7, a late endosome/lysosome-associated small GTPase. Moreover, Rab7 and mTOR were co-increased and co-localized to lysosomes and physically interacted in lal-/- ECs. Rab7 inhibition reversed lal-/- EC dysfunctions, including decreasing their enhanced migration and permeability of tumor-stimulatory myeloid cells, and suppressed EC-mediated stimulation of in vitro tumor cell transmigration, proliferation, and migration and in vivo tumor growth and metastasis. Finally, Rab7 inhibition reduced overproduction of reactive oxygen species and increased IL-6 and MCP-1 secretion in lal-/- ECs. Our results indicate that metabolic reprogramming resulting from LAL deficiency enhances the ability of ECs to stimulate tumor cell proliferation and metastasis through stimulation of lysosome-anchored Rab7 activity.

The pretubulysin-induced exposure of collagen is caused by endothelial cell retraction that results in an increased adhesion and decreased transmigration of tumor cells.

Microtubule-targeting agents (MTAs) are the most widely used chemotherapeutic drugs. Pretubulysin (PT), a biosynthetic precursor of the myxobacterial tubulysins, was recently identified as a novel MTA. Besides its strong anti-tumoral activities, PT attenuates tumor angiogenesis, exerts anti-vascular actions on tumor vessels and decreases cancer metastasis formation in vivo. The aim of the present study was to analyze the impact of PT on the interaction of endothelial and tumor cells in vitro to gain insights into the mechanism underlying its anti-metastatic effect. The influence of PT on tumor cell adhesion and transmigration onto/through the endothelium as well as its influence on cell adhesion molecules and the chemokine system CXCL12/CXCR4 was investigated. Treatment of human endothelial cells with PT increased the adhesion of breast cancer cells to the endothelial monolayer, whereas their transmigration through the endothelium was strongly reduced. Interestingly, the PT-induced upregulation of ICAM-1, VCAM-1 and CXCL12 were dispensable for the PT-evoked tumor cell adhesion. Tumor cells preferred to adhere to collagen exposed within PT-triggered endothelial gaps via β1-integrins on the tumor cell surface. Taken together, our study provides, at least in part, an explanation for the anti-metastatic potential of PT.

Abstract A53: Probing forces and modulation of cancer cell mechanical properties during transendothelial migration

Abstract For all types of cancer, the survival rate while the tumor is still localized is significantly higher than when cancer has metastasized. Though different therapeutic methods can be applied to successfully treat primary tumors, treatment of metastasised cancer is a great challenge due to its complex and systemic nature. A deeper understanding of how cancer initiates, grows and migrates is essential for creating successful therapies. Particularly, extravasation — a process during which streaming tumor cells (TCs) adhere to the blood vessels and traverse through the vascular endothelium into the surrounding tissue — is one of the crucial steps of cancer metastasis. While cancer research has mostly focused on the initial processes of metastasis, little is known about mechanisms of transmigration of tumor cells through the vascular wall. We found that during transendothelial migration (TEM), cancers cells undergo substantial shape changes transforming from semi-spherical (with a 2D contact with the endothelium) to a fully spread 3D morphology inside the ECM. This significant morphological change occurs due to forces generated intracellularly and distributed externally. During these morphological changes in transmigration, the cytoskeleton provides the force for shape change but the rate at which shape change occurs is dictated by the degree of deformability of the tumor cell, endothelial cells (ECs) and subendothelial tissue. Hence, the strength and distribution of forces, as well as mechanical properties of the cells and ECM, play a central role in the process of TEM. In this project, we investigate modulation of TCs and ECs rheological properties and the role of forces generated by the cytoskeleton and their transmission to adjacent cells and the ECM during extravasation. To address the role of mechanical modulation during TEM, we have developed in vitro assays and combed them with optical/confocal microscopy, Traction Force Microscopy (TFM) and Brillouin Confocal Microscopy (BCM). In particular, we recently designed a simple system in which ECs are cultured on a thin layer of collagen. Using a combination of different coating treatments on glass to produce surface hydrophobicity and maintain strong adhesion, we created a thin (~80 μm) layer of gel. Following seeding by ECs (HUVECs) to produce a tight and uniform EC monolayer we introduced TCs on top of the EC monolayer and monitored extravasation in real-time with high spatiotemporal resolution. Measurements of HUVEC monolayer permeability in this system show physiologically relevant values (8±3 10-8 cm2/s). We further tested our system by using different cancer cells with different invasiveness potentials and investigated their extravasation efficiencies. A major advantage of this platform is that it allows integration of TFM and BCM so it enabled us to characterize forces and modulation of cell mechanical properties during TEM. In particular, we demonstrated the feasibility of our system for application of BCM on cancer cells extravasating through a monolayer. BCM is an optical technique in which the mechanical information can be read out optically via spectral analysis of the scattered light; thanks to the interaction of light with intrinsic mechanical vibrations (phonons) of the material. Our BCM measurements indicate a significant decrease in elasticity of TCs at different stages; from 260±20 Pa for TCs that were in the process of transmigration to 195±17 Pa for those that were fully transmigrated. Our results shed a new light on the fundamental understanding of the extravasation mechanics. Furthermore, we anticipate that these studies will potentially enhance our ability to identify and screen for new therapies to inhibit the tendency for metastatic spread of disease. Citation Format: Emad Moeendarbary, Roger Kamm, Giuliano Scarcelli. Probing forces and modulation of cancer cell mechanical properties during transendothelial migration. [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 A53.

On-chip human microvasculature for real-time visualization and quantification of tumor cell extravasation and early metastatic seeding

Event Abstract Back to Event On-chip human microvasculature for real-time visualization and quantification of tumor cell extravasation and early metastatic seeding Michelle Chen1, Jordan A. Whisler1, Alexandra Boussommier1 and Roger D. Kamm1, 2 1 MIT, Mechanical Engineering, United States 2 MIT, Biological Engineering, United States Introduction: Metastasis - the cause of about 90% of cancer related deaths - condists of hematogenous dissemination of single tumor cells via the blood circulation to distant sites. However, the mechanisms involved in this early metastatic seeding remain poorly defined due to the technical challenges associated with detection, tissue isolation, imaging and analysis of single disseminated tumor cells in the vasculature and stroma of distant sites in vivo[1]. Here we present the application of an on-chip human microvasculature platform, developed and charaterized perviously[2], to allow real-time and quantitative study of the extravasation and early metastatic seeding microenvironment, including tumor cell interaction with host bloodbourne immune cells, initial arrest, extravasation, and survival/proliferation. Methods: The early seeding microenvironement is recapitulated by forming perfusable human microvascular networks with human umbilical cord vein cells and human lung fibroblasts as supporting stromal cells. Soft lithography techniques are used to form channels to separate HUVECs from NLFs, which are seeded in fibrin gels and contained via surface tension provided by microposts (Fig 1A). The microvasculature is then perfused with tumor cells and the dynamics of transendothelial migration can be readily visualized, scored and quantified at low magnifications (20X) via confocal microscopy (Fig 1B). Results: At higher magnifications (30-60X), cytoskeletal dynamics of both tumor and endothelial cells can be observed. Tumor cells protrude actin rich fillipodial-like projections into the surrounding stroma. Platelets and bone marrow derived cells (e.g. neutrophils, monocytes) can be isolated from human blood and perfused simultaneously with tumor cells to visualize and assess the importance of host-tumor interactions in the circulation and during extravasation. We demonstrate the addition of activated platelets and neutrophils separately increases both tumor cell transmigration efficiency and kinetics (Fig 2A). Tumor cell interactions with the endothelial basement membrane can also be visualized via immunofluorescent staining of different ECM proteins. Via siRNA interference, we find that extravasation is highly dependent on laminin receptors alpha 3 and alpha 6 integrins, which is supported by the close association of extravasated tumor cells to the laminin basement membrane layer (Fig 2B). Lastly, survival and proliferation of cells post-extravasation can be quantified on a single cell level up to 48 hours post seeding to mimic the dynamics of initial micrometastases formation (Fig 3). We show that extravasated cells continue to proliferate within the parenchyma while intravascular cells proliferate at a lesser extent, suggesting that cells which extravasate and gain access to the stroma are more prone to survival. Conclusion: We have demonstrated the numerous capabilities and potential of this on-chip microvacsulature platform to investigate and gain molecular mechanistic insights into the extravasation and early seeding cascades. Further, the platform has the potential to test and screen therapies targeted towards various aspects of hematogenous dissemination.

Quantification of Malignant Breast Cancer Cell MDA-MB-231 Transmigration Across Brain and Lung Microvascular Endothelium.

Tumor cell extravasation through the endothelial barrier forming the microvessel wall is a crucial step during tumor metastasis. However, where, how and how fast tumor cells transmigrate through endothelial barriers remain unclear. Using an in vitro transwell model, we performed a transmigration assay of malignant breast tumor cells (MDA-MB-231) through brain and lung microvascular endothelial monolayers under control and pathological conditions. The locations and rates of tumor cell transmigration as well as the changes in the structural components (integrity) of endothelial monolayers were quantified by confocal microscopy. Endothelial monolayer permeability to albumin P (albumin) was also quantified under the same conditions. We found that about 98% of transmigration occurred at the joints of endothelial cells instead of cell bodies; tumor cell adhesion and transmigration degraded endothelial surface glycocalyx and disrupted endothelial junction proteins, consequently increased P (albumin); more tumor cells adhered to and transmigrated through the endothelial monolayer with higher P (albumin); P (albumin) and tumor transmigration were increased by vascular endothelial growth factor, a representative of cytokines, and lipopolysaccharides, a typical systemic inflammatory factor, but reduced by adenosine 3',5'-cyclic monophosphate. These results suggest that reinforcing endothelial structural integrity is an effective approach for inhibiting tumor extravasation.

The choroid plexus may be an underestimated site of tumor invasion to the brain: an in vitro study using neuroblastoma cell lines.

BackgroundThe central nervous system (CNS) is protected by several barriers, including the blood–brain (BBB) and blood-cerebrospinal fluid (BCSFB) barriers. Understanding how cancer cells circumvent these protective barriers to invade the CNS is of crucial interest, since brain metastasis during cancer is often a fatal event in both children and adults. However, whereas much effort has been invested in elucidating the process of tumor cell transmigration across the BBB, the role of the BCSFB might still be underestimated considering the significant number of meningeal cancer involvement. Our work aimed to investigate the transmigration of neuroblastoma cells across the BCSFB in vitro.MethodsWe used an inverted model of the human BCSFB presenting proper restrictive features including adequate expression of tight-junction proteins, low permeability to integrity markers, and high trans-epithelial electrical resistance. Two different human neuroblastoma cell lines (SH-SY5Y and IMR-32) were used to study the transmigration process by fluorescent microscopy analysis.ResultsThe results show that neuroblastoma cells are able to actively cross the tight human in vitro BCSFB model within 24 h. The presence and transmigration of neuroblastoma cancer cells did not affect the barrier integrity within the duration of the experiment.ConclusionsIn conclusion, we presume that the choroid plexus might be an underestimated site of CNS invasion, since neuroblastoma cell lines are able to actively cross a choroid plexus epithelial cell layer. Further studies are warranted to elucidate the molecular mechanisms of tumor cell transmigration in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-015-0257-2) contains supplementary material, which is available to authorized users.

Quantification of Breast Cancer Cell Transmigration across Brain and Lung Microvascular Endothelium

Extravasation is a crucial step during tumor metastasis. Breast cancer cells prefer to extravasate into the lungs and brain. However, where and how tumor cells transmigrate through these organ-specific endothelial barriers to complete their extravasation remains unclear. Using an in vitro transwell model, we performed a transmigration assay of malignant breast tumor cells (MDA-MB-231) through lung or brain microvascular endothelial monolayers under control and pathological conditions. The locations and rates of tumor cell transmigration as well as the changes in structural components of endothelial monolayers were investigated by confocal microscopy. Endothelial monolayer permeability to albumin Palbumin was also quantified under the same conditions. Our results show: 1) More than 95% of transmigration occurs at the joints of endothelial cells instead of cell bodies; 2) Tumor cell adhesion and transmigration degrade endothelial surface glycocalyx and disrupt junctional proteins between adjacent endothelial cells, consequently increasing Palbumin; 3) The transmigration rate of breast tumor cells is higher through the lung endothelial monolayer with higher Palbumin; 4) Both Palbumin and tumor transmigration rates are increased by vascular endothelial growth factor (VEGF) and an inflammatory agent, lipopolysaccharides (LPS) treatments. These results suggest that reinforcing endothelial structural integrity, e.g. preserving surface glycocalyx and junction proteins, is an effective approach for inhibiting tumor extravasation. Supported by NIH SC1CA153325-01.

Abstract A04: Endothelial FAK activity controls vascular permeability and tumor metastasis

Abstract Tumor spread remains a primary cause of cancer mortality. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase co-activated by integrin and growth factor receptors in the control of vascular permeability. As FAK inhibitors are being tested in clinical trials, it is important to determine whether FAK signaling plays distinct roles in tumor versus stromal cell biology. Herein, we elucidate a new mechanism for endothelial cell (EC) FAK activity in the regulation of blood vessel barrier integrity and the passage of tumor cells through an EC barrier. We show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is phosphorylated by FAK in tumor-associated ECs. Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC Y658 phosphorylation. Both FAK inhibition and VEC Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. Although it is known that Src tyrosine kinase activity is important in controlling VEC tyrosine phosphorylation downstream of VEGF, we find that pharmacological Src inhibition prevents FAK activation. Moreover, FAK inhibition prevents both Src and FAK translocation to EC adherens junctions, but does not alter Src activation. Together, these results identify EC FAK as a key intermediate between Src and the regulation of EC barrier function controlling tumor metastasis. Citation Format: Christine Jean, Xiao Lei Chen, Isabelle Tancioni, Sean Uryu, Christine Lawson, Nichol Miller, Patric Turowski, Elisabetta Dejana, Sara Weis, David Cheresh, David D. Schlaepfer. Endothelial FAK activity controls vascular permeability and tumor metastasis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A04. doi:10.1158/1538-7445.CHTME14-A04

Abstract 3600: NanoChannel-mediated communication between tumor cells and endothelium mediates metastatic parasitism

Abstract Uncovering mechanisms underlying metastasis is the final frontier in cancer biology. Metastasis is a complex process with many discrete stages. While there has been much research on the seed (metastatic tumor cells) and the soil (colonized host tissue) hypothesis, limited knowledge exists on the interaction between metastatic cells and vascular endothelial cells, prerequisite for the development of metastatic disease. Here we describe nanoscale tubular structures, or nanoChannels (nCs), which mediate early intercellular communication between tumor epithelial cells and the endothelium in vitro and in vivo. These structures were examined in a 3D in vitro co-culture model system capturing molecular and physical interactions between metastatic cells and the endothelium. Unlike classical modes of cell-cell communication (i.e. paracrine, endocrine, and autocrine signaling), nCs allow for both rapid and directed transfer of intracellular contents from metastatic breast cancer cells to the endothelium. The ability to form nanoChannels correlates with metastatic potential of the tumor cells and enables direct tracking of both synthetic materials (chemicals and nanoparticles) and bioorganic macromolecules (proteins and miRNAs). As one specific example, we show that miR-132, which is implicated in pathological angiogenesis, can translocate from tumor cells to the endothelial cells through nCs, resulting in decreased p120RasGAP and activation of AKT in the recipient endothelial cells. Furthermore, nC-mediated communication resulted in upregulation of pathological angiogenesis markers, such as CD137 (26x, p<0.001) and CD276 (2x, p<0.001). The increase in these pathological angiogenesis markers could be reduced by pharmacological inhibition of nC formation. Combination of 500pM Docetaxel with 30nM Latrunculin A or 50nM Cytoclasin D resulted in a 3x (p<0.01) disruption of TNT structures, reducing TNT-mediated communication by 42% (p<0.0001) and 56.6% (p<0.0001), respectively. In summary, we describe a novel form of heterotypic intercellular communication between cancer cells and the host endothelium, where tumor cells can physically hijack and transform normal endothelial cells to a pathological angiogenesis phenotype. We define this process as metastatic parasitism. NanoChannel-mediated transfer of microRNAs may emerge as a new signaling mechanism in metastasis, which may function to prime the endothelium to facilitate transmigration of tumor cells and/or recruitment of vasculature to growing metastases. Elucidation of microRNAs transported through the nanoChannels presents a powerful opportunity to explain mechanisms underlying pathological angiogenesis and for the development of metastasis-specific therapeutics. Targeting the formation of these nCs and the resulting metastatic parasitism may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer. Citation Format: Yamicia D. Connor, Sarah Tekleab, Amjad Husain, Cherelle Walls, Bruce Zetter, Harold Dvorak, Shiladitya Sengupta. NanoChannel-mediated communication between tumor cells and endothelium mediates metastatic parasitism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3600. doi:10.1158/1538-7445.AM2014-3600

Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function

Pharmacological focal adhesion kinase (FAK) inhibition prevents tumor growth and metastasis, via actions on both tumor and stromal cells. In this paper, we show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is a target of FAK in tumor-associated endothelial cells (ECs). Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC-Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC-Y658 phosphorylation. Both FAK inhibition and VEC-Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. As pharmacological c-Src or FAK inhibition prevents VEGF-stimulated c-Src and FAK translocation to EC adherens junctions, but FAK inhibition does not alter c-Src activation, our experiments identify EC FAK as a key intermediate between c-Src and the regulation of EC barrier function controlling tumor metastasis.

Role of motility-related protein-1 in promoting the development of several types of cancer (Review)

Motility-related protein-1 (CD9), a type of cell surface glycoprotein comprising a four-pass transmembrane domain that forms multimeric complexes with other cell surface proteins, belongs to the tetraspanins family. From previous studies, we know that CD9 is considered to function primarily as a progression and metastasis suppressor in a variety of cancers, including breast, non-small cell lung colon and myeloma. However, an expanding body of literature has shown the contradictory outcome that tetraspanin CD9 is also vital in promoting cancer progression in several types of cancer. This review summarizes the recent studies on CD9 and concludes that it does not always act as a progression and metastasis suppressor. Conversely, in specific cases, CD9 may promote tumor progression through the following three aspects: Facilitating tumor cell transmigration, increasing tumor cell motility and hastening the growth of some cancers. In addition, CD9 appears to be an important marker of cancer stem cells in certain types of tumor.

Abstract B44: A role for endothelial αvβ3 integrin in breast tumor cell migration

Abstract Vascular endothelial growth factor (VEGF) and αvβ3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood and evasive resistance of antiangiogenic therapy has been reported. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in experimental models. DisBa-01 blocks αvβ3 integrin binding to vitronectin and inhibits integrin mediated downstream signaling cascades and cell migration. Recently, we reported that DisBa-01 strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. Here, DisBa-01 was submitted to adhesion under flow and transmigration assays, both using different DisBa-01 concentrations and β3-siMDA-MB-231 cells. In the first assay, labeled tumor cells incubated with Disba-01 perfused for 5 minutes through a chamber containing endothelial cells as coat. In the second, labeled tumor cells incubated with DisBa-01 were attracted by complete medium and transmigrated through an endothelial cell coat. Individually, assays were photographed and the cells quantified. Results show that DisBa-01 dose-dependently inhibited adhesion of MDA-MB-231 cells to human endothelial cells under flow conditions, similarly to the effect observed by β3-siMDA-MB-231 cells. In addition, the disintegrin or β3-silencing inhibited the transmigration of tumor cells through endothelial cells in vitro. These results demonstrate that endothelial αvβ3 integrin has a key role in tumor cell adhesion and transmigration through the endothelium under flow conditions. Support: Fapesp (grant no. 1998/14138-2), CNPq, and CAPES (Brazil) Citation Format: Cyntia Freitas Montenegro, Araceli Cristina Durante, Kelli Cristina Micocci, Antonio Carlos Manucci Pereira, Jr., Heloisa Sobreiro Selistre-de-Araujo. A role for endothelial αvβ3 integrin in breast tumor cell migration. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B44.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature