The interaction between CD44 on tumour cells and hyaluronan under physiologic flow conditions: implications for metastasis formation

The adhesion of tumour cells to the endothelial cells of blood vessels of the microcirculation represents a crucial step in haematogenous metastasis formation. Similar to leukocyte extravasation, selectins mediate initial tumour cell rolling on endothelium. An additional mechanism of leukocyte adhesion to endothelial cells is mediated by hyaluronan (HA). However, data on the interaction of tumour cells with hyaluronan under shear stress are lacking. The expression of the hyaluronan binding protein CD44 on tumour cell surfaces was evaluated using flow cytometry. The adhesion of tumour cells to HA with regard to adhesive events and rolling velocity was determined in flow assays in the human small cell lung cancer (SCLC) cell lines SW2, H69, H82, OH1 and OH3, the colon carcinoma cell line HT29 and the melanoma cell line MeWo. Hyaluronan deposition in human and mouse lung blood vessels was histochemically determined. MeWo adhered best to HA followed by HT29. SCLC cell lines showed the lowest CD44 expression on the cell surface and lowest number of adhesive events. While hyaluronan was deposited in patches in the microvasculature of the alveolar septum in the human lung, it was only present in the periarterial space in the mouse lung. Certain tumour entities bind to HA under physiological shear stresses so that HA can be considered a further ligand for cell extravasation in haematogenous metastasis. As hyaluronan is deposited within the pulmonary microvasculature, it may well serve as a ligand for its binding partner CD44, which is expressed by many tumour cells.     

Selectins Mediate Small Cell Lung Cancer Systemic Metastasis

Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC) patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181). However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.     

Crossing the endothelium

Diapedesis is a vital part of tumour metastasis, whereby tumour cells attach to and cross the endothelium to enter the circulation. Specific adhesion molecules, expressed by both the tumour and endothelial cells, mediate this process. This review summarises recent findings regarding the mechanisms by which colon cancer cells migrate through the endothelium under flow conditions mediated by E-selectin. Using a laminar flow chamber and a tissue engineered human blood vessel, E-selectin was found to regulate initial attachment and rolling of colon cancer cells and also the subsequent diapedesis through the endothelium. Three different mechanisms of diapedesis were reported to be regulated by E-selectin; the formation of a mosaic chimeric layer of tissue, paracellular diapedesis between endothelial cells and transcellular diapedesis, in which tumour cells were transported via large vacuoles within the endothelial cells. Moreover activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase by E-selectin was further required for paracellular diapedesis. This study is the first to report these observations under dynamic and shear stress conditions.     

Gangliosides expressed on breast cancer cells are E-selectin ligands

Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood flow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specifically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLe^x) and sialyl Lewis A (sLe^a) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to significantly alter the carbohydrate expression profiles, binding to soluble E-selectin–Ig chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectin–Ig chimera and adhesion of E-selectin transfected cells under physiological flow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis.     

Biophysics of selectin-ligand interactions in inflammation and cancer

Selectins (L-, E- and P-selectin) are calcium-dependent transmembrane glycoproteins that are expressed on the surface of circulating leukocytes, activated platelets, and inflamed endothelial cells. Selectins bind predominantly to sialofucosylated glycoproteins and glycolipids (E-selectin only) present on the surface of apposing cells, and mediate transient adhesive interactions pertinent to inflammation and cancer metastasis. The rapid turnover of selectin-ligand bonds, due to their fast on- and off-rates along with their remarkably high tensile strengths, enables them to mediate cell tethering and rolling in shear flow. This paper presents the current body of knowledge regarding the role of selectins in inflammation and cancer metastasis, and discusses experimental methodologies and mathematical models used to resolve the biophysics of selectin-mediated cell adhesion. Understanding the biochemistry and biomechanics of selectin-ligand interactions pertinent to inflammatory disorders and cancer metastasis may provide insights for developing promising therapies and/or diagnostic tools to combat these disorders.     

Circulating Tumor Cells from Prostate Cancer Patients Interact with E-Selectin under Physiologic Blood Flow

Hematogenous metastasis accounts for the majority of cancer-related deaths, yet the mechanism remains unclear. Circulating tumor cells (CTCs) in blood may employ different pathways to cross blood endothelial barrier and establish a metastatic niche. Several studies provide evidence that prostate cancer (PCa) cell tethering and rolling on microvascular endothelium via E-selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the development of metastases. However, it is unknown if CTCs from PCa patients interact with E-selectin expressed on endothelium, initiating a route for tumor metastases. Here we report that CTCs derived from PCa patients showed interactions with E-selectin and E-selectin expressing endothelial cells. To examine E-selectin-mediated interactions of PCa cell lines and CTCs derived from metastatic PCa patients, we used fluorescently-labeled anti-prostate specific membrane antigen (PSMA) monoclonal antibody J591-488 which is internalized following cell-surface binding. We employed a microscale flow device consisting of E-selectin-coated microtubes and human umbilical vein endothelial cells (HUVECs) on parallel-plate flow chamber simulating vascular endothelium. We observed that J591-488 did not significantly alter the rolling behavior in PCa cells at shear stresses below 3 dyn/cm². CTCs obtained from 31 PCa patient samples showed that CTCs tether and stably interact with E-selectin and E-selectin expressing HUVECs at physiological shear stress. Interestingly, samples collected during disease progression demonstrated significantly more CTC/E-selectin interactions than samples during times of therapeutic response (p=0.016). Analysis of the expression of sialyl Lewis X (sLe^x) in patient samples showed that a small subset comprising 1.9-18.8% of CTCs possess high sLe^x expression. Furthermore, E-selectin-mediated interactions between prostate CTCs and HUVECs were diminished in the presence of anti-E-selectin neutralizing antibody. CTC-Endothelial interactions provide a novel insight into potential adhesive mechanisms of prostate CTCs as a means to initiate metastasis.     

Integrin VLA-4 enhances sialyl-Lewisx/a-negative melanoma adhesion to and extravasation through the endothelium under low flow conditions

During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells. The capacity of tumor cells to form metastasis is related to their ability to interact with and extravasate through endothelial cell layers, which involves multiple adhesive interactions between tumor cells and endothelium (EC). Thus it is essential to identify the adhesive receptors on the endothelial and melanoma surface that mediate those specific adhesive interactions. P-selectin and E-selectin have been reported as adhesion molecules that mediate the cell-cell interaction of endothelial cells and melanoma cells. However, not all melanoma cells express ligands for selectins. In this study, we elucidated the molecular constituents involved in the endothelial adhesion and extravasation of sialyl-Lewis(x/a)-negative melanoma cell lines under flow in the presence and absence of polymorphonuclear neutrophils (PMNs). Results show the interactions of alpha(4)beta(1) (VLA-4) on sialyl-Lewis(x/a)-negative melanoma cells and vascular adhesion molecule (VCAM-1) on inflamed EC supported melanoma adhesion to and subsequent extravasation through the EC in low shear flow. These findings provide clear evidence for a direct role of the VLA-4/VCAM-1 pathway in melanoma cell adhesion to and extravasation through the vascular endothelium in a shear flow. PMNs facilitated melanoma cell extravasation under both low and high shear conditions via the involvement of distinct molecular mechanisms. In the low shear regime, beta(2)-integrins were sufficient to enhance melanoma cell extravasation, whereas in the high shear regime, selectin ligands and beta(2)-integrins on PMNs were necessary for facilitating the melanoma extravasation process.     

Rolling adhesion of leukocytes on soft substrates: Does substrate stiffness matter?

Cell rolling on vascular endothelium under hydrodynamic blood flow is critical for realization of many physiological and pathological processes, such as inflammatory response and tumor metastasis. The blood-borne cells are in direct contact with the inner layer of endothelium, formed by a highly compliant layer of endothelial cells. The effect of endothelial stiffness on the adhesion and motion of rolling cells is poorly understood. Inspired by recent in vitro studies, here we implemented a computational method to model the specific adhesion of a rolling cell onto a soft substrate, subjected to a creeping shear flow. The substrate is modeled as an elastic half-space, coated with P- and E-selectin receptors with specific affinity for the complementary ligands located on the moving cell. Of particular importance is to predict the effect of substrate stiffness on cell adhesion and its kinematics and kinetics of motion. Simulation results show that the effect of substrate compliance is minimal when coated with P-selectin. Conversely, the trajectory of rolling cells on E-selectin coated substrates is sensitive to the substrate compliance. This is attributed to the moderation of binding forces applied by the soft substrate which leads to a higher average translational velocity of cells.     

Human soluble E-selectin immunoadhesin inhibits leukemic monocyte adhesion to endothelial cells

Immunoadhesins are immunoglobulin (Ig)-like chimeric proteins comprised of target-binding regions fused to the Fc-hinge region of Ig, and are designed to have a long half-life and antibody-like properties. In an effort to find a good candidate for therapeutic use for inflammatory responses, we constructed a soluble human E-selectin immunoadhesin containing the extracellular region of human E-selectin fused to the Fc-hinge region of human IgG, and determined its effects on leukocyte adhesion and rolling in vitro. Our results revealed that the adhesion of leukocytes to endothelial cells was efficiently inhibited in the presence of 50 nM E-selectin immunoadhesin. In addition, the E-selectin immunoadhesin significantly inhibited leukocyte rolling on endothelial cells in perfusion experiments performed at 1.0 dyne/cm(2) wall shear stress. These findings indicate that our E-selectin immunoadhesin decreases leukocyte attachment and rolling in vitro, suggesting that this immunoadhesin may be a promising candidate for therapeutic anti-inflammatory use.     

Mac-2 binding protein is a novel e-selectin ligand expressed by breast cancer cells

Hematogenous metastasis involves the adhesion of circulating tumor cells to vascular endothelium of the secondary site. We hypothesized that breast cancer cell adhesion is mediated by interaction of endothelial E-selectin with its glycoprotein counter-receptor(s) expressed on breast cancer cells. At a hematogenous wall shear rate, ZR-75-1 breast cancer cells specifically adhered to E-selectin expressing human umbilical vein endothelial cells when tested in parallel plate flow chamber adhesion assays. Consistent with their E-selectin ligand activity, ZR-75-1 cells expressed flow cytometrically detectable epitopes of HECA-452 mAb, which recognizes high efficiency E-selectin ligands typified by sialofucosylated moieties. Multiple E-selectin reactive proteins expressed by ZR-75-1 cells were revealed by immunoprecipitation with E-selectin chimera (E-Ig chimera) followed by Western blotting. Mass spectrometry analysis of the 72 kDa protein, which exhibited the most prominent E-selectin ligand activity, corresponded to Mac-2 binding protein (Mac-2BP), a heretofore unidentified E-selectin ligand. Immunoprecipitated Mac-2BP expressed sialofucosylated epitopes and possessed E-selectin ligand activity when tested by Western blot analysis using HECA-452 mAb and E-Ig chimera, respectively, demonstrating that Mac-2BP is a novel high efficiency E-selectin ligand. Furthermore, silencing the expression of Mac-2BP from ZR-75-1 cells by shRNA markedly reduced their adhesion to E-selectin expressing cells under physiological flow conditions, confirming the functional E-selectin ligand activity of Mac-2BP on intact cells. In addition to ZR-75-1 cells, several other E-selectin ligand positive breast cancer cell lines expressed Mac-2BP as detected by Western blot and flow cytometry, suggesting that Mac-2BP may be an E-selectin ligand in a variety of breast cancer types. Further, invasive breast carcinoma tissue showed co-localized expression of Mac-2BP and HECA-452 antigens by fluorescence microscopy, underscoring the possible role of Mac-2BP as an E-selectin ligand. In summary, breast cancer cells express Mac-2BP as a novel E-selectin ligand, potentially revealing a new prognostic and therapeutic target for breast cancer.     

Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway

On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of beta 2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via beta 2integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears >/=0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that approximately 60% of the interacting cells remained firmly adherent, as compared with approximately 10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through beta 2 integrins binding to ICAM-1.     

Rac1-regulated endothelial radiation response stimulates extravasation and metastasis that can be blocked by HMG-CoA reductase inhibitors

Radiotherapy (RT) plays a key role in cancer treatment. Although the benefit of ionizing radiation (IR) is well established, some findings raise the possibility that irradiation of the primary tumor not only triggers a killing response but also increases the metastatic potential of surviving tumor cells. Here we addressed the question of whether irradiation of normal cells outside of the primary tumor augments metastasis by stimulating the extravasation of circulating tumor cells. We show that IR exposure of human endothelial cells (EC), tumor cells (TC) or both increases TC-EC adhesion in vitro. IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin. Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion. To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI). The data obtained show that TBI dramatically enhances tumor cell extravasation and lung metastasis. This pro-metastatic radiation effect was blocked by pre-treating mice with lovastatin, glycyrrhizic acid or NSC23766. TBI of mice prior to tumor cell transplantation also stimulated metastasis, which was again blocked by lovastatin. The data point to a pro-metastatic trans-effect of RT, which likely rests on the endothelial radiation response promoting the extravasation of circulating tumor cells. Administration of the widely used lipid-lowering drug lovastatin prior to irradiation counteracts this process, likely by suppressing Rac1-regulated E-selectin expression following irradiation. The data support the concern that radiation exposure might increase the extravasation of circulating tumor cells and recommend co-administration of lipid-lowering drugs to avoid this adverse effect of ionizing radiation.     

Carbohydrate-mediated cell adhesion involved in hematogenous metastasis of cancer

The carbohydrate determinants, sialyl Lewis A and sialyl Lewis X, which are frequently expressed on human cancer cells, serve as ligands for a cell adhesion molecule of the selectin family, E-selectin, which is expressed on vascular endothelial cells. These carbohydrate determinants are involved in the adhesion of cancer cells to vascular endothelium and thus contribute to hematogenous metastasis of cancer. The initial adhesion mediated by these molecules triggers activation of integrin molecules through the action of several cytokines and leads to the extravasation of cancer cells. Cancer cells also produce humoral factors that facilitate E-selectin expression on endothelial cells. The degree of expression of the carbohydrate ligands at the surface of cancer cells is well correlated with the frequency of hematogenous metastasis and prognostic outcome of patients with cancers. The alteration of glycosyltransferase activities that leads to the enhanced expression of these carbohydrate ligands on cancer cell surface are currently being investigated. This revised version was published online in November 2006 with corrections to the Cover Date.     

Dual role of melanoma cell adhesion molecule (MCAM)/CD146 in lymphocyte endothelium interaction: MCAM/CD146 promotes rolling via microvilli induction in lymphocyte and is an endothelial adhesion receptor

The melanoma cell adhesion molecule (MCAM)/CD146 is expressed as two isoforms differing by their cytoplasmic domain (MCAM long (MCAM-l) and MCAM short (MCAM-s)). MCAM being expressed by endothelial cells and activated T cells, we analyzed its involvement in lymphocyte trafficking. The NK cell line NKL1 was transfected by MCAM isoforms and submitted to adhesion on both the endothelial cell monolayer and recombinant molecules under shear stress. MCAM-l transfection reduced rolling velocity and increased NKL1 adhesion on the endothelial cell monolayer and VCAM-1. Scanning electron microscopy revealed that MCAM-l induced microvilli formation and extension. In contrast, MCAM short or mock transfection had no effect on adhesion of NKL1 cells and microvilli formation. As shown by mutagenesis, serine 32 of the MCAM-l cytoplasmic tail, belonging to a putative protein kinase C phosphorylation site, was necessary for MCAM-l-actin cytoskeleton interaction and microvilli induction. Accordingly, chelerythrine chloride, a protein kinase C inhibitor, abolished MCAM-l-induced microvilli and rolling of MCAM-l-transfected NKL1 cells. Inhibition of adhesion under shear stress by anti-MCAM Abs suggested that both lymphoid MCAM-l and endothelial MCAM were also directly involved in lymphocyte endothelium interaction. MCAM-l-transfected NKL1 and activated CD4 T cells adhered to rMCAM under shear stress whereas anti-MCAM Ab treatment inhibited this process. Taken together, these data establish that MCAM is involved in the initial steps of lymphocyte endothelium interaction. By promoting the rolling on the inflammation marker VCAM-1 via microvilli induction and displaying adhesion receptor activity involving possible homophilic MCAM-l-MCAM-l interactions, MCAM might be involved in the recruitment of activated T cells to inflammation sites.     

Different phenotypes of colon carcinoma cells interacting with endothelial cells: role of E-selectin and ultrastructural data

Adhesion molecules are intimately involved in the process of tumour progression. Among them, E-selectin is an inducible endothelial cell adhesion molecule that plays a role in the interactions of neoplastic cells with the endothelium. These interactions are required for the trans-endothelial migration of tumour cells that leads to the growth at the new sites. Since the detailed events in the early phase of metastasis still remain poorly defined, our study has undertaken an electron-microscopic analysis of the interactions of human colon carcinoma cells with endothelial cells as well as an analysis of the effect of recombinant purified E-selectin in the cell signalling involved in colon cancer cell malignant phenotype. Results revealed that SW480 and T84 colon cancer cell lines show different features, different adhesion kinetics, a different cytoskeletal organization, and a different tyrosine phosphorylation pattern when seeded on an endothelial cell monolayer or recombinant E-selectin. In particular T84 cancer cells adhere more efficiently to the E-selectin and this interaction is associated with pronounced morphological changes, actin redistribution and filopodial processes, and an increase in tyrosine phosphorylation of different proteins. These data support the hypothesis that E-selectin ligand is not only a cell-cell adhesion molecule but also initiates a signalling transduction pathway inside the cells.     

Neutrophil capture by selectins on endothelial cells exposed to cigarette smoke

We used a novel perfusion system to expose cultured human umbilical vein endothelial cells (HUVEC) to water-soluble components of cigarette smoke and study subsequent adhesion of flowing neutrophils. Neutrophils did not bind to HUVEC immediately after it had been exposed to cigarette smoke, but many adhered 90-150 min after exposure. The effect was reduced if the exposed medium was made serum-free, but this reduction was partially reversed if low density lipoprotein was added. Treatment of smoke-exposed HUVEC with antibodies against E-selectin or P-selectin reduced adhesion by approximately 50% or 75%, respectively; a combination of both antibodies essentially abolished adhesion. Enzyme-linked immunosorbent assay confirmed that exposure to smoke caused HUVEC to upregulate surface expression of E- and P-selectin. Thus, water-soluble constituent(s) of cigarette smoke cause efficient selectin-mediated capture of flowing neutrophils. This pro-inflammatory response may contribute to pathology associated with smoking, especially in tissues remote from the lung.     

Interleukin-1α Released from Epithelial Cells after Adenovirus Type 37 Infection Activates Intercellular Adhesion Molecule 1 Expression on Human Vascular Endothelial Cells

A key event in virus-induced inflammation (leukocyte extravasation through the endothelium) is the local activation of endothelial cells, as indicated by the expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin. In order to identify triggers of inflammation in adenovirus infection, we inoculated respiratory and ocular epithelial cells with adenovirus type 37 (Ad37), a human pathogen associated with keratoconjunctivitis as well as urogenital and respiratory infections. Fluids from virus-infected epithelial cells activated ICAM-1 (and to a lesser extent, VCAM-1) expression on cultured human umbilical vein endothelial cells. Blocking studies with anticytokine antibodies implicated interleukin-1alpha (IL-1alpha) as the epithelial cell-derived factor which activated endothelial cell ICAM-1 expression. The results thus identify epithelial cell-derived IL-1alpha as a potentially important activator of endothelial cells in Ad37-induced inflammation.     

Crossing the endothelium: E-selectin regulates tumor cell migration under flow conditions

Diapedesis is a vital part of tumor metastasis, whereby tumor cells attach to and cross the endothelium to enter the circulation. Specific adhesion molecules, expressed by both the tumor and endothelial cells, mediate this process. This review summarizes recent findings regarding the mechanisms by which colon cancer cells migrate through the endothelium under flow conditions mediated by E-selectin. Using a laminar flow chamber and a tissue engineered human blood vessel, E-selectin was found to regulate initial attachment and rolling of colon cancer cells and also the subsequent diapedesis through the endothelium. Three different mechanisms of diapedesis were reported to be regulated by E-selectin; the formation of a mosaic chimeric layer of tissue, paracellular diapedesis between endothelial cells and transcellular diapedesis, in which tumor cells were transported via large vacuoles within the endothelial cells. Moreover activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase by E-selectin was further required for paracellular diapedesis. This study is the first to report these observations under dynamic and shear stress conditions.Key words: E-selectin, endothelium, tumor cells, diapedesis, site-specific metastasisDuring metastasis, tumor cells must complete a number of stages to successfully migrate from one site in the body to another. These include detachment of cells from the primary tumor, degradation and migration through the surrounding extra cellular matrix, extravasation and survival in the circulation and arrest in distant capillaries.¹ Once at the secondary site, they must undergo the reverse process before initiating growth and angiogenesis to support their development. This is a highly inefficient process, with few cells completing all stages to establish metastases.²The process of extravasation involves the diapedesis of tumor cells across the endothelium (‘dia’ meaning through and ‘pèdan’ meaning to leap). Similar to neutrophil extravasation at sites of inflammation, tumor cells initially adhere to the endothelium forming loose interactions via adhesion molecules before rolling and finally forming firm attachments, prior to diapedesis. Site-specific metastasis has been described for a number of tumors, with specific interactions between the tumor and endothelial cells potentially playing a pivotal role. Expression of E-selectin by endothelial cells following activation by inflammatory cytokines for example, is reported to be involved with the homing of colon cancer cells to the liver.^3–5 This review summarizes recent findings by Tremblay et al. (2008), published in Cancer Research, in which three distinct mechanisms of transendothelial migration by colon cancer cells are described, ultilizing E-selectin adhesion molecules on the endothelium (Fig. 1): (1) paracellular diapedesis at endothelial junctions, (2) formation of mosaic chimeric tissue of tumor and endothelial cells and (3) transcellular diapedesis.⁶Open in a separate windowFigure 1Schematic representation of the three mechanisms that colon cancer cells use during diapedesis of the endothelium. Under flow conditions, tumor cells initially form loose attachments with the endothelium, prior to rolling and forming firm attachments, mediated by specific adhesion molecules such as E-selectin. Following firm adhesion, tumor cells initiate diapedesis by (1) migrating between the junctions of endothelial cells (paracellular diapedesis), (2) forming a mosaic chimeric layer of both cell types (although this may not lead to complete diapedesis) and (3) passaging through the endothelial cells in large vacuoles (transcellular diapedesis).To demonstrate the importance of E-selectin in the adhesion of colon cancer cells to the endothelium, a laminar flow chamber was initially used. Human umbilical vein endothelial cells (HUVECs), expressing WT or a truncated form of E-selectin (with a cytoplasmic domain deleted) (ES-ΔICD) grown as a monolayer on glass slides, were treated for four hours with IL-1β to induce E-selectin expression. Cultures were then placed in a laminar flow chamber with culture medium and shear stress applied. HT29 colon cancer cells were then injected into the flow chamber, video sequences were recorded after 10 and 30 minutes and the subsequent HT29 cells that had adhered to the HUVECs was counted. The results demonstrated that colon cancer cells adhered to the endothelium under flow conditions using E-selectin, but only following treatment with IL-β. Blocking E-selectin and mutating the intracellular domain of E-selectin both prevented tumor cell adhesion. Similar results were also generated using a tissue-engineered human blood vessel, to more closely mimic the in vivo situation.⁷ Using these systems, the authors verified that E-selectin activation by circulating colon cancer cells is involved in p38 and ERK activation, via the intracellular domain.Using HT29 cells labeled with Vybrant Di1, the authors next used time-lapse microscopy over a 24-hour period to investigate the mechanisms by which colon cancer cells migrate through the endothelium under flow conditions. With endothelial cells expressing WT and truncated E-selectin, E-selectin was found to mediate initial attachment and rolling of the HT29 cells on the endothelial layer, in support of their previous results. Interestingly, most adhered tumor cells penetrated the endothelium, with 75% inserting themselves within the endothelial cell layer, creating a chimeric mosaic (Fig. 1). Many of these cells however did not complete migration through the endothelium and this was especially the case when using endothelial cells expressing E-selectin with the truncated intracellular domain, suggesting that this region was required for tumor cells to complete diapedesis.Of the cells that penetrated the endothelium, only 25% of the colon cancer cells completed diapedesis. This process was observed by two different mechanisms (Fig. 1). Firstly, at the junction of three endothelial cells, HT29 cells migrated between the cells (i.e., paracellular diapedesis); this required E-selectin signaling to ERK. Secondly, transcellular migration was also observed, in which tumor cells attached to the endothelial cells (not at the cell junctions) and induced endothelial cell retraction and blebbing. As a result tumor cells were engulfed within large vacuoles in the endothelial cells and transported across the cell. This was accompanied by loss of endothelial contact with the extracellular matrix. Interestingly this process was not fatal for all endothelial cells and some remained in the culture medium.In conclusion, this is the first paper to demonstrate three different mechanisms by which colon cancer cells migrate through the endothelium under conditions of shear stress, in an E-selectin-dependent fashion. Although a large proportion of tumor cells remained within the endothelium as a mosaic layer and did not complete diapedesis, the authors concluded that such cells form a subpopulation of tumor cells that are capable of initiating, but not completing extravasation, potentially due to nature of the tight junctions between the endothelial cells.^8,9 If proliferation is then stimulated, such cells may further be responsible for the development of local metastases within the vessel. A small percentage of tumor cells however completed diapedesis. This occurred either at the junction of endothelial cells (paracellular) or of particular interest whereby tumor cells crossed through the endothelial cells (transcellular). Similar data have been described previously for neutrophils,¹⁰ but this is the first study to demonstrate this phenomenon with tumor cells via E-selectin. This work provides new insights in understanding the mechanisms by which tumor cells cross blood vessel walls and the metastatic process.     

SDA,a DNA Aptamer Inhibiting E- and P-Selectin Mediated Adhesion of Cancer and Leukemia Cells,the First and Pivotal Step in Transendothelial Migration during Metastasis Formation

Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K _d value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies.