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.     

Differential binding of hyaluronan on the surface of tissue-specific endothelial cell lines

Tissue-specific heterogeneity of endothelial cells, both structural and functional, plays a crucial role in physiologic as well as pathologic processes, including inflammation, autoimmune diseases and tumor metastasis. This heterogeneity primarily results from the differential expression of adhesion molecules that are involved in the interactions between endothelium and circulating immune cells or disseminating tumor cells. Among these molecules present on endothelial cells is hyaluronan (HA), a glycosaminoglycan that contributes to primary (rolling) interactions through binding to its main receptor CD44 expressed on leukocytes and tumor cells. While the regulation of CD44 expression and function on either leukocytes or tumor cells has been well characterized, much less is known about the ability of endothelial cells to express HA on their surface. Therefore, in these studies we analyzed HA levels on tissue-specific endothelium. We used endothelial cell lines of different origin, including lung, skin, gut and lymph nodes that had been established previously as model lines to study interactions between the endothelium and leukocytes/tumor cells. Our results indicate that HA is accumulated on the surface of all endothelial cells examined. Moreover, retention of endogenous HA differs between the lines and may depend on their tissue origin. Analysis of binding of exogenous HA reveals the presence of specific HA binding sites on all endothelial cell lines tested. However, the retention of endogenous HA and the binding of exogenous HA is mediated through a CD44-independent mechanism.     

Molecular events during leukocyte diapedesis

The recruitment of leukocytes from the circulation into tissues requires leukocyte migration through the vascular endothelium. The mechanisms by which leukocytes attach and firmly adhere to the endothelial cell surface have been studied in detail. However, much less is known about the last step in this process, the diapedesis of leukocytes through the vascular endothelium. This minireview focuses on the interactions between leukocyte and endothelial cell adhesion molecules that are important during leukocyte extravasation. In the past few years a series of endothelial cell surface and adhesion molecules have been identified that are located at endothelial cell contacts and found to participate in leukocyte diapedesis. These junctional cell adhesion molecules are believed to have an active role in controlling the opening and closure of endothelial cell contacts to allow the passage of leukocytes between adjacent endothelial cells. Alternatively, leukocytes can cross the endothelium at nonjunctional locations, with leukocytes migrating through a single endothelial cell. Further work is clearly needed to understand, in greater detail, the molecular mechanisms that allow leukocytes to cross the endothelium via either the paracellular or the transcellular pathway.     

Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis

Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.     

Induction of Endothelial Cell Apoptosis by Solid Tumor Cells

The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performedin vivoandin vitrodemonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two- to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation.     

Involvement of p38alpha mitogen-activated protein kinase in lung metastasis of tumor cells

To study the role of p38 mitogen-activated protein kinase (p38) activity during the process of metastasis, p38alpha(+/-) mice were subjected to an in vivo metastasis assay. The number of lung colonies of tumor cells intravenously injected in p38alpha(+/-) mice was markedly decreased compared with that in wild-type (WT) mice. On the other hand, the time-dependent increase in tumor volume after subcutaneous tumor cells transplantation was comparable between WT and p38alpha(+/-) mice. Platelets of p38alpha(+/-) mice were poorly bound to tumor cells in vitro and in vivo compared with those of WT mice. E- and P-selectin mRNAs were markedly induced in the lung after intravenous injection of tumor cells. However, the induction of these selectin mRNAs in p38alpha(+/-) mice was weaker than that in WT mice. Furthermore, the resting expression levels of E-selectin in lung endothelial cells and P-selectin in platelets of p38alpha(+/-) mice were suppressed compared with those of WT mice. The number of tumor cells attached on lung endothelial cells of p38alpha(+/-) mice was significantly reduced compared with that of WT mice. The transmigrating activity of tumor cells through lung endothelial cells of p38alpha(+/-) mice was similar to that of WT mice. These results suggest that p38alpha plays an important role in extravasation of tumor cells, possibly through regulating the formation of tumor-platelet aggregates and their interaction with the endothelium involved in a step of hematogenous metastasis.     

VE-Cadherin-Independent Cancer Cell Incorporation into the Vascular Endothelium Precedes Transmigration

Metastasis is accountable for 90% of cancer deaths. During metastasis, tumor cells break away from the primary tumor, enter the blood and the lymph vessels, and use them as highways to travel to distant sites in the body to form secondary tumors. Cancer cell migration through the endothelium and into the basement membrane represents a critical step in the metastatic cascade, yet it is not well understood. This process is well characterized for immune cells that routinely transmigrate through the endothelium to sites of infection, inflammation, or injury. Previous studies with leukocytes have demonstrated that this step depends heavily on the activation status of the endothelium and subendothelial substrate stiffness. Here, we used a previously established in vitro model of the endothelium and live cell imaging, in order to observe cancer cell transmigration and compare this process to leukocytes. Interestingly, cancer cell transmigration includes an additional step, which we term ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs, leading to the dislocation of EC VE-cadherin away from EC junctions bordering cancer cells, and spread into the monolayer. In some cases, ECs completely detach from the matrix. Furthermore, cancer cell incorporation occurs independently of the activation status and the subendothelial substrate stiffness for breast cancer and melanoma cells, a notable difference from the process by which leukocytes transmigrate. Meanwhile, pancreatic cancer cell incorporation was dependent on the activation status of the endothelium and changed on very stiff subendothelial substrates. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation is one of the earliest steps.     

In Vitro Model of Tumor Cell Extravasation

Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium.     

Evidence of a functional role for interaction between ICAM-1 and nonmuscle alpha-actinins in leukocyte diapedesis

ICAM-1 is involved in both adhesion and extravasation of leukocytes to endothelium during inflammation. It has been shown that the ICAM-1 cytoplasmic domain is important for transendothelial migration of leukocytes but the precise molecular mechanisms involving the intracytoplasmic portion of ICAM-1 is not known. To characterize precisely the molecular scaffolding associated with ICAM-1, we have used the yeast two-hybrid system, and we have identified six different proteins interacting with the ICAM-1 cytoplasmic domain. In this study, we report that the two forms of nonmuscle alpha-actinin (i.e., alpha-actinin 1 and alpha-actinin 4) associate with ICAM-1, and that these interactions are essential for leukocyte extravasation. These interactions were further confirmed by coimmunoprecipitation and immunofluorescence in endothelial cells and in ICAM-1-transfected Chinese hamster ovary cells. The function of these interactions was analyzed by point mutation of charged amino acids located on ICAM-1 cytoplasmic domain. We have identified three charged amino acids (arginine 480, lysine 481, and arginine 486) which are essential in the binding of alpha-actinins to the ICAM-1 cytoplasmic tail. Mutation of these amino acids completely inhibited ICAM-1-mediated diapedesis. Experiments with siRNA inhibiting specifically alpha-actinin 1 or alpha-actinin 4 on endothelial cells indicated that alpha-actinin 4 had a major role in this phenomenon. Thus, our data demonstrate that ICAM-1 directly interacts with cytoplasmic alpha-actinin 1 and 4 and that this interaction is required for leukocyte extravasation.     

Adhesion molecules and their role in cancer metastasis

This article describes various adhesion molecules and reviews evidence to support a mechanistic role for adhesion molecules in the process of cancer metastasis. A variety of evidence supports the involvement of specific adhesion molecules in metastasis.

1. For example, some cancer cells metastasize to specific organs, irrespective of the first organ encountered by the circulating cancer cells. This ability to colonize a specific organ has been correlated with the preferential adhesion of the cancer cells to endothelial cells derived from the target organ. This suggests that cancer cell/endothelial cell adhesion is involved in cancer cell metastasis and that adhesion molecules are expressed on the endothelium in an organ-specific manner.
2. Further, inclusion of peptides that inhibit cell adhesion, such as the YIGSR- or RGD-containing peptides, is capable of inhibiting experimental metastasis.
3. Metastasis can be enhanced by acute or chronic inflammation of target vessels, or by treatment of animals with inflammatory cytokines, such as interleukin-1. In vitro, cancer cell/endothelial cell adhesion can be enhanced by pretreating the endothelial cell monolayer with cytokines, such as interleukin-1 or tumor necrosis factor-α. This suggests that, in addition to organ-specific adhesion molecules, a population of inducible endothelial adhesion molecules is involved and is relevant to metastasis.
4. Further support for this model is found in the comparison to leukocyte/endothelial adhesion during leukocyte trafficking. Convincing evidence exists, both in vivo and in vitro, to demonstrate an absolute requirement for leukocyte/endothelial adhesion before leukocyte extravasation can occur. The relevance of this comparison to metastasis is reinforced by the observation that some of the adhesion molecules involved in leukocyte/endothelial adhesion are also implicated in cancer cell/endothelial adhesion. The involvement of adhesion molecules suggests a potential therapy for metastasis based on interrupting adhesive interactions that would augment other treatments for primary tumors.

     

Robert Feulgen Lecture 1998

Attachment of leukocytes to the blood vessel wall initiates leukocyte extravasation. This enables leukocytes to migrate to and accumulate at sites of tissue injury or infection where they execute host-defense mechanisms. A series of vascular cell adhesion molecules on leukocytes and on endothelial cells mediate leukocyte attachment to the endothelium in a stepwise process. A large panel of about 40 known human chemokines is able to specifically activate certain leukocytes and attract them to migrate across the endothelial barrier and within tissue. The specific combination of molecular signals provided by the diversity of cytokines, adhesion molecules, and chemokines regulates the specificity and selectivity of the recruitment of certain subpopulations of leukocytes in vivo. This review will focus on selectins and chemokines which initiate the cell contact and regulate activation and chemoattraction of leukocytes. Accepted: 20 May 1999     

High affinity interaction of integrin alpha4beta1 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) enhances migration of human melanoma cells across activated endothelial cell layers

The capacity of tumor cells to form metastatic foci correlates with their ability to interact with and migrate through endothelial cell layers. This process involves multiple adhesive interactions between tumor cells and the endothelium. Only little is known about the molecular nature of these interactions during extravasation of tumor cells. In human melanoma cells, the integrin alphavbeta3 is involved in transendothelial migration and its expression correlates with metastasis. However, many human melanoma cells do not express beta3 integrins. Therefore, it remained unclear how these cells undergo transendothelial migration. In this study we show that human melanoma cells with different metastatic potency, which do not express beta2 or beta3 integrins, express the VCAM-1 receptor alpha4beta1. VCAM-1 is up-regulated on activated endothelial cells and is known to promote transendothelial migration of leukocytes. Interestingly, despite comparable cell surface levels of alpha4beta1, only the highly metastatic melanoma cell lines MV3 and BLM, but not the low metastatic cell lines IF6 and 530, bind VCAM-1 with high affinity without further stimulation, and are therefore able to adhere to and migrate on isolated VCAM-1. Moreover, we demonstrate that function-blocking antibodies against the integrin alpha4beta1, as well as siRNA-mediated knock-down of the alpha4 subunit in these highly metastatic human melanoma cells reduce their transendothelial migration. These data imply that only high affinity interactions between the integrin alpha4beta1 on melanoma cells and VCAM-1 on activated endothelial cells may enhance the metastatic capacity of human beta2/beta3-negative melanoma cells.     

Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes

The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.     

Cytokines and Cell Adhesion Molecules in Tumor-Endothelial Cell Interaction and Metastasis

Metastasis is a multistep process in which a metastatic tumor cell detaches from the primary tumor, invades the surrounding tissues, passes through supporting structures such as interstitial stroma and extracellular matrix, and enters the lymphatic or blood circulation (Poste and Fidler, 1980). Only a few of the neoplastic cells released into the circulation, that survive hemodynamic pressure and host defense mechanisms, will form metastases. The arrest of tumor cells in the capillary bed of secondary organs through an interaction with vascular or lymphatic endothelium and subendothelial basement membrane is followed by their extravasation into the tissue parenchyma, and then micro-metastasis formation. Therefore cell-cell and cell-substrate adhesions occur at different moments in this process. With the recent identification and characterization of cell surface molecules, it has become of particular interest to clarify their role in tumor progression and metastasis (Albelda, 1993).     

Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis

VEGF is unique among angiogenic growth factors because it disrupts endothelial barrier function. Therefore, we considered whether this property of VEGF might contribute to tumor cell extravasation and metastasis. To test this, mice lacking the Src family kinases Src or Yes, which maintain endothelial barrier function in the presence of VEGF, were injected intravenously with VEGF-expressing tumor cells. We found a dramatic reduction in tumor cell extravasation in lungs or livers of mice lacking Src or Yes. At the molecular level, VEGF compromises the endothelial barrier by disrupting a VE-cadherin-beta-catenin complex in lung endothelium from wild-type, but not Yes-deficient, mice. Disrupting the endothelial barrier directly with anti-VE-cadherin both amplifies metastasis in normal mice and overcomes the genetic resistance in Yes-deficient mice. Pharmacological blockade of VEGF, VEGFR-2, or Src stabilizes endothelial barrier function and suppresses tumor cell extravasation in vivo. Therefore, disrupting Src signaling preserves host endothelial barrier function providing a novel host-targeted approach to control metastatic disease.     

Basement Membrane-Rich Organoids with Functional Human Blood Vessels Are Permissive Niches for Human Breast Cancer Metastasis

Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention.     

Interactions between endothelial cells and leukocytes

We present evidence that specific receptors are utilized by neutrophils to control their interaction with endothelial cells at sites of acute inflammation and that these receptors are related if not identical to lymphocyte "homing receptors" for lymphoid tissue high endothelium. We speculate that such receptors play a fundamental but not exclusive role in controlling the extravasation and tissue localization of all bone marrow-derived nucleated cells. In addition, we emphasize the active role of endothelial cells in the process of lymphocyte migration and leukocyte extravasation. By the expression of as yet unidentified organ-specific determinants for lymphocyte recognition, endothelial cells control the exit of particular lymphocyte subsets into mucosal versus nonmucosal sites, thus helping to determine the unique features of mucosal versus nonmucosal immune responses. Furthermore, we argue that endothelial cells are exquisitely responsive to local immune reactivity and present evidence that specific lymphokines, including gamma-interferon, play an important role in inducing postcapillary venules to express differentiated features required for the support of lymphocyte traffic into lymphoid organs and into sites of chronic inflammation. Leukocytes, endothelial cells, and probably other tissue cell classes appear to interact at multiple levels by a variety of mechanisms to regulate the local extravasation of immune effector cells.     

Cytokines and Cell Adhesion Molecules in Tumor-Endothelial Cell Interaction and Metastasis

Metastasis is a multistep process in which a metastatic tumor cell detaches from the primary tumor, invades the surrounding tissues, passes through supporting structures such as interstitial stroma and extracellular matrix, and enters the lymphatic or blood circulation (Poste and Fidler, 1980). Only a few of the neoplastic cells released into the circulation, that survive hemodynamic pressure and host defense mechanisms, will form metastases. The arrest of tumor cells in the capillary bed of secondary organs through an interaction with vascular or lymphatic endothelium and subendothelial basement membrane is followed by their extravasation into the tissue parenchyma, and then micro-metastasis formation. Therefore cell-cell and cell-substrate adhesions occur at different moments in this process. With the recent identification and characterization of cell surface molecules, it has become of particular interest to clarify their role in tumor progression and metastasis (Albelda, 1993).     

The blood-brain barrier, chemokines and multiple sclerosis

The infiltration of leukocytes into the central nervous system (CNS) is an essential step in the neuropathogenesis of multiple sclerosis (MS). Leukocyte extravasation from the bloodstream is a multistep process that depends on several factors including fluid dynamics within the vasculature and molecular interactions between circulating leukocytes and the vascular endothelium. An important step in this cascade is the presence of chemokines on the vascular endothelial cell surface. Chemokines displayed along the endothelial lumen bind chemokine receptors on circulating leukocytes, initiating intracellular signaling that culminates in integrin activation, leukocyte arrest, and extravasation. The presence of chemokines at the endothelial lumen can help guide the movement of leukocytes through peripheral tissues during normal immune surveillance, host defense or inflammation. The expression and display of homeostatic or inflammatory chemokines therefore critically determine which leukocyte subsets extravasate and enter the peripheral tissues. Within the CNS, however, infiltrating leukocytes that cross the endothelium face additional boundaries to parenchymal entry, including the abluminal presence of localizing cues that prevent egress from perivascular spaces. This review focuses on the differential display of chemokines along endothelial surfaces and how they impact leukocyte extravasation into parenchymal tissues, especially within the CNS. In particular, the display of chemokines by endothelial cells of the blood brain barrier may be altered during CNS autoimmune disease, promoting leukocyte entry into this immunologically distinct site. Recent advances in microscopic techniques, including two-photon and intravital imaging have provided new insights into the mechanisms of chemokine-mediated capture of leukocytes within the CNS.     

Proteolysis during Tumor Cell Extravasation In Vitro: Metalloproteinase Involvement across Tumor Cell Types

To test if proteolysis is involved in tumor cell extravasation, we developed an in vitro model where tumor cells cross an endothelial monolayer cultured on a basement membrane. Using this model we classified the ability of the cells to transmigrate through the endothelial cell barrier onto the underlying matrix, and scored this invasion according to the stage of passage through the endothelium. Metalloproteinase inhibitors reduced tumor cell extravasation by at least 35%. Visualization of protease and cell adhesion molecules by confocal microscopy demonstrated the cell surface localization of MMP-2, MMP-9, MT1-MMP, furin, CD44 and α_vβ₃, during the process of transendothelial migration. By the addition of inhibitors and bio-modulators we assessed the functional requirement of the aforementioned molecules for efficient migration. Proteolytic digestion occurred at the cell-matrix interface and was most evident during the migratory stage. All of the inhibitors and biomodulators affected the transition of the tumor cells into the migratory stage, highlighting the most prevalent use of proteolysis at this particular step of tumor cell extravasation. These data suggest that a proteolytic interface operates at the tumor cell surface within the tumor-endothelial cell microenvironment.