Enhanced tumor cell adhesion to the subendothelial matrix resulting from 12(S)-HETE-induced endothelial cell retraction

A 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), which is produced by platelets and tumor cells, was tested for its ability to induce retraction of endothelial cell monolayers. The induction of endothelial cell retraction is a critical step in tumor cell metastasis. Endothelial cells demonstrated reversible retraction in response to 12(S)-HETE, but did not respond to the stereoisomer 12(R)-HETE or to unrelated 5-lipoxygenase (i.e., 5[S]-HETE) or 15-lipoxygenase (i.e., 15[S]-HETE) metabolites. Endothelial cells did not demonstrate loss of viability in response to 12(S)-HETE. The induction of retraction was both dose and time dependent. Scanning electron microscopy confirmed that 12(S)-HETE induced endothelial cell retraction and revealed collapsed filopodia on their surface, the appearance of spaces between endothelial cells and the underlying subendothelial matrix, in addition to large gaps between adjacent endothelial cells. Tumor cell adhesion to endothelial cell monolayers was enhanced 1 h after pretreatment of monolayers with 12(S)-HETE but not after pretreatment with other lipoxygenase metabolites. Tumor cell adhesion to endothelial cell monolayers 36 h after pretreatment with 12(S)-HETE was not different from adhesion to untreated monolayers. Therefore we suggest that 12(S)-HETE generated during tumor cell-platelet-endothelial cell interactions may induce reversible endothelial cell retraction, allowing tumor cell access to the subendothelial matrix, which is a critical step in their eventual extravasation from the microvasculature during hematogenous metastasis.     

Atomic Force Microscopy Reveals a Role for Endothelial Cell ICAM-1 Expression in Bladder Cancer Cell Adherence

Cancer metastasis is a complex process involving cell-cell interactions mediated by cell adhesive molecules. In this study we determine the adhesion strength between an endothelial cell monolayer and tumor cells of different metastatic potentials using Atomic Force Microscopy. We show that the rupture forces of receptor-ligand bonds increase with retraction speed and range between 20 and 70 pN. It is shown that the most invasive cell lines (T24, J82) form the strongest bonds with endothelial cells. Using ICAM-1 coated substrates and a monoclonal antibody specific for ICAM-1, we demonstrate that ICAM-1 serves as a key receptor on endothelial cells and that its interactions with ligands expressed by tumor cells are correlated with the rupture forces obtained with the most invasive cancer cells (T24, J82). For the less invasive cancer cells (RT112), endothelial ICAM-1 does not seem to play any role in the adhesion process. Moreover, a detailed analysis of the distribution of rupture forces suggests that ICAM-1 interacts preferentially with one ligand on T24 cancer cells and with two ligands on J82 cancer cells. Possible counter receptors for these interactions are CD43 and MUC1, two known ligands for ICAM-1 which are expressed by these cancer cells.     

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host^1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors^4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process⁶. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes^7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance^9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.Download video file.^{(52M, mov)}     

26S proteasome activity is down-regulated in lung cancer stem-like cells propagated in vitro

Cancer stem cells (CSCs) are a small subset of cancer cells capable of self-renewal and tumor maintenance. Eradicating cancer stem cells, the root of tumor origin and recurrence, has emerged as one promising approach to improve lung cancer survival. Cancer stem cells are reported to reside in the side population (SP) of cultured lung cancer cells. We report here the coexistence of a distinct population of non-SP (NSP) cells that have equivalent self-renewal capacity compared to SP cells in a lung tumor sphere assay. Compared with the corresponding cells in monolayer cultures, lung tumor spheres, formed from human non-small cell lung carcinoma cell lines A549 or H1299, showed marked morphologic differences and increased expression of the stem cell markers CD133 and OCT3/4. Lung tumor spheres also exhibited increased tumorigenic potential as only 10,000 lung tumor sphere cells were required to produce xenografts tumors in nude mice, whereas the same number of monolayer cells failed to induce tumors. We also demonstrate that lung tumor spheres showed decreased 26S proteasome activity compared to monolayer. By using the ZsGreen-cODC (C-terminal sequence that directs degradation of Ornithine Decarboxylase) reporter assay in NSCLC cell lines, only less than 1% monolayer cultures were ZsGreen positive indicating low 26S proteasome, whereas lung tumor sphere showed increased numbers of ZsGreen-positive cells, suggesting the enrichment of CSCs in sphere cultures.     

Breakdown of the endothelial barrier function in tumor cell transmigration

The ability of tumor cells to metastasize is associated with a poor prognosis for cancer. During the process of metastasis, tumor cells circulating in the blood or lymph vessels can adhere to, and potentially transmigrate through, the endothelium and invade the connective tissue. We studied the effectiveness of the endothelium as a barrier against the invasion of 51 tumor cell lines into a three-dimensional collagen matrix. Only nine tumor cell lines showed attenuated invasion in the presence of an endothelial cell monolayer, whereas 17 cell lines became invasive or showed a significantly increased invasion. Endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-β. Expression of the IL-8 and Gro-β receptor, CXCR2, was upregulated in invasive tumor cells. Addition of IL-8 or Gro-β increased tumor cell invasiveness by more than twofold. Tumor cell variants selected for high CXCR2 expression were fourfold more invasive in the presence of an endothelial cell layer, whereas CXCR2 siRNA knock-down cells were fivefold less invasive. We demonstrate that Gro-β and IL-8 secreted by endothelial cells, together with CXCR2 receptor expression on invasive tumor cells, contribute to the breakdown of the endothelial barrier by enhancing tumor cell force generation and cytoskeletal remodeling dynamics.     

Secretome derived from breast tumor cell lines alters the morphology of human umbilical vein endothelial cells

Metastases, responsible for most of the solid tumor associated deaths, require angiogenesis and changes in endothelial cells. In this work, the effect of the secretomes of three breast tumor cell lines (MCF-7, MDA-MB-231 and ZR-75-30) on human umbilical vein endothelial cells (HUVEC) morphology was investigated. HUVEC treated with secretomes from breast cells were analyzed by confocal and time-lapse microscopy. Secretomes from ZR-75-30 and MDA-MB-231 cells modify the morphology and adhesion of HUVEC. These changes may provoke the loss of endothelial monolayer integrity. In consequence, tumor cells could have an increased access to circulation, which would then enhance metastasis.     

Retraction of cultured endothelial cell monolayer by human breast cancer cells, MCF-7.

A human breast cancer cell line (MCF-7), when sealed on confluent bovine pulmonary aortic endothelial cell (CPAE) monolayers, induced morphological changes (retraction) in CPAE cells. The area of retraction depended on the incubation time and the number of MCF-7 cells, suggesting that MCF-7 cells had the capacity to retract CPAE cells. This capacity was reduced by 60% by pretreatment of MCF-7 cells with 17β-estradiol (E) and progesterone (Pg). The extent of retraction was not affected by the addition of various protease inhibitors. CPAE retraction was induced also by adding conditioned medium (CM) from the culture of MCF-7 cells. Considerably less activity was detected in the CM obtained from MCF-7 cells cultured in the presence of E and Pg. The retraction was reversed in 24 h by culturing the monolayer in fresh medium without CM and was not induced by trypsin treatment of the CM.     

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.     

Expression level of neural cell adhesion molecule (NCAM) inversely correlates with the ability of neuroblastoma cells to adhere to endothelium in vitro

The precise function of cell adhesion molecules in the hematogenous phase of neuroblastoma metastasis is poorly understood. The aim of this study was to investigate whether neural cell adhesion molecule (NCAM) modulates neuroblastoma cell (NB) adhesion and transendothelial penetration in a coculture model. Our data, assessed on 11 NB cell lines, demonstrate an inverse correlation between NCAM expression and NB cell adhesion. Transfection of the NB cell line UKF-NB-4 with a cDNA encoding the human NCAM-140 kD isoform enhanced NCAM expression and the amount of tumor cell aggregates, reduced the amount of single tumor cells, and diminished initial NB cell adhesion to an endothelial cell monolayer. Treatment of UKF-NB-4 with NCAM antisense oligonucleotides reduced NCAM surface level, increased the number of single tumor cells, and induced up-regulation of NB cell adhesion to endothelium. Modulation of NCAM expression had no effect on transendothelial penetration. Fluorescence analysis revealed a down-regulation of NCAM in single tumor cells, prior to NB adhesion. The data support the view that low levels of NCAM are necessary for NB cells to leave a tumor cell aggregate and adhere to endothelial cells.     

Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), has been shown to increase potently the permeability of endothelium and is highly expressed in breast cancer cells. In this study, we investigated the role of VEGF/VPF in breast cancer metastasis to the brain. Very little is known about the role of endothelial integrity in the extravasation of breast cancer cells to the brain. We hypothesized that VEGF/VPF, having potent vascular permeability activity, may support tumor cell penetration across blood vessels by inducing vascular leakage. To examine this role of VEGF/VPF, we used a Transwell culture system of the human brain microvascular endothelial cell (HBMEC) monolayer as an in vitro model for the blood vessels. We observed that VEGF/VPF significantly increased the penetration of the highly metastatic MDA-MB-231 breast cancer cells across the HBMEC monolayer. We found that the increased transendothelial migration (TM) of MDA-MB-231 cells resulted from the increased adhesion of tumor cells onto the HBMEC monolayer. These effects (TM and adhesion of tumor cells) were inhibited by the pre-treatment of the HBMEC monolayer with the VEGF/VPF receptor (KDR/Flk-1) inhibitor, SU-1498, and the calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)ester. These treatments of the HBMEC monolayer also inhibited VEGF/VPF-induced permeability and the cytoskeletal rearrangement of the monolayer. These data suggest that VEGF/VPF can modulate the TM of tumor cells by regulating the integrity of the HBMEC monolayer. Taken together, these findings indicate that VEGF/VPF might contribute to breast cancer metastasis by enhancing the TM of tumor cells through the down-regulation of endothelial integrity.     

Differences in the biological activity of TNF alpha and TNF beta correlate with their different abilities for binding to the target cells.

TNF alpha and TNF beta were compared regarding their binding to different types of target cells, cytotoxic/cytostatic activity against murine and human tumor cell lines as well as human capillary endothelial cells, their ability to induce differentiation in myeloid leukemia cell lines, and induction of hemorrhagic tumor necrosis and tumor regression as well as lethal toxicity in tumor-bearing mice. The results show considerable quantitative differences in the biological activity between TNF alpha and TNF beta depending on the type of target cell which has been used. TNF beta was 3 fold more cytotoxic than TNF alpha against murine L929 fibroblasts and 3-5 times more active concerning the induction of hemorrhagic tumor necrosis, complete tumor regression and more toxic in tumor-bearing mice. In contrast to this, TNF beta was markedly less cytotoxic against human capillary endothelial cells and the human mammary carcinoma cell line MCF7 and much less cytostatic against the human myeloid leukemia cell lines HL60 and U937. The lesser antiproliferative effect of TNF beta correlated with a lower ability for induction of differentiation in these cell lines. Competitive radioligand binding assays showed that TNF beta was about 4 fold more effective than TNF alpha in competing with 125I-labeled TNF alpha for the binding to murine L929 fibroblasts. But it was 15-20 times less effective in binding to the human MCF7 cells and the human myeloid leukemia cell lines HL60 and U937. This revealed that, at least for these targets, the differences in the biological activity between TNF alpha and TNF beta are due to different abilities for binding to the target cells. Possible mechanisms for these different binding abilities are discussed.     

TEL Induces Aggregation in Transformed Cells and Induces Tube Formation in NIH3T3-UCLA Cells

TEL/ETV6 is the frequent target of translocations associated with lymphoid and myeloid leukemias and solid tumors. We show that TEL induces aggregation of immortalized and transformed fibroblasts, endothelial cells and astrocytes. These aggregates form cellular cords in NIH3T3-UCLA by a cell autonomous process, which occurs when the monolayer is made up of over 75% of cells expressing exogenous TEL. Cords with a diameter of 15-25 microm contain a lumen and occur as tube structures. The possible relevance for vasculogenic mimicry is discussed. By contrast TEL did not induce aggregation of regular NIH3T3 cells, an effect that could only be induced by co-expression of oncogenic RAS/Lys12. Also transduction of TEL and RAS retroviral vectors into the endothelial MS1 cell line and TEL alone in the highly transformed glioblastoma cell lines EH-A and EH-B resulted in extensive aggregation. Thus, the induction of cellular aggregation by TEL correlates with transformation.     

Cell Density Impacts Epigenetic Regulation of Cytokine-Induced E-Selectin Gene Expression in Vascular Endothelium

Growing evidence suggests that the phenotype of endothelial cells during angiogenesis differs from that of quiescent endothelial cells, although little is known regarding the difference in the susceptibility to inflammation between both the conditions. Here, we assessed the inflammatory response in sparse and confluent endothelial cell monolayers. To obtain sparse and confluent monolayers, human umbilical vein endothelial cells were seeded at a density of 7.3×10³ cells/cm² and 29.2×10³ cells/cm², respectively, followed by culturing for 36 h and stimulation with tumor necrosis factor α. The levels of tumor necrosis factor α-induced E-selectin protein and mRNA expression were higher in the confluent monolayer than in the sparse monolayer. The phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase or nuclear factor-κB activation was not involved in this phenomenon. A chromatin immunoprecipitation assay of the E-selectin promoter using an anti-acetyl-histone H3 antibody showed that the E-selectin promoter was highly and specifically acetylated in the confluent monolayer after tumor necrosis factor α activation. Furthermore, chromatin accessibility real-time PCR showed that the chromatin accessibility at the E-selectin promoter was higher in the confluent monolayer than in the sparse monolayer. Our data suggest that the inflammatory response may change during blood vessel maturation via epigenetic mechanisms that affect the accessibility of chromatin.     

Functional involvement of P-glycoprotein in blood-brain barrier.

P-glycoprotein, an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in various normal tissues, including brain capillaries. To study the physiological function of P-glycoprotein expressed in brain capillary endothelium, we established nine mouse brain capillary endothelial cell (MBEC) lines and examined the transport of antitumor agents across the monolayer of MBEC epithelia. In the MBECs, the activities of alkaline phosphatase and gamma-glutamyl transpeptidase, specific markers for brain capillary endothelial cells, were about three times higher than those in other cells including human umbilical vein endothelial cells. By immunoblot analysis, P-glycoprotein was detected in all of the nine MBEC clones. The P-glycoprotein expressed in MBECs specifically bound [125I]iodoaryl azidoprazosin as that in multidrug-resistant cells, and efflux of vincristine was observed in the MBECs. When MBECs were grown on a porous filter membrane, they formed a monolayer of epithelium. By immunoelectron microscopic analysis, P-glycoprotein in MBEC epithelia was shown to be localized to the apical surface of the cells. Moreover, the unidirectional transepithelial transport of vincristine from basal side to apical side was demonstrated in vitro. These observations indicate that P-glycoprotein in brain capillary endothelium prevents vincristine from entering the central nervous system and thus may be one of the functional components of the blood-brain barrier.     

星形胶质细胞可以诱导内皮细胞系紧密连接的形成

为探索星形胶质细胞在血脑屏障内皮细胞紧密连接形成中的重要意义,通过内皮细胞系ECV304与星形胶质细胞体外接触共培养的方法,采用电镜及内皮细胞紧密连接的银染观察星形胶质细胞对内皮细胞系紧密连接的诱导作用。运用Millipore-ERS系统检测紧密连接的功能状况。结果发现,星形胶质细胞可以诱导内皮细胞系形成广泛而连续的紧密连接并产生较高的跨内皮阻抗(transendothelial electrical resistance,TER),于第10d可达321．3Ωcm^2。提示,星形胶质细胞可以诱导ECV304细胞产生紧密连接。同时,ECV304细胞与星形胶质细胞的体外共培养可以作为研究血脑屏障紧密连接结构与功能的一种可靠而简便的体外实验方法。     

Quantitative analysis of autocrine-regulated, matrix-induced, and tumor cell-stimulated endothelial cell migration using a silicon template compartmentalization technique.

Autocrine-regulated, matrix-induced, and tumor cell-stimulated endothelial cell migration was quantitatively analyzed using a two-dimensional, two-compartment coculture system. Silicon templates were used to subdivide 35-mm tissue culture dishes into two separate compartments. Endothelial cells were grown to confluence in the inner compartment and released from growth arrest by removal of the silicon template. The distance of endothelial cell outgrowth from the monolayer was measured in 24-h intervals. Endothelial cells from different vascular beds migrated with different migration rates (large vessel endothelial cells greater than hemangioendothelioma cells greater than microvessel endothelial cells). Prior coating of tissue culture wells with fibronectin, type I collagen, or type IV collagen and increasing serum concentrations strongly enhanced endothelial cell migration. Seeding tumor cells into the outer compartment prior to removal of the silicon template permitted the direct coculture analysis of tumor cell-induced endothelial cell migration. Microvascular endothelial cell migration was stimulated in a tumor cell number-dependent fashion, whereas large vessel endothelial cells could not consistently be stimulated by coculture with tumor cells. It is concluded that silicon templates offer a useful approach for the quantitative study of migration of anchorage-dependent cells, permitting follow-up measurements over several days, the study of matrix effects, and the direct coculture analysis of cell migration.     

Use of multicellular tumor spheroids to dissect endothelial cell-tumor cell interactions: a role for T-cadherin in tumor angiogenesis

This study addresses establishment of an "in vitro" melanoma angiogenesis model using multicellular tumor spheroids (MCTS) of differentiated (HBL) or undifferentiated (NA8) melanoma cell lines. DNA microarray assay and qRT-PCR indicated upregulation of pro-angiogenic factors IL-8, VEGF, Ephrin A1 and ANGPTL4 in NA8-MCTSs (vs. monolayers) whereas these were absent in MCTS and monolayer cultures of HBL. Upon co-culture with endothelial cell line HMEC-1 NA8-MCTS attract, whereas HBL-MCTS repulse, HMEC-1. Overexpression of T-cadherin in HMEC-1 leads to their increased invasion and network formation within NA8-MCTS. Given an appropriate angiogenic tumor microenvironment, T-cadherin upregulation on endothelial cells may potentiate intratumoral angiogenesis.     

Cytoplasmic dye transfer between metastatic tumor cells and vascular endothelium

Metastatic colonization of a secondary organ site is initiated by the attachment of blood-borne tumor cells to organ-specific adhesion molecules expressed on the surface of microvascular endothelial cells. Using digital video imaging microscopy and fluorescence activated cell sorting techniques, we show here that highly metastatic cells (B16-F10 murine melanoma and R3230AC-MET rat mammary adenocarcinoma cells) previously labeled with the fluorescent dye BCECF begin to transfer dye to endothelial cell monolayers shortly after adhesion is established. The extent of BCECF transfer to endothelial cell monolayers is dependent upon the number of BCECF-labeled tumor cells seeded onto the endothelial cell monolayer and the time of coculture of the two cell types, as visualized by an increase in the number of BCECF-positive cells among cells stained with an endothelial cell-specific mAb. Dye transfer to BAEC monolayers proceeds with a progressive loss of fluorescence intensity in the BCECF-labeled tumor cell population with time of coculture. The transfer of dye is bidirectional and sensitive to inhibition by 1-heptanol. In contrast, poorly metastatic B16-F0 melanoma cells and non-metastatic R3230AC-LR mammary adenocarcinoma cells do not efficiently couple with vascular endothelial cells. It is inferred from these experiments and from the amounts of connexin43 mRNA expressed by tumor cells that tumor cell/endothelial cell communication is mediated by gap junctional channels and that this interaction may play a critical role in tumor cell extravasation at secondary sites.