Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2005) 29, 497–505 (Printed in Great Britain)
Role of reactive oxygen species (ROS), metalloproteinase-2 (MMP-2) and interleukin-6 (IL-6) in direct interactions between tumour cell spheroids and endothelial cell monolayer
Roman Paducha*1, Adam Walter‑Croneckb, Barbara Zdzisińskaa, Agnieszka Szuster‑Ciesielskaa and Martyna Kandefer‑Szerszeńa
aDepartment of Virology and Immunology, Institute of Microbiology and Biotechnology, Maria Curie-Skłodowska University, ul. Akademicka 19, 20-033 Lublin, Poland
bDepartment of Haematooncology and Bone Marrow Transplantation, Medical University, Jaczewskiego 8, 20-950 Lublin, Poland


Metastasis is a multistep process involving a variety of direct cell–cell, cell–matrix and paracrine interactions. In the present study, we examined some consequences of direct interaction between tumour cells and endothelial cells in vitro. When multicellular spheroids of two human tumour cell lines (HeLa and Hep-2) were transferred onto a human umbilical vein endothelial cell (HUVEC) monolayer, a peri-spheroidal zone of damaged endothelial cells was observed after 24h co-culture. To determine the cause of this damage, the production levels of superoxide anion (O2), interleukin-6 (IL-6) and metalloproteinase-2 (MMP-2) were measured both in co-culture and in monocultures of the tumour cell spheroids and endothelial cells. Attachment of HeLa and Hep-2 cellular spheroids to the HUVEC monolayer resulted in 1.6-fold and 2.1-fold increases in O2 release, respectively. Also, the MMP-2 level was five times greater in the co-culture than in the tumour spheroid monoculture. The increase of IL-6 in the co-culture model, on the other hand, was only slight. However, a 2h preincubation of endothelial cells with LPS (10μg/ml) prior to the transfer of spheroids induced a significant increase in the production of this cytokine compared to an appropriate control (an LPS-activated endothelial cell monolayer). These results strongly suggest that both ROS and MMP-2 are involved in endothelial cell injury when tumour cells cross the endothelial barrier. Moreover, IL-6, which participates in the inflammatory response, may also be involved in the extravasation of tumour cells.

Keywords: Tumour spheroids, Endothelium, Metastasis, Reactive oxygen species, Metalloproteinase-2, Interleukin-6.

1The Foundation for Polish Science scholarship holder.

*Corresponding author. Tel.: +48 81 537 59 42; fax: +48 81 537 59 59.

1 Introduction

Tumour metastasis involves migration in the blood or lymph stream, attachment and migration through the vascular wall, and proliferation in sites distant from the primary tumour. This process depends not only on cell adhesion molecules, which are necessary for direct cell-to-cell interaction, but is also mediated by reactive oxygen species (ROS), angiogenic growth factors and their receptors and proteolytic enzymes (Liekens et al., 2001).

ROS are involved in the pathogenesis of several blood vessel diseases, e.g., sepsis and arteriosclerosis (Lorenz et al., 1998); they cause oxidation of membrane lipids, DNA breakdown and decomposition of proteins in endothelial cells (Lorenz et al., 1998; Yen et al., 2001). Tumour cells may produce ROS that injure endothelial cells and promote metastasis (Offner et al., 1996).

The continued growth and dissemination of solid tumours require proteolysis of the ECM as well as endothelial cell injury. Members of the matrix metalloproteinase (MMP) family are believed to play an important part in this process. MMPs are a group of secreted or transmembrane enzymes that can digest basement membrane and ECM components (Chambers and Matrisian, 1997). They are secreted in a latent form, so they require to be activated (Foda and Zucker, 2001). The MMP family comprises at least 20 enzymes, which can be divided into four subclasses: collagenases, stromelysins, gelatinases and the matrix type-MMPs (MT-MMPs) (Jackson and Nguyen, 1997). MMP-9 (gelatinase B/type IV collagenase) and MMP-2 (gelatinase A/type IV collagenase) are postulated to play crucial roles in tumour invasion (Fridman et al., 1995). The 72kDa MMP-2 protein is the most widely distributed member of the MMPs family and is constitutively expressed in epithelial, endothelial and other cell types. The activated enzyme can degrade collagen types IV, V, VII and X, gelatin, laminin, fibronectin and elastin (Foda and Zucker, 2001; Kräling et al., 1999). Some polypeptide fragments released after cleavage of matrix components by MMP-2 possess new biological properties; for instance, cleavage products of laminin-5 promote the migration of normal and tumour cells (Foda and Zucker, 2001; Chang and Werb, 2001).

IL-6 is a central proinflammatory cytokine, an important inducer of acute phase proteins and a regulator of the immune response, and it is implicated in cancer development (Lin-Hung et al., 2001). It is multifunctional and is produced by endothelial cells as well as macrophages and T cells. Endothelial-derived IL-6 inhibits tumour cell growth in premalignant and early stages of cancer. However, in intermediate and advanced stages, substantial microenvironmental levels of this cytokine stimulate primary tumour angiogenesis and metastasis (Rak et al., 1996).

A multicellular tumour spheroid (MCTS) is a three-dimensional tumour cell cluster. Its well-defined geometry, or the definite number of cells forming its mass, make the MCTS useful for analysing the mechanisms involved in tumour–endothelium interactions (Hamilton, 1998). It is well known that the adhesion of tumour cells to the luminal surface of the blood vessel wall can modulate the secretive capacity of endothelial cells and, by inducing apoptosis of these cells, alter endothelial integrity (Kebers et al., 1998). Clinical observations as well as in vitro studies indicate that injury to the vascular endothelium can facilitate and promote tumour cell metastasis (Offner et al., 1992). However, the mechanisms involved in endothelial injury are poorly understood.

The present study was conducted to investigate whether direct contact between human tumour cell spheroids and monolayers of endothelial cells induce changes in MMP-2, ROS and IL-6 production in comparison to tumour and endothelial cells cultivated separately.

2 Materials and methods

2.1 Tumour cell lines

Cells of the human cervical carcinoma line (HeLa: ECACC No 85060701) and the human laryngeal carcinoma line (Hep-2: ECACC No 86030501) were used in this study. They were grown as monolayers in 25-cm3 culture flasks (Nunc., Roskilde, Denmark) in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS) (Gibco™, Paisley, UK) and antibiotics (100U/ml penicillin, 100μg/ml streptomycin) at 37°C in a humidified atmosphere with 5% CO2.

2.2 Tumour cell spheroid preparation

Tumour cell spheroids were prepared by the liquid overlay method (Offner et al., 1993). An aliquot of tumour cell suspension (200μl, 1×104cells/ml) in RPMI 1640 medium supplemented with 10% FBS was plated on 1% agarose-coated 96-multiwell culture plates (2000cells/well). After 4 days' incubation at 37°C in a humidified atmosphere with 5% CO2, the cells formed spheroids of about 250μm in diameter. Only tight and round-shaped aggregates were used in the co-culture tests.

2.3 Endothelial cells

HUVEC were isolated from human umbilical cords obtained from healthy donors up to 4h after birth. The umbilical vein was washed with warm PBS containing 200U/ml penicillin, 200μg/ml streptomycin and 0.5μg/ml amphotericin B. Endothelial cells were isolated from the umbilical vein by a 10min digestion with 0.2% collagenase type I (Sigma, St. Louis, MO, USA), suspended in CS-C medium (Sigma) supplemented with 10% FBS, and centrifuged at 200×g for 10min. The pellet was resuspended in CS-C medium containing 10% FBS and 75μg/ml endothelial cell growth factor (ECGF) (Sigma), and the cells were grown in 25cm3 tissue culture flasks (Nunc). Cells were identified by their cobblestone morphology and by immunofluorescence after staining with antibodies against factor VIII-related von Willebrandt antigen (Dakopatts, Glostrup, Denmark). Cells were used in the experiments at passages 1 and 2.

2.4 Skin fibroblasts

HSF cells were isolated from freshly excised tissue fragments from healthy donors. The explants were washed twice with RPMI 1640 medium containing 200U/ml penicillin, 200μg/ml streptomycin and 0.5μg/ml amphotericin B. The fragments were overlaid with a 1% agarose. The culture was maintained by adding medium (RPMI 1640 supplemented with 10% FBS) and incubating at 37°C in a humidified atmosphere with 5% CO2. After three weeks, outgrowths of fibroblasts were separated and cultured.

2.5 Tumour cell spheroid – HUVEC co-cultures

Tumour spheroids were harvested with glass pipettes from agarose-coated microplates and transferred into a Petri dish filled with warm RPMI 1640 medium. After washing for 5min, 15 spheroids were transferred onto a confluent HUVEC monolayer in 24-well tissue culture plates in 1ml CS-C medium supplemented with 2% FBS and ECGF, and incubated at 37°C in a humidified atmosphere with 5% CO2. In parallel experiments, a HUVEC monolayer was preactivated for 2h with 10μg/ml LPS from Escherichia coli, serotype 0111:B4 (Sigma). After 24h co-culture, supernatants were collected and stored at −20°C until the MMP-2 or IL-6 assay was performed.

2.6 Staining of spheroid-HUVEC co-culture

A stock solution containing 0.1mg/ml 2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole (Hoechst No 33342) and 1mg/ml propidium iodide in ethanol/HBSS (2% v/v ethanol) was prepared and stored at −20°C. Working dilutions (1:100) were prepared in Hank's buffered salt solution (HBSS) immediately before use. Cells were stained with the dye mixture for 2min at room temperature and were analysed under a fluorescence microscope (Olympus BX 51) at 380nm. Hoechst 33342-stained cells were blue while the propidium iodide penetrated cells with damaged cytoplasmic membranes (necrotic cells) conferred red fluorescence on the nuclei.

2.7 Confocal microscopy

Co-cultures of tumour spheroids with HUVEC were stained with 3,6-bis(dimethylamino)acridinium chloride (1mg/ml) in ethanol/HBSS (2% v/v ethanol) for 2min and analysed under a laser confocal microscope (LSM 5 PASCAL) at 488nm. Cross-sectional reconstruction was accomplished using the LSM 5 Image Examiner (Zeiss) program.

2.8 Measurement of superoxide anion production by cytochrome c reduction assay

A HUVEC suspension at 5×105cells/ml was added to each well (100μl/well) of a 96-well microplate. After 24h, 1 tumour cell spheroid was added to each well and the microplate was incubated at 37°C, 5% CO2 for the next 24h. Thereafter, the culture medium was discarded and replaced with 243.5μl of a mixture of HBSS without phenol red (226μl), 12.5μl cytochrome c solution in HBSS (final concentration 75μM) and 5μl of either SOD solution (final concentration 60U/well) or HBSS. The plate was incubated again at 37°C, 5% CO2 for 60min and 200μl of the solution was transferred onto a new 96-well plate. The absorbance values at 550nm (differences in OD between samples with and without SOD) were read in a microplate reader. The results were expressed as nanomoles of O2, on the basis of the following formula: nmol O2 per well=ΔOD550nm×14.88 (conversion coefficient calculated on the basis of reagent concentration, incubation time and sample volume) (Colowick and Kaplan, 1986).

2.9 Zymography

MMP activity was measured using SDS-PAGE zymography. Gelatin (300μg/ml) (Sigma) was incorporated into a 7.5% polyacrylamide gel. Samples were mixed with equal volumes of 2× sample buffer (0.25M Tris–HCl pH 6.8, 10% (v/v) glycerol, 0.05% (w/v) bromophenol blue and 5% (w/v) SDS) and separated by electrophoresis. The gel was washed in distilled water, incubated for 1h at room temperature with 2.5% (v/v) Triton X-100, washed again in distilled water and incubated for 24h at 37°C with developing buffer (0.05M Tris–HCl pH 7.4, 0.01M CaCl2 and 0.02% (w/v) NaN3). After washing, the gel was stained for 1h with 0.2% Coomassie blue in 50% (v/v) methanol and 10% (v/v) acetic acid. It was destained with 20% (v/v) methanol and 10% (v/v) acetic acid. Bands of digested substrate (lytic zones) indicated metalloproteinase activity. Semiquantitative densitometric analysis of MMP levels on the gels was carried out using the Bio-Profil Bio-1D Windows Application V.99.03 program.

2.10 MMP-2 and IL-6 assay

The levels of MMP-2 and IL-6 were tested immunoenzymatically (ELISA) using commercially available kits (R&D, Minneapolis, MN and Oncogene, Darmstadt, Germany), according to the manufacturers' instructions. Briefly, samples of supernatants were added to 96-well microplates coated with murine monoclonal antibodies against human MMP-2 or IL-6 and incubated for 2h at room temperature. After three washes in the appropriate buffer, polyclonal secondary antibodies conjugated to horseradish peroxidase were added and incubated for 2h at room temperature. The optical density was measured at 450nm using a microplate reader (Molecular Devices Corp., Emax, Menlo Park, CA, USA), and MMP-2 or IL-6 concentrations were calculated from standard curves prepared from recommended substrates. The detection limits were: <1pg/ml of human IL-6 and 0.1ng/ml of human MMP-2. Amounts of cytokine below the detection limit were considered as 0 for the purposes of analysis.

2.11 Statistical analyses

Significance levels were calculated using student's t-test. P-values lower than 0.05 were considered statistically significant.

3 Results

Four days after the single-tumour cell suspension was seeded onto 1% agarose-coated microplate wells, tightly packed, rounded spheroids were obtained, approximately 250–300μm in diameter. Stained with the fluorochrome mixture of Hoechst 33342 and propidium iodide, the spheroid displayed a heterogeneous, concentric structure (Fig. 1). At the periphery there was a thick rim of viable cells; the inner zone consisted of dead cells forming a necrotic core. Confocal microscopy revealed that during co-culture of the spheroids with HUVEC, the necrotic core collapsed or the cells from the upper side of the spheroid migrated downwards. When the slides were examined by confocal microscopy and the images were analysed in 13.6μm slices, a ring of living cells and an inner zone consisting of necrotic cells was observed in the upper part of the spheroid (Fig. 2). Moreover, adhesion of the tumour spheroids to the endothelium induced degradation of the endothelial cells in the centre of the contact zone; a local effect was observed as a circular peri-spheroidal zone of damaged endothelial cells. When the co-culture incubation was extended to 24h, the tumour cells migrated from the spheroids and formed a circular rim of about 300μm in diameter surrounding the spheroid (Fig. 3).

Fig. 1

The structure of a HeLa-cell spheroid. Membrane integrity IP-H assay, analysed under fluorescence inversion microscope (Olympus BX 51). Bar 50μm. Hoechst 33342 (H) penetrates viable cells, freely crossing the intact plasma membrane and intercalates with double-stranded DNA, resulting in a blue fluorescence of the nuclei. Propidium iodide (IP) is a negatively charged, high polar dye that can penetrate only cells with damaged plasma membranes exhibiting a red fluorescence of the nuclei. Viable cells do not take it up. The cells were examined at a wavelength of 380nm.

Fig. 2

Confocal microscope analysis of co-culture of HeLa spheroids with HUVEC. The co-culture was stained for 2min with AO (acridine orange). Tumour cells inside the spheroid were non-viable and the spheroid was attached to the HUVEC monolayer only by a rim of living cells.

Fig. 3

Migration zone of HeLa cells on HUVECs. Co-culture after 24h. Tumour cells have migrated from the spheroid and formed a circular monolayer zone around the aggregate. The diameter of this zone differs depending on the direction of tumour cell migration and the shape of the spheroid. Light microscope. Bar 200μm.

In order to identify the factors responsible for cell damage, we tested superoxide generation and metalloproteinase production during direct interactions between tumour cell spheroids and endothelial cell monolayers. After 24h co-culture of HeLa and Hep-2 spheroids with endothelial cells we found 1.6-fold and 2.1-fold increases in O2 anion production, respectively, compared to the HUVEC monoculture (Fig. 4A). As a control, normal human skin fibroblast (HSF) spheroids were attached to a HUVEC monolayer; no increase in O2 anion production was observed. HUVEC activation by LPS pretreatment caused an additional increase in superoxide anion production (Fig. 4B). The interaction between tumour cell spheroids and endothelial cell monolayers altered the paracrine activity of the co-cultured cells as well as induced morphological changes and generated free radicals. Zymographic analysis of the supernatant samples indicated production of at least two different MMPs (Fig. 5). Densitometric analysis revealed a 1.7-fold increase in the enzymatic activity of the 72kDa protein in the co-culture model compared to tumour spheroid or HUVEC monocultures. Quantitative (ELISA) analysis of the total MMP-2 protein level revealed that after 24h co-cultivation of HeLa and Hep-2 cell spheroids with the endothelial cell monolayer, MMP-2 production was increased about 5-fold in comparison to the HUVEC monoculture (Fig. 6).

Fig. 4

(A) Superoxide anion (O2) production during 24-h co-culture of tumour cell spheroids with HUVEC. (B) The influence of LPS-stimulated HUVEC activation on superoxide anion (O2) production during 24h co-culture of tumour cell spheroids with HUVEC.

Fig. 5

SDS-PAGE analysis of metalloproteinase (MMP) production. Lane 1 – MMPs in culture medium supplemented with 2% FCS; lane 2 – molecular weight marker; lane 3 – MMPs produced by HUVEC monoculture; lanes 4 and 5 – MMPs produced in co-culture of the HUVEC monolayer with HeLa and Hep-2 cell line spheroids, respectively; lanes 6 and 7 – MMPs produced by HeLa and Hep-2 cell line spheroids monocultures, respectively. Values in the table represent densitometrically quantitated volumes of bands.

Fig. 6

Metalloproteinase-2 (MMP-2) production during 24h co-culture of tumour cell spheroids with HUVEC.

Immunoenzymatic analysis showed that the endothelium and tumour spheroids alone produced low levels of IL-6 (Fig. 7A). In the co-culture model, the quantities of the cytokine were significantly higher than in the HUVEC monoculture, but they did not exceed the sum of IL-6 production by endothelial cells and tumour spheroids cultured separately. However, after preincubation with LPS, IL-6 production increased significantly in HUVECs and their co-culture with tumour spheroids (Fig. 7B).

Fig. 7

(A) Interleukin-6 (IL-6) production during 24h co-culture of tumour cell spheroids with HUVEC. (B) The influence of LPS-stimulated HUVEC activation on interleukin-6 (IL-6) production during 24h co-culture of tumour cell spheroids with HUVEC.

4 Discussion

Metastasis is a multistep process involving the escape of cells from the primary tumour, intravasation, dissemination via blood or lymphatic vessels, adhesive interactions with the endothelium, proteolytic degradation of the basement membrane and ECM components, extravasation, and secondary tumour growth in distant sites (Chambers and Matrisian, 1997). The attachment of the tumour cells to the internal blood vessel wall depends crucially on the expression of adhesive molecules on endothelial cell surfaces. Cytokines and growth factors are also implicated in the metastatic process. Moreover, interactions between tumour cell clusters and endothelial cells often occur where inflammatory reactions develop and where proinflammatory cytokines, which play a crucial role in cell–cell adhesions, are released (Takahashi et al., 2001). Cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) are overexpressed in the inflammatory state and play an important role in tumour development, influencing the metastatic potential of the tumour cells (Johnson, 1991; Maruo et al., 2002). ICAM-1 expression is induced by proinflammatory cytokines including interferon-γ (IFN-γ), interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α) (Kirnbauer et al., 1992).

Direct cell–cell interactions and cytokine release are not sufficient for extravasation of tumour cells or their migration into the connective tissue (Voura et al., 1998). The development of an invasive tumour cell phenotype also entails changes in cell motility (Siegel and Malmstein, 1997) and the capacity to evoke local proteolysis. The ability of tumour cells to migrate across the endothelium and basement membrane is considered one of the major characteristics of malignancy (Werner et al., 2002), involving the paracrine production of matrix metalloproteinases, cytokines, and growth factors (Rak et al., 1996). Therefore, in this study, we analysed ROS, MMP-2 and IL-6 production after direct interaction of tumour cell spheroids (HeLa and Hep-2 cell lines) with an endothelial cell (HUVEC) monolayer as an in vitro model of the direct and paracrine interactions between malignant cells and the blood vessel endothelium.

After 12h co-culture of the tumour spheroids with HUVEC, we observed a peri-spheroidal zone of damaged endothelial cells. Moreover, a significant increase in O2 was observed when HUVEC cells were co-cultured for 24h with HeLa and Hep-2 tumour spheroids, but not with normal human skin fibroblast (HSF) cells. Therefore, our results are in agreement with those of Offner et al. (1993), who showed that endothelial damage is closely associated with tumour cell attachment. Moreover, these authors demonstrated that tumour cells can produce increased amounts of superoxide anion, which is particularly injurious to the local endothelium. Wartenberg et al. (2001) showed that ROS generation is elevated in small tumour spheroids; and that large spheroids with extended central necrosis, very similar to the spheroids used in our experiments, also produce ROS. Moreover, we can exclude that degradation products from the necrotic core of the spheroid could significantly influence peri-spheroidal zone of endothelial cells. They should, however, diffuse in high enough concentration through the living rim of tumour cells that form external zone of the spheroid to influence endothelial cells viability. Therefore, if degradation products of tumour cells could influence and degrade endothelial cells they should also destroy spheroid external cellular rim but it was not observed. Moreover, spheroids formed by normal human skin fibroblasts (HSF) not producing ROS did not form “the halos” of damaged endothelial cells surrounding the spheroid.

Therefore, the question arises: what are the inducers of ROS production in tumour cells adhering to the endothelium? ROS are generated (inter alia) during cellular respiration, and their most important sources are mitochondria (Jackson and Loeb, 2001). ROS release is stimulated and controlled by cytokines and growth factors, e.g. IL-1β or TNF-α, and these in turn influence the regulatory transcription factors that mediate apoptosis (Zapolska-Downar et al., 2002; Haddad, 2002). Therefore, some cytokines have been shown in several cell models to induce oxidative stress. Also, it has been reported that mitochondrial-derived ROS contribute to tumour necrosis factor-α (TNF-α)-induced cytotoxicity and nuclear factor-κB (NF-κB) activation. NF-κB activation is necessary for the expression of TNF-α-induced cellular adhesion molecules (E-selectin, VCAM-1) (Zapolska-Downar et al., 2002; Haddad, 2002), and these molecules facilitate direct cell–cell interactions. There are also several interdependencies between ROS and IL-6 production. ROS induce IL-6 production that is highly responsive to inflammation. IL-6, in turn, increases superoxide release by neutrophils, hepatocytes or fibroblast-like synoviocytes (Koren et al., 2000; Sung et al., 2000). Therefore, we suspect that co-culture of tumour spheroids with endothelial cells involves the following sequence of events: initial attachment of tumour spheroids to the HUVEC cell monolayer may signal ROS release, then ROS stimulates the expression of endothelial adhesive molecules that facilitate tumour implantation and IL-6 production. IL-6 is one of the cytokines that influence the capacity of tumour cells to migrate.

Oxidative stress is stimulated by LPS as well as by cytokines. Moreover, oxidative stress increases the production of proinflammatory cytokines from endothelial cells, e.g. IL-1β, TNF-α and IL-6, and these are potent oxidative stress mediators (Haddad, 2002; Legrand-Poels et al., 1997). IL-6 is a multifunctional, central proinflammatory cytokine crucial in the acute phase response. It is produced mainly not only by monocytes, macrophages and fibroblasts but is also produced by endothelial cells (Saba et al., 1996). In this study, we observed that IL-6 was produced in HUVEC monocultures and in co-cultures with tumour spheroids, but its level was very low. LPS is one of the most potent stimulants of endothelial IL-6 expression. Von Asmuth et al. (1991) showed that the stimulation of endothelial cells with LPS significantly increases IL-6 release. We found that pretreatment of endothelial cells with LPS increased both superoxide anion release and IL-6 production. The positive correlation between proinflammatory IL-6 production and superoxide anion release strongly suggests that IL-6 is involved in the generation of oxidative stress; this, in turn, enhances proinflammatory cytokine production. Considering the causes of enhanced IL-6 production, we can also speculate that cell-to-cell contact or soluble factors are responsible for the phenomenon. It is well known that sIL-6R strongly sensitises endothelial cells. Endothelial cells do not express membrane-bound IL-6R and therefore cannot be stimulated by IL-6 alone (Rose-John, 2003; Dowdall et al., 2002). In contrast to endothelial cells, cancer cells can express IL-6R and secrete the soluble IL-6R form (Matsuo et al., 2003). Therefore, the increase in IL-6 in our co-culture model may be at least partially a result of co-operation between IL-6 and tumour-derived sIL-6R, which may directly or indirectly enhance IL-6 production in the endothelial and tumour cells.

Apart from ROS, an essential role in tumour metastasis and invasion is also played by MMP expression. Metastasis requires proteolytic degradation of ECM components by MMPs to facilitate the invasion of malignant cells through the basement membrane and, subsequently, the connective tissue. In our experiments, we did not cover the cell-culture surfaces with any extracellular matrix element. Nevertheless, a significant increase in MMP production was observed after interaction between the tumour cell spheroids and the endothelial cell monolayer. Therefore, we suggest that endothelial cell-derived ECM elements and direct cell–cell contact are enough to stimulate an increased MMPs production by tumour cells. It is well known that the expression of metalloproteinases is regulated by various growth factors, cytokines and oncogene products (FGF-2, EGF, TNF-α or Ras) (Hah and Lee, 2003). It has also been shown that enhanced expression of IL-6 during inflammation may augment the expression of MMP-2 (Yano et al., 2003). There is also a significant positive correlation between the activated form of MMP-2 and the expression of vascular endothelial growth factor (VEGF). In consequence, enhanced VEGF expression stimulates tumour vascularization and motility of tumour cells (Kurizaki et al., 1998; Garzetti et al., 1999). Experiments addressing the role of cytokines in tumour cell–endothelium interactions are in progress.

In conclusion, the present study reveals that tumour cell spheroids induce local damage after direct contact with the endothelial monolayer, and the damage is mediated by overproduction of reactive oxygen species (ROS) and increased MMP-2 activity. In addition, the activation of endothelial cells with LPS, as an in vitro model of inflammation, significantly increases superoxide anion and IL-6 production in co-cultures of tumour cell spheroids and HUVEC.


This research was supported by grant No 6 PO4C 048 18 from State Committee for Scientific Research.


Chambers AF, Matrisian, LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 1997:89:1260-70
Crossref   Medline   1st Citation   2nd  

Chang CH, Werb, Z. The many faces of metalloproteinases: cell growth, invasion, angiogenesis and metastasis. Trends Cell Biol 2001:11:S37-43
Medline   1st Citation  

Chang CH, Werb, Z. . Colowick SP, Kaplan NO. Methods in enzymology. In: Di Sabato G, Everse J, editors. Phagocytosis and cell-mediated cytotoxicity, 132, Part J; 1986. p. 410–17.
1st Citation  

Dowdall JF, Winter, DC, Andrews, E, Laug, WE, Wang, JH, Redmond, HP. Soluble interleukin-6 receptor (sIL-6R) mediates colonic tumor adherence to the vascular endothelium: a mechanism for metastatic initiation? J Surg Res 2002:107:1-6
Medline   1st Citation  

Foda HD, Zucker, S. Matrix metalloproteinases in cancer invasion, metastasis and angiogenesis. Drug Discov Today 2001:6:478-82
Crossref   Medline   1st Citation   2nd   3rd  

Fridman R, Toth, M, Pena, D, Mobashery, S. Activation of progelatinase B (MMP-9) by gelatinase A (MMP-2). Cancer Res 1995:55:2548-55
Medline   1st Citation  

Garzetti GG, Ciavattini, A, Lucarini, G, Pugnaloni, A, De nictolis, M, Amati, S. Expression of vascular endothelial growth factor related to 72-kilodalton metalloproteinase immunostaining in patients with serous ovarian tumors. Cancer 1999:85:2219-25
Crossref   Medline   1st Citation  

Haddad JJ. Redox regulation of pro-inflammatory cytokines and IκB-α/NF-κB nuclear translocation and activation. Biochem Biophys Res Commun 2002:296:847-56
Crossref   Medline   1st Citation   2nd   3rd  

Hah N, Lee, S-T. An absolute role of the PCK-dependent NF-κB activation for induction of MMP-9 in hepatocellular carcinoma cells. Biochem Biophys Res Commun 2003:305:428-33
Crossref   Medline   1st Citation  

Hamilton G. Multicellular spheroids as an in vitro tumor model. Cancer Lett 1998:131:29-34
Crossref   Medline   1st Citation  

Jackson AL, Loeb, LA. The contribution of endogenous sources of DNA damage to the multiple mutations in cancer. Mutat Res 2001:477:7-21
Medline   1st Citation  

Jackson ChJ, Nguyen, M. Human microvascular endothelial cells differ from macrovascular endothelial cells in their expression of matrix metalloproteinases. Int J Biochem Cell Biol 1997:29:1167-77
Crossref   Medline   1st Citation  

Johnson JP. Cell adhesion molecules of the immunoglobulin supergene family and their role in malignant transformation and progression to metastatic disease. Cancer Metastasis Rev 1991:10:11-22
Crossref   Medline   1st Citation  

Kebers F, Lewalle, J-M, Desreux, J, Munaut, C, Devy, L, Foidart, J-M. Induction of endothelial cell apoptosis by solid tumor cells. Exp Cell Res 1998:240:197-205
Crossref   Medline   1st Citation  

Kirnbauer R, Charvat, B, Schauer, E, Kock, A, Urbanski, A, Forster, E. Modulation of intercellular adhesion molecule-1 expression on human melanocytes and melanoma cells: evidence for a regulatory role of IL-6, IL-7, TNF-β, and UVB light. J Invest Dermatol 1992:98:320-6
Crossref   Medline   1st Citation  

Koren R, Rocker, D, Kotestiano, O, Liberman, UA, Ravid, A. Synergistic anticancer activity of 1,25-dihydroxyvitamin D3 and immune cytokines: the involvement of reactive oxygen species. J Steroid Biochem Mol Biol 2000:73:105-12
Crossref   Medline   1st Citation  

Kräling BM, Wiederschain, DG, Boehm, T, Rehn, M, Mulliken, JB, Moses, MA. The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro. J Cell Sci 1999:112:1599-609
Medline   1st Citation  

Kurizaki T, Toi, M, Tominaga, T. Relationship between matrix metalloproteinase expression and tumor angiogenesis in human breast cancer. Oncol Rep 1998:5:673-7
Medline   1st Citation  

Legrand-Poels S, Maniglia, S, Boelaert, JR, Piette, J. Activation of the transcription factor NF-κB in lipopolysaccharide-stimulated U937 cells. Biochem Pharmacol 1997:53:339-46
Crossref   Medline   1st Citation  

Liekens S, De Clercq, E, Neyts, J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol 2001:61:253-70
Crossref   Medline   1st Citation  

Lin-Hung W, Min-Liang, K, Chi-An, Ch, Wen-Fang, Ch, Shao-Pei, Ch, Fon-Jou, H. Interleukin-6 in cervical cancer: the relationship with vascular endothelial growth factor. Gynecol Oncol 2001:82:49-56
Crossref   Medline   1st Citation  

Lorenz B, Schlüter, T, Bohnensack, R, Pergande, G, Müller, WEG. Effect of flupirtine on cell death of human umbilical vein endothelial cells induced by reactive oxygen species. Biochem Pharmacol 1998:56:1615-24
Crossref   Medline   1st Citation   2nd  

Maruo Y, Gochi, A, Kaihara, A, Shimamura, H, Yamada, T, Tanaka, N. ICAM-1 expression and the soluble ICAM-1 level for evaluating the metastatic potential of gastric cancer. Int J Cancer 2002:100:486-90
Crossref   Medline   1st Citation  

Matsuo K, Oka, M, Murase, K, Soda, H, Isomoto, H, Takeshima, F. Expression of interleukin 6 and its receptor in human gastric and colorectal cancers. J Int Med Res 2003:31:69-75
Medline   1st Citation  

Offner FA, Bigalke, I, Schiefer, J, Wirtz, HCh, Klosterhalfen, B, Feichtinger, H. Interaction of human malignant melanoma tumor spheroids with endothelium and reconstituted basement membrane: modulation by RGDS. Int J Cancer 1993:54:506-12
Crossref   Medline   1st Citation   2nd  

Offner FA, Schiefer, J, Wirtz, HCh, Bigalke, I, Pavelka, M, Hollweg, G. Tumor-cell–endothelial interactions: free radicals are mediators of melanoma-induced endothelial cell damage. Virchows Arch 1996:428:99-106
Medline   1st Citation  

Offner FA, Wirtz, HCh, Schiefer, J, Bigalke, I, Klosterhalfen, B, Bittinger, F. Interaction of human malignant melanoma (ST-ML-12) tumor spheroids with endothelial cell monolayer. Am J Pathol 1992:141:601-10
Medline   1st Citation  

Rak J, Filmus, J, Kerbel, RS. Reciprocal paracrine interactions between tumour cells and endothelial cells: the ‘angiogenesis progression’ hypothesis. Eur J Cancer 1996:32A:2438-50
Medline   1st Citation   2nd  

Rose-John S. Interleukin-6 biology is coordinated by membrane bound and soluble receptors. Acta Biochim Pol 2003:50:603-11
Medline   1st Citation  

Saba AA, Kaidi, AA, Godziachvili, V, Dombi, GW, Dawe, EJ, Libcke, JH. Effect of interleukin-6 and its neutralizing antibodies on peritoneal adhesion formation and wound healing. Am Surg 1996:62:569-72
Medline   1st Citation  

Siegel G, Malmstein, M. The role of the endothelium in inflammation and tumor metastasis. Int J Microcirc Clin Exp 1997:17:257-72
Crossref   Medline   1st Citation  

Sung J-Y, Hong, J-H, Kang, H-S, Choi, I, Lim, S-D, Lee, J-K. Methotrexate suppresses the interleukin-6 induced generation of reactive oxygen species in the synoviocytes of rheumatoid arthritis. Immunopharmacology 2000:47:35-44
Crossref   Medline   1st Citation  

Takahashi T, Hato, F, Yamane, T, Fukumasu, H, Suzuki, K, Ogita, S. Activation of human neutrophils by cytokine-activated endothelial cells. Circ Res 2001:88:422-9
Medline   1st Citation  

Von Asmuth EJ, Leeuwenberg, JF, Ceska, M, Buurman, WA. LPS and cytokine-induced endothelial cell IL-6 release and ELAM-1 expression; involvement of serum. Cytokine Netw 1991:2:291-7
1st Citation  

Voura EB, Sandig, M, Siu, CH. Cell–cell interactions during transendothelial migration of tumor cells. Microsc Res Tech 1998:43:265-75
Crossref   Medline   1st Citation  

Wartenberg M, Ling, FC, Schallengerg, M, Bäumer, AT, Petrat, K, Hescheler, J. Down-regulation of intrinsic P-glycoprotein expression in multicellular prostate tumour spheroids by reactive oxygen species. J Biol Chem 2001:276:17420-8
Crossref   Medline   1st Citation  

Werner JA, Rathacke, IO, Mandic, R. The role of matrix metalloproteinases in squamous cell carcinomas of the head and neck. Clin Exp Metastasis 2002:19:275-82
Crossref   Medline   1st Citation  

Yano S, Nokihara, H, Yamamoto, A, Goto, H, Ogawa, H, Kanematsu, T. Multifunctional interleukin-1β promotes metastasis of human lung cancer cells in SCID mice via enhanced expression of adhesion-, invasion- and angiogenesis-related molecules. Cancer Sci 2003:94:244-52
Crossref   Medline   1st Citation  

Yen CH, Hsieh, CC, Chou, SY, Lau, YT. 17β-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells. Life Sci 2001:70:403-13
Crossref   Medline   1st Citation  

Zapolska-Downar D, Zapolski-Downar, A, Naruszewicz, M, Siennicka, A, Krasnodēbska, B, Kołodziej, B. Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes. Life Sci 2002:71:2897-908
Crossref   Medline   1st Citation   2nd  

Received 14 September 2004/18 December 2004; accepted 23 January 2005


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)