Oualid Haddad
email: oualid_haddad@yahoo.fr

Roxana Chotard-Ghodsnia

Claude Verdier

Alain Duperray

Tumor cell/endothelial cell tight contact upregulates endothelial adhesion molecule expression mediated by NFκB: differential role of the shear stress

Keywords: metastasis, NFκB, endothelium, flow, cancer, adhesion molecule, interaction

Cancer metastasis is a multistep process involving cell-cell interactions, but little is known about the adhesive interactions and signaling events during extravasation of tumor cells (TCs). In this study, cell adhesion molecule (CAM) expression was investigated using an in vitro assay, in which TCs were seeded onto an endothelial cell (ECs) monolayer and cocultured during 5 hours. Flow cytometry, confocal microscopy as well as western blot analysis indicated that endothelial ICAM-1 (Inter Cellular Adhesion Molecule-1), VCAM-1 (Vascular Adhesion Molecule-1) and E-selectin were up-regulated after TC-EC coculture, whereas no change was observed for CAMs expression in tumor cells. This increased CAMs expression required tight contact between TCs and ECs. Incubation of ECs with the pyrrolidine-dithiocarbamate NFκB inhibitor prior to coculture, fully prevented cocultureinduced expression of endothelial CAMs. Using specific blocking antibodies we showed an implication of ICAM-1 and VCAM-1 for TCs extravasation and VCAM-1 for adhesion. Moreover, fluid flow experiments revealed that high shear stress totally abolished cocultureinduced as well as TNFα-induced CAMs over-expression. This study suggests that TCs could act as a potent inflammatory stimulus on ECs by inducing CAMs expression via NFκB activation, and that this action can be modulated by shear stress.

Introduction:

Hematogenous cancer metastasis is a complex multistep process that involves the detachment of TCs from the primary tumor, migration through tumor-derived vessels into blood stream, survival in blood circulation, extravasation (migration through vascular endothelium to target organs), and finally establishment of secondary tumors [START_REF] Fidler | The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited[END_REF]. All these steps involve close interactions between Tumor Cells (TCs) and Endothelial Cells (ECs) which can be mediated by soluble factors as well as Cell Adhesion Molecules (CAMs). The progression of tumor malignancies is often associated with changes in CAMs profile [START_REF] Christofori | Changing neighbours, changing behaviour: cell adhesion moleculemediated signalling during tumour progression[END_REF], indicating that CAMs play an important role in cancer metastasis. To date, little is known about the details of tumor metastasis and the precise molecular mechanisms involved when TCs interact with the EC layer.

The process of leukocyte diapedesis can serve as a model for describing the interactions between circulating TCs and ECs [START_REF] Takada | Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium[END_REF]. This process occurs in several major steps: leukocytes rolling on the endothelial surface in the direction of blood flow, cell activation, adhesion, locomotion to and protrusions extension through interendothelial junctions, and finally transendothelial migration [START_REF] Schreiber | Shear flow-dependent integration of apical and subendothelial chemokines in T-cell transmigration: implications for locomotion and the multistep paradigm[END_REF]. ECs provide an interface between blood and the vessel wall, and are constantly subjected to hemodynamic forces. It is well established that leukocyte interactions with ECs as well as leukocyte extravasation into inflamed tissues are affected by flow [START_REF] Osborn | Leukocyte adhesion to endothelium in inflammation[END_REF]. Similarly to leukocytes, TC rolling before arrest on the endothelial surface has already been observed under in vitro flow conditions [START_REF] Giavazzi | Rolling and adhesion of human tumor cells on vascular endothelium under physiological flow conditions[END_REF]. However other studies have shown that initial arrest of TCs occurs primarily by size restriction in capillaries [START_REF] Chambers | Early steps in hematogenous metastasis of B16F1 melanoma cells in chick embryos studied by highresolution intravital videomicroscopy[END_REF] without cell rolling on the endothelium [START_REF] Orr | Interactions between cancer cells and the endothelium in metastasis[END_REF]. In some case, TC adhesion to the endothelium can be leukocytes dependent. Strell et al. showed that neutrophil granulocytes mediate the adhesion of breast cells to the endothelium in flow condition [START_REF] Strell | Surface molecules regulating rolling and adhesion to endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interaction[END_REF], and that. the immunoglobulin ICAM-1 (Inter Cellular Adhesion Molecule-1) and its beta2-integrin ligand are likely implicated.

Shear stress is known to induce ECs alignment and elongation [START_REF] Dewey | The dynamic response of vascular endothelial cells to fluid shear stress[END_REF], cytoskeleton rearrangement [START_REF] Galbraith | Shear stress induces spatial reorganization of the endothelial cell cytoskeleton[END_REF] and modification of gene expression [START_REF] Chiu | Shear stress increases ICAM-1 and decreases VCAM-1 and Eselectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells[END_REF][13][START_REF] Tsuboi | Involvement of integrins, MAPK, and NF-kappaB in regulation of the shear stress-induced MMP-9 expression in endothelial cells[END_REF] with activation of the Nuclear Factor Kappa B (NFκB) signaling pathway [START_REF] Imberti | Shear stress-induced cytoskeleton rearrangement mediates NF-kappaB-dependent endothelial expression of ICAM-1[END_REF].

NFκB is a dimeric transcription factor formed by members of a family of proteins, mediating its functional effects through the transcriptional regulation of NFκB target genes. In most cells, NFκB complexes are inactive, residing primarily in the cytoplasm complexed with one member of the family of inhibitory κB (IκB) proteins such as IκBα and IκBβ. Upon activation, IB proteins are degraded, allowing the NFκB complex enter the nucleus and bind to specific sequences located in the promoter region of target genes to modulate their expression [START_REF] Scherbarth | Intravital videomicroscopic evidence for regulation of metastasis by the hepatic microvasculature: effects of interleukin-1alpha on metastasis and the location of B16F1 melanoma cell arrest[END_REF]. Numerous genes are regulated by NFκB, e.g. chemokines MCP-1 and IL-8 [START_REF] Baldwin | Series introduction: the transcription factor NF-kappaB and human disease[END_REF], cytokines TNFα and IFN-γ [START_REF] Trede | Transcriptional activation of the human TNF-alpha promoter by superantigen in human monocytic cells: role of NF-kappa B[END_REF], and adhesion molecules ICAM-1 [START_REF] Chen | Tumor necrosis factor alpha-induced activation of downstream NF-kappaB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway[END_REF], VCAM-1 [START_REF] Iademarco | Characterization of the promoter for vascular cell adhesion molecule-1 (VCAM-1)[END_REF][START_REF] Neish | Functional analysis of the human vascular cell adhesion molecule 1 promoter[END_REF] and E-selectin [START_REF] Whelan | An NF kappa B-like factor is essential but not sufficient for cytokine induction of endothelial leukocyte adhesion molecule 1 (ELAM-1) gene transcription[END_REF].

We previously characterized T24 cell line (Human bladder carcinoma) spreading on an EC monolayer and we demonstrated that shear stress levels modulate TCs spreading patterns [START_REF] Chotard-Ghodsnia | Morphological analysis of tumor cell/endothelial cell interactions under shear flow[END_REF]. The purpose of the present study is to further investigate the effect of T24 cells-EC contact on CAMs expression, as well as to characterize the influence of shear stress levels on ECs response.

Materials and methods

Cell lines and reagents

Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase digestion of umbilical veins from fresh cords [START_REF] Jaffe | Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria[END_REF]. The cells were plated on gelatin-coated culture dishes in M199 medium containing 20% heat-inactivated fetal bovine serum (FBS), ECGS (Endothelial Cells Growth Supplement, 50 µg/mL), heparin (100 µg/mL) and antibiotics. Replicated cultures were obtained by trypsinization and only early passages (1-4) were used.

Eight TC lines were used in this study: T24 cell line and TCCSUP (bladder carcinoma), MCF7 and MDA-MB-435S cells (breast adenocarcinoma), A549 and Calu-6 cells (lung carcinoma) and RKO cells (colon adenocarcinoma) were obtained from ATTC (Rockville, USA). DAG-1 (bladder carcinoma) was kindly provided by Dr Pierre Champelovier [START_REF] Champelovier | Dag-1 carcinoma cell in studying the mechanisms of progression and therapeutic resistance in bladder cancer[END_REF].

Bladder, lung and colon cell lines TCs were cultured respectively in RPMI 1640 and DMEM medium supplemented with 10% fetal bovine serum and antibiotics. Breast cell lines were cultured in DMEM F-12 medium supplemented with 10% fetal bovine serum. Cultures were kept at 37°C in a 5% CO 2 humidified atmosphere. Coculture experiments were performed in HUVEC medium because primary endothelial cells are more sensitive to medium change than tumor cell lines. We make sure that TCs culture in M199 medium had no effect on CAM expression compared to the usual TC medium (data not shown).

TC as well as TC-EC coculture conditioned medium were prepared either by incubating T24 cells in M199 medium (~1.6 × 10 5 cells/ml) for 5 h, or by incubating HUVEC-T24 cells coculture in M199 medium (~3.3 × 10 5 cells/ml) for 5 h. The medium was then removed, centrifuged (1300 rpm, 5 min) and filtered with a 0.22-μm filter. HUVECs were then incubated with the corresponding conditioned medium for 5 h. All cells were cultured at 37°C in a humidified atmosphere with 5% CO 2 .

The NFκB inhibitor pyrrolidine-dithiocarbamate (PDTC, 100µM) was purchased from Sigma Aldrich and used at 100µM concentration. The recombinant human TNFα was purchased from Strathmann biotec AG (Germany). The cytoplasmic dye Cell Tracker Green (CTG) was purchased from Invitrogen (USA).

Antibodies, plasmids

Mouse monoclonal antibodies to VCAM-1 and E-selectin (Becton Dickinson, France), ICAM-1 [START_REF] Languino | Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition[END_REF] and IκBα (sc-1643, Santa Cruz, CA) were used at a 5µg/mL concentration. The plasmid expressing GFP (pEGFP-N2) was obtained from Clontech BD and transfected into T24 cells using lipofectamine 2000 (Invitrogen).

Immunoassay for TNFα

TCs were incubated for 12, 24 and 48h in complete culture medium. At the end of the incubation time, supernatants were collected, centrifuged at room temperature for 10 min at 1500g in order to remove cellular debris, and stored at -80°C until analysis of TNFα levels.

Concentrations of human TNFα in cell culture supernatants of tumor cell lines were determined in triplicates with a colorimetric sandwich enzyme linked immunosorbent assay (PromoCell, Heidelberg, Germany), following the manufacturer's instructions.

FACS analysis of CAMs expression in TC/EC cocultures

To easily distinguish TCs from ECs, TCs were labeled with the cytoplasmic dye Cell Tracker Green (20µM, 20min). For T24 cells, we have developed a stable transfectant cell line expressing GFP which was used for the majority of experiments performed in this paper.

Labeled TCs (5.10 5 cells) were cocultured on an endothelial monolayer (5.10 5 cells) for 5 hours in HUVEC medium. Cells were trypsinized, then incubated with appropriate antibodies (1h), followed by phycoerythrin-conjugated goat anti-mouse antibody (30min). FACS analysis (FACScan flow cytometer Becton Dickinson) was performed to examine CAM levels on the EC and TC surfaces. Quantification was made by measuring the geometric mean fluorescence.

Determination of cell adhesion by flow cytometry

HUVECs (5.10 5 ) were seeded in triplicates in a 6-well plate precoated with gelatin 2% and grown to confluency for 48h. In some cases HUVECs were stimulated with TNFα (0.5 U/mL, 5h), and with the appropriate blocking antibodies (20µg/mL, 30min). Subsequently 5.10 5 T24-GFP cells were added per well and incubated for 5 hours (37 °C and 5% CO 2 ) in the presence of the same blocking antibody. At the end of the assay, wells were washed twice with Phosphate Buffered Saline (PBS). Samples were trypsinized and the number of adherent T24-GFP cells was determined using flow cytometry.

Transendothelial cell migration

Cell migration was assayed using a modified Boyden chamber assay. HUVECs (1,5.10 4 ) were grown to confluence (48h) on a 3µm-pore size polycarbonate membrane (treated with fibronectin at 20µg/mL) separating the two compartments of a migration chamber (Transwell, Becton Dickinson, 6.5mm diameter). HUVECs were treated or not with 100U/mL TNFα (24h). Thereafter, EC monolayers were rinsed before T24-GFP addition. After 5h-coculture, cells on the upper face of the membrane were scraped using a cotton swab. TCs that migrated to the lower face of the filter were lysed by PBS-Triton 1%. The migrating cells were quantified by measuring the GFP fluorescence emission using a fluorometer (Fluoroskan Ascent, Labsystems).

Microscopy and immunofluorescence staining

T24 cells were incubated on a HUVEC monolayer for 5 hours. Cells were fixed with 2% paraformaldehyde, then permeabilized with 0.5% Triton X-100, stained with anti ICAM-1, and incubated with Alexa fluor 488-conjugated goat anti-mouse IgG (Molecular Probes) before confocal microscopy observations (Zeiss LSM510, NLO).

Immunoblot Analysis

5.10 5 ECs were cultured on a gelatin-coated 6-well plate (48h). An equal number of TCs was deposited on the endothelial monolayer and cocultured for 5 h. After washing, about 50% of the total added TCs adhered to the EC monolayer which represents a 1 TC for 2 ECs ratio.

Therefore, to prepare samples for 0h of coculture, TCs and ECs were collected separately, and then mixed together (with a ratio of 1 TC for 2 ECs) before lysis and immunoblot analysis.

Protein samples were separated by SDS-PAGE 10%. Blots were probed with antibodies and developed with ECL reagents. For quantification, the blots were analyzed using ImageJ software (NIH Image, Bethesda, USA) and quantification was based on the pixel value of each band.

Laminar flow assays

A parallel-plate flow chamber providing a laminar flow was used, as previously described [START_REF] Chotard-Ghodsnia | A new flow chamber for the study of shear stress and transmural pressure upon cells adhering to a porous biomaterial[END_REF]. The wall shear stress ranged from 0.9±0.2 to 18±4 dynes/cm². To test the regulatory effect of shear stress on the coculture-induced CAM expressions in ECs, HUVECs monolayers were incubated under static conditions for 24h or exposed to flow for 24h, or exposed to flow for 19h before coculture with T24-GFP for 5h under different flow conditions (0.9 to 18 dynes/cm 2 ). The final concentration (4.10 6 cells/mL) of the T24-GFP suspension injected in the flow loop was calculated to obtain a constant ratio of 1EC for 1 TC in the flow chamber during coculture experiments. TCs were allowed to settle down and adhere to the EC monolayer for 15min under static conditions and then exposed to flow. In additional experiments, the effect of shear stress on the TNFα-induced protein expression was studied by subjecting ECs to flow for 19h before TNFα-stimulation (100U/mL) for 5h under identical flow conditions. To determine the implication of NFκB signaling pathways in the cocultureinduced CAMs over-expression under flow, ECs were presheared 18h at 9 dynes/cm², then treated with PDTC (100µM, 1h) under the same flow condition, prior to 5h-coculture at 9 dynes/cm². At the end of the flow assays, cells were trypsinized, and the CAM levels on cell surfaces were determined using flow cytometry and western blot analysis.

Results

Endothelial ICAM-1 is up-regulated during HUVEC-T24 bladder carcinoma cells coculture

To investigate the effect of TC-EC interaction on CAM expression, bladder carcinoma cell line T24 was added to a Human Umbilical Vein Endothelial Cell (HUVEC) monolayer and cocultured for five hours. After washing, cells were collected and probed with an anti-ICAM-1 monoclonal antibody. FACS analysis (Figure 1a-c) was carried out to determine ICAM-1 expression levels for each cell population and to quantify adherent TCs. Fluorescent TCs were used to easily distinguish each cellular type (cf. materials and methods). Both cell types express a low level of ICAM-1 under basal conditions (Figure 1a andb), this expression being increased by cytokines such as TNFα.

Figure 1c represents the pattern obtained by flow cytometry from a 5h-coculture of ECs and T24-GFP. In this typical experiment, T24-GFP adherent cells represent 44% of total initially added TCs. Figure 1d shows the endothelial ICAM-1 levels before (black) and after (gray) coculture; the fluorescence intensity of the gray curve is significantly higher than the fluorescence intensity of the black one, indicating an increase of ICAM-1 expression in ECs after coculture. Figure 1e shows the tumoral ICAM-1 levels before (black) and after (gray) coculture; in this case, fluorescence intensity is similar for both curves, indicating no change of ICAM-1 expression in TCs after coculture. In conclusion, a fourfold increase of ICAM-1 expression was observed on ECs (Figure 1d), but no change was detected on TCs (Figure 1e).

Using confocal microscopy, resting ECs displayed a weak ICAM-1 expression (Figure 1f), whereas, after 5h-coculture, a strong expression of endothelial ICAM-1 was detected (Figure 1g). Western blot analysis confirmed this up-regulation in ICAM-1 expression after coculture (Figure 1h), with a fourfold increase when compared to ICAM-1 expression on resting HUVECs. These data clearly indicate that HUVEC-T24 cells interactions stimulate the expression of ICAM-1 on ECs, but not on T24 cells.

Endothelial VCAM-1 and E-selectin are also over-expressed during HUVEC-T24 coculture

Considering that endothelial ICAM-1 expression was induced by T24-EC coculture (Figure 1), we further investigated the modulation of other CAMs expression. HUVECs and T24 cells cocultured for 5h were collected and then analyzed for E-selectin, ICAM-1, VCAM-1, PECAM-1, VE-and N-cadherin, β1 and β3 integrin expression by FACS analysis. No change in tumoral CAM expression was observed after 5h-coculture (supplementary data, Figure S1), whereas only three endothelial CAMs were up-regulated: ICAM-1, VCAM-1 and E-selectin (Figure 2).

Characterization of CAM up-regulation induced by HUVEC-T24 coculture

We next investigated whether this CAM over-expression depends on the number of TCs (T24) in contact with ECs. Therefore, we analyzed ICAM-1, VCAM-1 and E-selectin expression levels for different numbers of TCs added (from 5.10 4 to 1.10 6 TCs) to HUVEC monolayers, and cocultured for 5h (Figure 3a). Protein expression levels were determined by FACS analysis. As shown in Figure 3a, endothelial ICAM-1, VCAM-1 and E-selectin expression increased as a function of TC number. E-selectin reached its maximum expression for 4.10 5 TCs whereas ICAM-1 and VCAM-1 reached their maximum expression for roughly 8.10 5 TCs. For all the following experiments, a TC/EC ratio of one was used (5.10 5 TCs for 5.10 5 ECs).

We then determined ICAM-1, VCAM-1 and E-selectin expression kinetics during TC-EC coculture (Figure 3b). CAM over-expression started after 2h coculture, reaching a plateau after 5h. In contrast, PECAM-1 levels were not changed during this 5h experiment. This coculture-induced CAM over-expression is very similar to that observed in HUVEC stimulated with cytokines such as LPS or TNFα [START_REF] Kayal | Listeriolysin Odependent activation of endothelial cells during infection with Listeria monocytogenes: activation of NF-kappa B and upregulation of adhesion molecules and chemokines[END_REF], suggesting the involvement of the same signaling pathway. Therefore, we investigated whether coculture-induced CAM overexpression kinetics could be reproduced with a low concentration of TNFα. For this experiment, confluent HUVEC monolayers were stimulated with various concentrations of TNFα, ranging from 0.2 to 200 U/mL. ICAM-1 expression was determined after 5 hours of TNFα stimulation by FACS analysis (supplementary data, Figure S2). We determined that a concentration of 0.5 U/mL of TNFα induced a similar ICAM-1 increase (about fourfold increase) as the one obtained under coculture conditions, while the maximal expression of ICAM-1 was reached for 12.5 U/mL of TNFα.

Endothelial ICAM-1 expression kinetics were then determined either with the selected dose of 0.5 U/mL TNFα, or after TC-EC coculture. Both stimulation modes induced similar kinetics pattern with a plateau beginning after 5h (Figure 3c). Similar results were obtained for VCAM-1 and E-selectin kinetics (supplementary data, Figure S2).

ICAM-1, VCAM-1 and E-selectin over-expression requires a close HUVEC-T24 contact

Since coculture-induced CAMs over-expression kinetics was similar to TNF-induced CAMs kinetics, we hypothesized that a soluble molecule might be involved. To investigate whether TC-EC contact induces cytokines production, confluent HUVECs were cultured in insert with a T24-HUVEC coculture in the lower compartment (coculture+HUVEC in insert) for 5h. TCs production of cytokines was tested by separating ECs from TCs in the Transwell (T24+HUVEC in insert). Then the selected ECs (dotted line in Figure 4) were analyzed for CAM expression by flow cytometry. Figure 4 shows that endothelial ICAM-1, VCAM-1 and E-selectin were up-regulated only when TCs were in tight contact with the HUVEC monolayer. We also tested if a low concentration of TNFwhich might be secreted by TCs or TC-EC contact, was able to stimulate ECs in another compartment. HUVECs were cultured on the upper compartment of a Transwell (0.4µm pore, 6-well plate) with TNFα (0.5 U/mL) inside the lower compartment. Five hours later, HUVECs displayed a significant overexpression of ICAM-1, VCAM-1 and E-selectin (Figure 4), showing that in our test, a low concentration of soluble molecules can cross the filter and stimulate ECs. Similar results were obtained after 24 h using the same test (supplementary data, Figure S4). To further confirm that the ICAM-1 increase expression was not induced by a soluble factor released by TCs, we performed two additional experiments: we prepared conditioned medium either from TCs alone or from a TC-EC coculture. These conditioned media were then added to ECs for 5 h.

In both cases, no increase in ICAM-1 expression was detected (supplementary data, Figure S5). We also quantified TNFα secretion by TCs. TNFα protein concentrations in culture supernatants were assessed by ELISA after a 12, 24 or 48h incubation time. Four cell lines were tested for TNFα production. T24 and MDA, which were able to increase endothelial ICAM-1 expression after coculture (as shown in table 1), and TCC-Sup and DAG-1, which did not induce any modification in endothelial ICAM-1 expression in coculture. TNFα protein concentrations remained below the detection limit of the ELISA assay (<15pg/ml for 10 6 cells) for all cell lines (data not shown). Therefore, as strongly suggested by the transwell assays (Fig. 4

HUVEC-T24 coculture-induced CAM over-expression depends on NFκB signaling pathway

ICAM-1, VCAM-1 and E-selectin are mainly regulated by the transcription factor NFκB [START_REF] May | Signal transduction through NF-kappa B[END_REF].

To determine whether NFκB is activated in HUVECs after contact with TCs, HUVECs were treated with the NFκB inhibitor pyrrolidine-dithiocarbamate (PDTC), prior to 5h-coculture, and the ICAM-1 expression was analyzed by flow cytometry. PDTC is an antioxidant which has been shown to be an effective inhibitor of NFκB activation, presumably working through the inhibition of IκB degradation [START_REF] Heitmeier | Double-stranded RNA-induced inducible nitoric-oxide synthase expression and interleukin-1 release by murine macrophage requires NFκB activation[END_REF][START_REF] Nakao | Tumor necrosis factor alpha (TNF-alpha)-induced prostaglandin E2 release is mediated by the activation of cyclooxygenase-2 (COX-2) transcription via NFkappaB in human gingival fibroblasts[END_REF][START_REF] Crisostomo | Human mesenchymal stem cells stimulated by TNF-alpha, LPS, or hypoxia produce growth factors by an NF kappa B-but not JNK-dependent mechanism[END_REF]. Treatment of HUVECs with PDTC fully prevented the expression of endothelial ICAM-1 induced by coculture or by TNFα treatment (Figure 5). This suggests that both coculture and TNFα may trigger similar pathways via the activation of the NFκB transcription factor.

Effect of CAM expression on T24 cell adhesion and transendothelial migration

We then studied the involvement of endothelial ICAM-1, VCAM-1 and E-selectin on the adhesion of T24 cells to HUVEC monolayers. HUVECs stimulated or not with 0.5 U/mL TNFα were preincubated with the appropriate blocking antibodies: anti-ICAM-1, anti-VCAM-1 and anti-E-selectin. Suspensions of T24-GFP cells were added to monolayers for 5h in the presence of the same blocking antibody and adherent T24-GFP cells were quantified using flow cytometry. Note that in these experiments we define the "percentage of adherent TCs" as the percentage of TC which attached or spread on the endothelial monolayer after 5h coculture.

With unstimulated HUVECs (Figure 6a), inhibition of endothelial ICAM-1, VCAM-1 and Eselectin had no effect on T24 cell adhesion, with about 60% of adherent cells. TNFα treatment of HUVEC monolayers induced a 20% increase in the number of adherent TCs when compared to unstimulated HUVECs. VCAM-1 inhibition resulted in a detectable decrease of 25% of T24 adherent cells to TNFα-stimulated ECs, whereas ICAM-1 and E-selectin inhibition had no influence on TC adhesion (Figure 6a).

We next investigated whether ICAM-1, VCAM-1 or E-selectin could also be involved in T24 cells transmigration (Figure 6b). With unstimulated HUVECs (Figure 6b), inhibition of endothelial ICAM-1 resulted in a significant decrease of the percentage of TCs transmigrated (19±3% versus 26±3%). VCAM-1 or E-selectin inhibition had no effect on TC migration through untreated EC monolayers.

With stimulated HUVECs, TC transmigration increased (39±6% in TNFα treated HUVECs versus 26±3% in unstimulated HUVECs) and this transendothelial migration was strongly reduced when endothelial ICAM-1 was blocked (19±5% versus 39±6%), suggesting a major role for ICAM-1 in T24 cells transmigration. Furthermore, inhibition of VCAM-1 significantly reduced the percentage of T24 cells transmigration through TNFα-stimulated HUVECs (27±5% versus 39±6%). This is likely due to the involvement of VCAM-1 in TC adhesion on TNFα-stimulated HUVECs (Figure 6a). On the other hand, E-selectin inhibition had no effect on transmigration.

Effect of other carcinoma cell lines on endothelial CAM expression

Throughout this study, our data indicated that direct contact between HUVECs and bladder carcinoma cell line T24 induced endothelial ICAM-1, VCAM-1 and E-selectin up-regulation via NFκB transcription factor activation. We next explored whether other human carcinoma cell lines could induce endothelial CAM over-expression during coculture. Therefore, two bladder carcinoma cell lines (DAG-1 and TCCSUP), two breast adenocarcinoma cell lines (MCF7 and MDA-MB-435S), two lung carcinoma cell lines (A549 and Calu-6), and one colon adenocarcinoma cell line (RKO) were tested (cf. materials and methods). HUVEC monolayers were cocultured during 5h in contact with the different fluorescently labeled TCs and ICAM-1 expression was analyzed by flow cytometry.

Among this panel of TC lines, only T24 cells and MDA-MB-435S were able to induce endothelial CAM over-expression after coculture, with a fourfold increase on average (Table 1). This suggests that the observed CAM over-expression is cell type dependent.

Shear stress differentially modulates the HUVEC-T24 coculture-induced CAM expression in ECs

Under physiological conditions, ECs are constantly subjected to hemodynamic forces. To investigate the effect of shear stress on coculture-induced CAM expression, we used a parallel-plate flow chamber (cf. materials and methods). The EC monolayer was inserted into the flow chamber and presheared at 0.9, 9 or 18 dynes/cm² for 19h. A suspension of T24 cells was then perfused through the chamber, allowed to settle down for 15min, and cells were exposed to a constant shear stress (0.9, 9 or 18 dynes/cm²) for 5h. The effect of flow on the TNFα-induced CAM expression was also studied under the same conditions, with TNFα added for the last 5h. At the end of assays, ICAM-1, VCAM-1 and E-selectin expressions were analyzed by flow cytometry. As shown in Figure 7a, with unstimulated ECs in the absence of TCs, ICAM-1 expression increased as a function of shear stress intensity, whereas VCAM-1 or E-selectin expression levels were unchanged (Figure 7b-c). Under Moderate Shear Stress (MSS) conditions (≤ 9 dynes/cm²), 5h-coculture induced a significant increase in endothelial ICAM-1, VCAM-1 and E-selectin levels (Figures 7a-c) as already observed under static conditions (Figure 2). In contrast, under High Shear Stresses (HSS) of 18 dynes/cm², TC-EC coculture did not induce any modification of CAMs expression (Figure 7a-c), as confirmed by the western blot analysis for ICAM-1 (Figure 7e). In accordance with our previous report [START_REF] Chotard-Ghodsnia | Morphological analysis of tumor cell/endothelial cell interactions under shear flow[END_REF], the percentage of attached TCs on the EC monolayer after 5h hours coculture significantly decreased (from 57±8% to 21±9%) when the shear flow increased from 0.9 to 9 dynes/cm², and remained stable (from 21±9% to 22±7%) when the shear increased from 9 to 18 dynes/cm², (supplementary data S3). This indicates that the absence of cocultureinduced CAM expression under HSS was not due to a lack of TCs attached to the endothelial layer. Furthermore, HSS also inhibited the TNFα-induced CAMs over-expression, whereas the TNFα-stimulated HUVECs under MSS displayed a significant increase in ICAM-1, VCAM-1 and E-selectin expressions (Figure 7a-c).

Our findings thus identify HSS as an inhibitor of CAM expression in TNFα-stimulated HUVECs as well as in ECs cocultured with T24 cells. This confirms the existence of a shear stress threshold above which flow acts as an anti-inflammatory agent [START_REF] Chiu | Shear stress inhibits adhesion molecule expression in vascular endothelial cells induced by coculture with smooth muscle cells[END_REF]. As already observed under static conditions (Figure 5), the over-expression under MSS was dependent upon the activation of the NFκB signaling pathway, since PDTC treated HUVECs cocultured with TCs and exposed to a shear stress of 9 dynes/cm² did not display any CAMs upregulation (Figure 7d). Preshearing of ECs at 18 dynes/cm² (but not at 9 dynes/cm², nor under static condition) significantly inhibited the decrease in IκBα protein expression induced by TC-EC coculture (Figure 7f). When compared to static ECs, inhibition of endothelial NFκB signaling pathway by PDTC blocked IκBα degradation after TC-EC coculture under flow conditions.

Discussion

Extravasation of TCs is a complex process involving adhesive interactions [START_REF] Sandig | Role of cadherins in the transendothelial migration of melanoma cells in culture[END_REF][START_REF] Voura | Involvement of integrin alpha(v)beta(3) and cell adhesion molecule L1 in transendothelial migration of melanoma cells[END_REF].

Stimulation of ECs induced by contact with other cells has already been reported by several

authors. Kokura et al. [START_REF] Kokura | Postanoxic T lymphocyteendothelial cell interactions induce tumor necrosis factor-alpha production and neutrophil adhesion: role of very late antigen-4/vascular cell adhesion molecule-1[END_REF] showed that close contact between T lymphocytes and HUVECs induced endothelial TNFα and IL-8 production in a post anoxic model. Damle et al. [START_REF] Damle | Van der Vieren, Direct interaction with primed CD4+ CD45R0+ memory T lymphocytes induces expression of endothelial leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1 on the surface of vascular endothelial cells[END_REF] showed that interactions between ECs and subpopulations of human T cells induced endothelial CAMs expression, and this required direct intercellular contact. Other studies showed that the coculture of ECs and TCs induced tumoral N-cadherin over-expression [START_REF] Qi | Transendothelial migration of melanoma cells involves N-cadherin-mediated adhesion and activation of the beta-catenin signaling pathway[END_REF], and transient down-regulation of tumoral α2, α3 and α5 integrins [START_REF] Jones | Transient down-regulation of beta1 integrin subtypes on kidney carcinoma cells is induced by mechanical contact with endothelial cell membranes[END_REF], altering cell migration.

In our model, no change in tumoral CAM expression was observed after 5h-coculture, whereas endothelial ICAM-1, VCAM-1 and E-selectin were highly increased under static and MSS conditions. Other studies have shown that TCs are able to induce cell activation in heterotypic coculture. Mierke et al [START_REF] Mierke | Breakdown of the endothelial barrier function in tumor cell transmigration[END_REF] showed that endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-. Peng et al [START_REF] Peng | Regulation of interleukin-8 expression in melanoma-stimulated neutrophil inflammatory response[END_REF] demonstrated that IL-8 is up-regulated in PMNs upon co-culturing with melanoma cells, and this facilitates melanoma cell migration through the endothelium under flow conditions. Since heterotypic cell-cell interactions between T24 cells and ECs stimulate the expression of endothelial CAMs, we postulated that this phenomenon may influence T24 cell adhesion and transmigration. Using monoclonal antibodies, we showed that ICAM-1 and VCAM-1 facilitate T24 cells transendothelial migration whereas VCAM-1 plays a role in T24 cells adhesion. It is likely that VCAM-1 implication in both transmigration and adhesion are linked. It is now established that CAMs mediate TC and leukocyte diapedesis through EC monolayers. For example, VCAM-1 was up-regulated on activated ECs and promoted transendothelial migration of leukocytes [START_REF] Chan | VLA-4 integrin mediates lymphocyte migration on the inducible endothelial cell ligand VCAM-1 and the extracellular matrix ligand fibronectin[END_REF]. Recently, Klemke et al. [START_REF] Klemke | 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[END_REF] demonstrated that transendothelial migration of highly metastatic melanoma cell lines depended on VCAM-1/VLA-4 interaction. Yang et al. [START_REF] Yang | ICAM-1 regulates neutrophil adhesion and transcellular migration of TNF-alpha-activated vascular endothelium under flow[END_REF] showed that ICAM-1 regulates neutrophil transendothelial migration of vascular endothelium under dynamic conditions.

In our conditions, ICAM-1, VCAM-1 and E-selectin expressions displayed similar kinetic patterns in HUVECs cocultured with T24 cells and in TNFα-stimulated HUVECs, suggesting the involvement of a common signaling pathway. It is well known that vascular endothelium is a major target for proinflammatory cytokines such as TNFα. Many signaling events are initiated by the binding of TNFα to one of its two cell surface receptors TNFR-1 and TNFR-2 [START_REF] Wallach | Tumor necrosis factor receptor and Fas signaling mechanisms[END_REF] leading to nuclear translocation of NFκB and transcription of target genes [START_REF] Hoffmann | Transcriptional regulation via the NF-kappaB signaling module[END_REF]. After TNFα stimulation of ECs, NFκB binds to a specific region in the ICAM-1, VCAM-1 and Eselectin gene promoters, inducing their respective transcription [START_REF] May | Signal transduction through NF-kappa B[END_REF][START_REF] Roy | Intercellular adhesion molecule-1 (ICAM-1) gene expression in human T cells is regulated by phosphotyrosyl phosphatase activity. Involvement of NF-kappaB, Ets, and palindromic interferon-gamma-responsive element-binding sites[END_REF]. Here, we showed that the NFκB signaling pathway is likely involved in coculture-induced endothelial ICAM-1, VCAM-1 and E-selectin expressions under static and MSS conditions. This is in agreement with Ohno et al. [START_REF] Ohno | Inhibition of cellular adhesion in human umbilical vein endothelial cells by NF-kappaB inhibitor DHMEQ under flow[END_REF] who showed similarly that NFκB inhibition in ECs reduced the adhesion between HUVEC and HT-29 colon cancer cells under static and flow conditions (6 dynes/cm²).

In addition, we observed that flow-induced endothelial ICAM-1 up-regulation was dependent on the level of shear stress. This is in agreement with Tsuboi et al. [START_REF] Tsuboi | Involvement of integrins, MAPK, and NF-kappaB in regulation of the shear stress-induced MMP-9 expression in endothelial cells[END_REF] who demonstrated a modulating effect of shear stress on flow-induced endothelial ICAM-1 expression. An important finding of the current study is that the level of shear stress (HSS: 18 dynes/cm² versus MSS: 0.9 and 9 dynes/cm²) modulates the response of EC gene expression to TC coculture as well as to TNFα stimulation. HSS totally ruled out the coculture-induced as well as TNFα-induced expressions of ICAM-1, VCAM-1, and E-selectin in ECs; this suggests that HSS, in addition to its anti-inflammatory role, regulates the response of T24 adherent cells. Such a phenomenon has already been shown for TNFα. Sheikh et al [START_REF] Sheikh | Exposure to fluid shear stress modulates the ability of endothelial cells to recruit neutrophils in response to tumor necrosis factor-alpha: a basis for local variations in vascular sensitivity to inflammation[END_REF] demonstrated that HSS (10-20dynes/cm²) reduced the TNFα-induced expression of endothelial selectins by 66% and this abolished the adhesion of neutrophils to the endothelial monolayer. The mechanism by which HSS inhibits EC gene expression induced by coculture or TNFα stimulation is unclear but is likely to be multifactorial. Chiu et al. [START_REF] Chiu | Shear stress inhibits adhesion molecule expression in vascular endothelial cells induced by coculture with smooth muscle cells[END_REF] showed that HSS (12 dynes/cm²) inhibited ICAM-1, VCAM-1 and E-selectin expression in vascular ECs, induced by coculture with smooth muscle cells. In this model, the authors demonstrated that the coculture-induced CAMs up-regulation was not dependent on close contact between EC and muscle cells but involved soluble factors (IL-1β and IL-6), acting at a small distance. This led to activation of the c-Jun-NH2-terminal kinase (JNK) and p38 MAPK and increased the NFκB promoter binding activity in ECs [START_REF] Chiu | Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress[END_REF]. In our study, the endothelial ICAM-1 upregulation was dependent on TC/EC close contact and was not mediated by a soluble factor such as TNFα secreted by TCs. Indeed, in 5 and 24h-transwell experiments, TC conditioned medium experiments, as well as TNFα immunodosage assays confirmed a TC-EC contact at the origin of this coculture-induced CAM overexpression. Recent studies [START_REF] Partridge | Laminar shear stress acts as a switch to regulate divergent functions of NF-kappaB in endothelial cells[END_REF] showed that preshearing of ECs (16 dynes/cm² for 16h) significantly altered transcriptional responses to TNFα stimulation by suppressing pro-inflammatory transcripts (E-selectin, VCAM-1, IL-8) and enhanced the expression of cytoprotective molecules (Bcl-2, MnSOD, GADD45). Mohan et al. [START_REF] Mohan | Low shear stress preferentially enhances IKK activity through selective sources of ROS for persistent activation of NF-kappaB in endothelial cells[END_REF] also showed a differential effect of the shear stress level in human aortic ECs. These authors demonstrated that MSS (2 dynes/cm²) induced a strong and persistent activation of NFκB (+100% vs. static control, over 2h). In contrast HSS (16 dynes/cm²) induced a weak activation of NFκB (+10%) at the early times of shear stress application, and this activation dramatically decreased (-70% vs. static control) after a few minutes. All these studies are in favour of the existence of a shear stress threshold above which flow acts as an antiinflammatory agent [START_REF] Chiu | Shear stress inhibits adhesion molecule expression in vascular endothelial cells induced by coculture with smooth muscle cells[END_REF]. Such observations correlate with the fact that veins and capillaries (in which MSS are predominant) are preferential targets for metastasis [START_REF] Yi | Tumors of the pulmonary vasculature[END_REF].

Several cell lines were tested for coculture in the presence of ECs, but only the T24 (bladder carcinoma) and MDA-MB-435S cell lines (breast adenocarcinoma) were able to induce CAMs up-regulation (Table 1). T24 and MDA cells share several common characteristics.

Both cell lines express ICAM-1 [START_REF] Ali | Intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and regulated on activation normal T cell expressed and secreted are expressed by human breast carcinoma cells and support eosinophil adhesion and activation[END_REF][START_REF] Roche | Fibrinogen mediates bladder cancer cell migration in an ICAM-1-dependent pathway[END_REF], N-cadherin [START_REF] Mialhe | Expression of E-, P-, N-cadherins and catenins in human bladder carcinoma cell lines[END_REF][START_REF] Sillanpaa | CYP1A1 and CYP1B1 genetic polymorphisms, smoking and breast cancer risk in a Finnish Caucasian population[END_REF], and the transcription factor E2A [START_REF] Perez-Moreno | A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions[END_REF]. It has also been reported that T24 and MDA cells, which derive from epithelial tissues, do not express any E-cadherins [START_REF] Perez-Moreno | A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions[END_REF]. It is known that the invasion process involves the loss of cell-cell interactions with the gain of migratory properties [START_REF] Perl | A causal role for E-cadherin in the transition from adenoma to carcinoma[END_REF]. E-cadherin is the main adhesion molecule of epithelia [START_REF] Aberle | Cadherin-catenin complex: protein interactions and their implications for cadherin function[END_REF]61], and it has been implicated in carcinogenesis because it is frequently lost in human epithelial tumors [START_REF] Birchmeier | Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness[END_REF][START_REF] Takeichi | Cadherins in cancer: implications for invasion and metastasis[END_REF]. Perez-Moreno et al. [START_REF] Perez-Moreno | A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions[END_REF] indeed showed an implication of E2A transcription factor as a repressor of E-cadherin expression during tumor progression in MDA and T24 cells. The cytoplasmic domain of Ecadherin generates complexes with many signaling molecules, including α-, β-, and γ-catenin, p120ctn, and Shc. This common phenotype shared by T24 and MDA cell lines may explain their similar coculture-induced endothelial CAM over-expression. Further investigations using gene transfection are needed to determine the potential implication of E-cadherin in this phenomenon.

To sum up, our study indicates that tight contact between T24 and HUVECs upregulates ICAM-1, VCAM-1 and E-selectin expression under static and MSS conditions, whereas, the application of HSS inhibits these coculture-induced as well as TNFα-induced CAMs overexpressions. This CAM upregulation is NFκB-dependent and increases T24 cell adhesion and transmigration. Further investigations are needed to understand the molecular mechanisms involved in this coculture-induced CAM expression. and HUVEC monolayer cocultured with T24-GFP cells during 5 hours (g) stained for ICAM-1 (gray). Immunoblots showing ICAM-1 expression after coculture (h). Samples were taken from resting HUVECs (EC), resting T24 (TC), or HUVEC-T24 coculture (Co). TCs and ECs were also cultured separately and mixed together (with a ratio of 1 TC for 2 EC) before lysis and immunoblot analysis (EC+TC) (cf materials and methods). Blots were probed with antibody against actin as a loading control; immunoblot quantification is shown under each band. ECs were exposed to 9 dynes/cm 2 for 24h (HUVECs), or presheared for 19h before 5hcoculture at 9 dynes/cm² (Coculture), or presheared for 18h at 9 dynes/cm², then treated with PDTC (100µM) for 1h under flow prior to 5h-coculture at 9 dynes/cm² (PDTC+Coculture).

The diagram shows mean fluorescence ± SD of two independent experiments at least. Immunoblots for ICAM-1 (e) and IκBα (f) expression before and after coculture are also presented. Samples were taken from resting HUVECs (EC) and TNFα stimulated HUVECs (TNF) 100 U/mL for 30min (f) or 24h (e). ECs were presheared 19h at 9 dynes/cm² before coculture with TCs at the same flow (Co 9 dynes/cm²) for 30min (f) or 5h (e). Same experiments were prepared for coculture under 18 dynes/cm 2 shear stress (Co 18 dynes/cm²).

ECs were also presheared 18h at 9 dynes/cm² then treated with PDTC (100µM) for 1h under flow prior to 5h-coculture at 9 dynes/cm² (Co 9 dynes/cm²+PDTC) for 5h (e) or 30min (f). In (f) TC-EC were cocultured for 30min under static condition (Co static). T24 cells and ECs were also cultured separately then mixed together (with a ratio of 1 TC for 2 ECs) before lysis and immunoblot analysis (EC+TC) (control of ICAM-1 level without coculture, as described in material and methods). Blots were also probed with antibody against actin as a loading control; immunoblot quantification is shown under each band. Statistical significance was calculated using the Wilcoxon test (* p<0.001 vs. presheared HUVEC control at the same shear stress; # p<0.03 vs. 0.9 dynes/cm² presheared HUVEC control at 0.9 dynes/cm²).

Table 1: FACS analysis of the endothelial ICAM-1 expression after 5 hour-contact

between a carcinoma cell line and HUVECs. HUVEC monolayers were cocultured for 5 hours in contact with, DAG-1, T24 or TCCSUP cells (bladder carcinoma), MCF7 or MDA cells (breast adenocarcinoma), Calu-6 or A549 cells (lung carcinoma) and RKO cells (colon adenocarcinoma) stained with 20µM of Cell Tracker Green. Quantification of the ICAM-1 level was carried out for endothelial cells by flow cytometry. Fold increase was calculated as the ratio of endothelial ICAM-1 after coculture on ICAM-1 expression on resting HUVECs. HUVECs were either cocultured (with a ratio of 1 TC for 1 EC for 5 h, "Coculture") or incubated 5 h with a 5 h-TC-EC coculture conditioned medium (HUVEC + coculture medium), or incubated 5 h with a 5 h-TC conditioned medium (HUVEC + T24 medium).

Titles and legends to figures for supplementary data

ICAM-1 expression was analyzed by flow cytometry (cf. "materials and methods"). The diagram shows mean fluorescence ± SD of three independent experiments. Each experiment was carried out in duplicate. Statistical significance was calculated using the Wilcoxon test (* p<0.01 vs. resting HUVEC).

Tables Table 1

EC ICAM-1fold increase induced by coculture DAG-1 0,8 ± 0,1 T24 4,2 ± 0,1 TCCSUP 1,1 ± 0,1 MCF7 0,9 ± 0,1 MDA 4,1 ± 0,3 Calu-6 0,9 ± 0,1 A549

1,0 ± 0,2 RKO 0,8 ± 0,1

Cell lines

  and Fig S4) and the conditioned medium experiment (Fig S5), the increase in endothelial ICAM-1 expression is not mediated by TNFα secreted by TCs. All together, these data clearly demonstrate that coculture-induced CAM overexpression required a tight contact between TCs and HUVECs and was not due to a soluble factor such as TNFα.

Figure 1 :

 1 Figure 1: TC-EC coculture induces endothelial ICAM-1 over-expression. FACS analysis of the mixed cell population (HUVEC and T24-GFP) collected from cocultures (a-e). ICAM-1 was stained with phycoerythrin (PE)-conjugated secondary antibody (gray). (a) Endothelial cells and (b) T24-GFP cells before coculture were stained for ICAM-1. (c) A mixture of EC and T24-GFP cells (with a ratio of 1 TC for 1 EC) after 5h-coculture. Quantification of ICAM-1 levels before (black) and after (gray) coculture was carried out for both cell types: (d) endothelial cells; (e) T24-GFP cells. Confocal microscopic images of resting HUVECs (f)

Figure 2 :

 2 Figure 2: Expression of CAMs induced by TC-EC coculture. FACS analysis of the mixed cell population (HUVEC and T24-GFP) collected from cocultures. E-selectin, ICAM-1, VCAM-1, PECAM-1, VE-and N-cadherin, β1 and β3 integrin were stained with phycoerythrin (PE)-conjugated antibody (red). Quantification of the CAM levels before coculture (resting HUVEC) and after 5 hours of coculture was carried out for HUVECs. The diagram shows mean fluorescence ± SD of two independent experiments representative of duplicate experiments with similar results.

Figure 3 :

 3 Figure 3: Characterization of CAM over-expression: (a). Dose-response curves for ICAM-1 (circles), VCAM-1 (squares) and E-selectin (triangles) expression in HUVEC cocultured with 0.5 to 10.10 5 T24-GFP cells. (b) Time-course of ICAM-1, VCAM-1, PECAM-1 and E-selectin expressions in endothelial cells cocultured with T24-GFP cells (EC/TC ratio of 1) during 1 to 6h. Time-course of ICAM-1 expression (c) on HUVEC stimulated with 0.5 U/mL of TNFα or cocultured with TCs. The fluorescence measured for resting cells (no TC added) was subtracted from each fluorescence point. The diagram shows fluorescence increase ± SD of three independent experiments analyzed by flow cytometry.Each experiment was carried out in duplicate.

Figure 4 :

 4 Figure 4: FACS analysis of the endothelial ICAM-1, VCAM-1, E-selectin and PECAM-1 expression after 5 hours coculture of T24-GFP and HUVECs. HUVECs monolayers were cocultured for 5 hours either in contact with T24-GFP or separated from the T24-GFP cells by 0.4µm Transwell filters. The fold increase measured for resting HUVECs was arbitrarily set to 1. The five sketches illustrate the experimental set up. Tumor cells are represented as black circles whereas endothelial cells are in white. The dotted line shows the analyzed ECs. The diagram shows mean fluorescence ± SD of three independent experiments. Each experiment was carried out in duplicate. Statistical significance was calculated using the Wilcoxon test (* p<0.001 vs. resting HUVEC).

Figure 5 :

 5 Figure 5: Analysis of the endothelial ICAM-1 expression in cocultured or in TNFαstimulated HUVECs after treatment with the NFκB inhibitor (PDTC). HUVECs were treated with PDTC (100µM) 1 hour prior to experiment. Then HUVECs were either cocultured (with a ratio of 1 TC for 1 EC, 5 h) or stimulated with TNFα (0.5 U/mL, 5 h). ICAM-1 expression was analyzed by flow cytometry (cf. materials and methods). The diagram shows mean fluorescence ± SD of three independent experiments. Each experiment

Figure 6 :

 6 Figure 6: (a) Adhesion of T24 cells to HUVEC monolayer. Percentage of adherent T24 cells to either untreated or TNFα stimulated HUVEC monolayer (0.5 U/mL) in the presence of 20µg/mL of the appropriate blocking antibody (cf. materials and methods). The percentage of adherent T24 cells compared to untreated cells (normalized to 100%) was estimated by flow cytometry after 5 hours of TC-EC contact. (b) Transendothelial migration of T24 cells. HUVEC monolayers were either untreated or stimulated with 100 U/mL TNFα. Then T24-GFP cells (in suspension in M199 culture medium) were added to HUVEC monolayers previously treated with the appropriate mAb (20µg/mL during 30min) (cf. materials and methods). After 5 hours, the TCs that have migrated to the lower face of the filter were quantified. The diagram shows mean fluorescence ± SD of three independent experiments. Each experiment was carried out in duplicate. Statistical significance was calculated using the Wilcoxon test (* p<0.02 vs. control).

Figure 7 :

 7 Figure 7: High shear stress inhibits endothelial expression of ICAM-1, VCAM-1 and Eselectin induced by coculture with T24 cells as well as TNFα-induced CAMs upregulation. ECs were exposed to a shear stress of 0.9, 9 or 18 dynes/cm² for 24h hours (HUVECs) or were presheared 19h before addition of T24-GFP cells in the flow loop (coculture) or were presheared 19h before the TNFα treatment (TNF). Shear stress was applied to ECs simultaneously with the addition of TCs or TNFα (100 U/mL) for 5h (see materials and methods). Quantification of the ICAM-1 (a), VCAM-1 (b) and E-selectin (c) levels were carried out for endothelial cells by flow cytometry. (d) In additional experiments

Figure S1 :

 S1 Figure S1: Tumor cell expression of CAMs induced by TC-EC coculture. FACS analysis of the mixed cell (HUVEC and T24) collected from cocultures. ICAM-1, N-cadherin, β1 and β3 integrin were stained with phycoerythrin (PE)-conjugated antibody (red). Quantification of the CAM levels before coculture (resting TC) and after 5 hours of coculture was carried out for T24 cells. The diagram shows mean fluorescence ± SD of two independent experiments in duplicate.

Figure S2 :

 S2 Figure S2: CAMs over-expression kinetics. Dose-response curves for ICAM-1 expression in HUVEC cocultured with various concentrations of TNFα, from 0.2 to 200 U/mL (a). Timecourse of E-selectin (b), and VCAM-1 expression (c) on HUVEC stimulated with 0.5 U/mL of TNFα or cocultured with TCs. The fluorescence measured for resting HUVECs (time 0h) was subtracted from each fluorescence point. The diagrams show mean fluorescence ± SD of three independent experiments analyzed by flow cytometry. Each experiment was carried out in duplicate.

Figure S3 :

 S3 Figure S3: Effect of shear flow on the number of attached TCs. Percentage of attached TCs on the EC monolayer (relative to the number of TCs remaining on the EC monolayer at the end of the 15-min incubation time).

Figure S4 :

 S4 Figure S4: FACS analysis of endothelial ICAM-1 expression after 24 hours coculture of T24-GFP and HUVECs. HUVECs monolayers were cocultured for 24 hours either in contact with T24-GFP or separated from the T24-GFP cells by 0.4µm Transwell filters. The four sketches illustrate the experimental set up. Tumor cells are represented as black circles whereas endothelial cells are in white. The dotted line shows the analyzed ECs. The diagram shows mean fluorescence ± SD of two independent experiments. Each experiment was carried

Figure S5 :

 S5 Figure S5: Effect of TC conditioned medium on the endothelial ICAM-1 expression.

Figure

  Figure 3

  Figure 3

Acknowledgements

We gratefully acknowledge Remy Pedeux for helpful discussions and critical reading of the manuscript and Alexeï Grichine for his assistance with the confocal microscope. We thank the workshop of "Laboratoire de Spectrométrie Physique" for the flow chamber design. This work has been partly supported by "La ligue contre le cancer" and the RTN project funded by the EU (contract N° CT-2000-00105).


Karolina Orcid

Koc-Michalska

Darren Lilleker

Damian Guzek

Marton Bene

Larissa Doroshenko

Miloš Gregor

CEE countries faced significant political, economic, social, and technological transformations over the last four decades. Democratic processes, after relative stabilisation, tremble again around polarizing values, populist leaders, or nationalistic ideologies. Online communication, especially social media platforms, play a vital role in shaping how citizens interact with the state, political actors, media, and other citizens. The collection of manuscripts focuses on some of the challenges democratic institutions in the region face, in transforming and sustaining civil society and attempts to capture how the digital media environments mitigate or exacerbate those challenges. Included manuscripts focus on the role that online platforms play in the satisfaction with democracy in the CEE region, the interactions between journalists and political actors, the strategic media coverage of elections, affective polarisation and political antagonism, and discursive attempts to discourage young people from civic engagement.

Introduction

The countries in Central and Eastern Europe (CEE) have been going through tremendous changes and developments over the last forty years. The collapse of the Soviet Union saw these countries gain, or seize, their independence in largely bloodless transitions of power. Economic, social and political developments profoundly changed how their societies have been functioning. The nations are largely flourishing market economies, but growing inequalities have fueled migration. Democratic institutions have been created, yet parties are not fully embedded and are often the political vehicles for charismatic personalities (see studies within Eibl & Gregor, 2019). The processes of transformation and development are well advanced in certain countries (especially those that joined the European Union in its enlargements of 2004 and 2013), but are still not fully achieved in others. Some nations did not democratise (Belarus), others struggled to resolve their independence or geopolitical situation (Ukraine, Kosovo, Northern Macedonia), many witness high political polarisation and civic protest (Poland, Romania), and there are numerous examples where the CEE nations have witnessed a rise in support for populist politicians (Hungary and Poland).

The transitions to democratic market economies occurred at the same time as the nations of Central and Eastern Europe witnessed sharp technological development. Just as democracy was becoming embedded, citizens gained access to a range of platforms that simplified interactions. New media, Internet-based platforms or social media, facilitated citizens' opportunities to express their opinions, engage in or connect for collective action. However, there remain sharp digital divides in many CEE nations. Although access to the Internet is widespread, it is still lower than in the Western parts of Europe (especially broadband1 access). There are also sharp divides between rural and urban areas, as well as skills divides [START_REF] Esteban-Navarro | The rural digital divide in the face of the COVID-19 pandemic in Europerecommendations from a scoping review[END_REF]. Certain groups of privileged citizens have unfettered access, while others have been left behind. The digital divide, often a consequence of infrastructural, educational and financial inequalities, can complement incomprehension and polarization within the society. Restrictions in access to information, differential availability of internet affordances to spread civic knowledge, unequal abilities to participate in political discussion or to be active in political discourse lead to divided societies [START_REF] Dragulanescu | Social impact of the "Digital Divide" in a Central-Eastern European country[END_REF].

It is perhaps all too easy to view the nations of Central and Eastern Europe as being in a predevelopment phase, held back by weak governance, insufficient resources and the migration of many of the younger and brighter citizens. However, this important region is pivotal in terms of understanding how civil society can be built and maintained, and how democratic norms of thinking and acting can become embedded within society. It is equally important for understanding the challenges that democracy faces. The problems confronted by citizens in CEE countries are not unique to this region, they are to differing extents shared by many nations the continent of Europe and indeed globally [START_REF] Berman | Populism and the decline of social democracy[END_REF]. The CEE region has faced severe challenges during the pandemic, but even more so since early 2022, as many of these nations now have a war on their doorsteps and are coping with an influx of refugees displaced due to Russia's invasion of Ukraine. Facing these challenges successfully requires good governance and a strong, vibrant and inclusive civil society. All the CEE nations are classified by the Democracy Index (EIU, 2023) as flawed democracies; they hold free and fair elections and basic civil liberties are respected, but there remain significant weaknesses in systems of governance, an underdeveloped political culture and low levels of political participation. Of the sixteen nations which constitute Central and Eastern Europe, our studies include the highest performing nation, Czechia, two mid-table nations, Poland and Hungary, and lower performer Albania. Kosovo does not have a separate entry within the index. The data shows that most of these nations perform overall better than CEE average but there are key weaknesses, not only in Albania but also deficiencies in civil liberties in Poland and Hungary, and with political participation in Hungary. The Democracy Index scores out of ten show a mixed picture for the region, although it is worth noting that in terms of their global rank Czechia is just behind France, equal with Greece and ahead of Portugal and the USA. Poland and Hungary are ranked closely with nations of South America (Brazil and Argentina) as well as Indonesia and Thailand, Albania has a similar ranking to countries such as Ghana and Suriname. This offers a perception of the range of challenges faced in these nations. It is equally interesting to note that over the history of the Democracy Index 2006-2022 (Table 2), we notice a minor improvement for Albania but also minor decreases in performance for the other three nations. While minor changes, these demonstrate the fragility of democracy in the region. With attacks on media freedom, civil liberties, rights to free speech and protest being infringed, there is a need for a strong civil society to defend core principles of democracy. Though, since a democratic regime is considered embedded within societies, it is time to examine the relationship between technological developments and the sustainability of civil society within this region. The aim of this special edition is to raise questions regarding the ways in which digital technologies, and specific affordances of platforms, offer the potential for enhancing democratic culture and in what ways they undermine principles of pluralism, having a well-informed citizenry who are confident and able to debate political issues and reach a consensus.

The special CEE collection

The proposed special issue is composed of five papers covering CEE as a region [START_REF] Placek | Social media, quality of democracy, and citizen satisfaction with democracy in central and eastern Europe[END_REF], and specific case studies from Albania and Kosovo [START_REF] Camaj | The audience logic in election news reporting on Facebook: What drives audience engagement in transitional democracies of Albania and Kosovo[END_REF], Czechia and Hungary [START_REF] Vochocová | Soros's soldiers, slackers, and pioneers with no expertise? Discursive exclusion of environmental youth activists from the digital public sphere in Hungary and Czechia[END_REF][START_REF] Macková | One way or another? Discussion disagreement and attitudinal homogeneity on social networking sites as pathways to polarization in Czechia[END_REF], and Poland [START_REF] Adamczewska | Interactive Election Campaigns on Social Media? Flow of Political Information Among Journalists and Politicians as an Element of the Communication Strategy of Political Actors[END_REF].

Placek ( 2023) offers a broad perspective drawing on six waves of the Eurobarometer data from 2014 to 2019 analysing the relationship between social media use and its employment for information acquisition with the variation in satisfaction with democracy. The author indicates that those who are searching for information on social media are more critical towards democratic standards. They tend to be satisfied with the functioning of the democracy when the liberal democracy index and the electoral democracy index (v-dem.net) per country are above .7 and .8, respectively (with the score .6 for those not searching). These relations alter depending on the strength or backsliding of the democratic regime.

Adamczewska (2023) examines relations on social media platforms between journalists and political actors in the context of the Polish parliamentary elections of 2015 and 2019. Surprisingly, the density of the links between media representatives and politicians dropped in time. The relations were measured based on reactions to posts by the journalists rather than on interactive exchanges of ideas.

Between the two electoral periods, the most visible politicians, mainly from the opposition parties, amplified their online presence and visibility. The growing absence of political actors from the ruling party and journalists linked to public media is observed. [START_REF] Camaj | The audience logic in election news reporting on Facebook: What drives audience engagement in transitional democracies of Albania and Kosovo[END_REF] study the public's engagement with news posted by media in Albania and Kosovo. The authors build on the media logic and audience logic theoretical approaches to examine the reasons (interviews with editors) and effects (text analysis) of strategy-or issue-based, personalization and negativity positing tactics within Facebook profiles of twelve news organizations.

The audience tends to engage less with negative attacking posts and with posts focusing on issues, except for those covering social welfare, health and immigration. The findings suggest that the audience-oriented modus operandi employed by the editors involves closely monitoring the community's reactions towards publications. The audience-oriented logic sets the tone for news choices by media but also the strategic behaviour of political actors. [START_REF] Macková | One way or another? Discussion disagreement and attitudinal homogeneity on social networking sites as pathways to polarization in Czechia[END_REF] examine the relations between social networking sites, affective polarization and political antagonism in Czechia. The authors find substantial contrasts among the predictors: only the level of attitudinal homogeneity has the same effect on both polarization and antagonism, while other explanatory variables (experienced negativity, political unfriending, or political interest) are more context depending. The study suggests that a homogenous pattern of social media contribution to polarization may not exist and that the relationship is more nuanced.

The special issue ends with a paper studying the discursive representation of the youth climate changeoriented movement in Czechia and Hungary. [START_REF] Vochocová | Soros's soldiers, slackers, and pioneers with no expertise? Discursive exclusion of environmental youth activists from the digital public sphere in Hungary and Czechia[END_REF] provide a study on normative assumptions around the exclusion of the youth from participating in the public sphere expressed via comments on the main newspapers' profiles on Facebook. Findings show the general willingness to dismiss young people from politics. Two argumentation lines are prominent: firstly, denying expertise, experience and rationality for political participation, and secondly, accusing youth of errant values and thus normatively incapable of participating in the democratic processes.

Young people seem to be rather discouraged from political activities than being bolstered for further civic engagement.

This special issue is an attempt to bring together a collection dedicated to Central and Eastern Europe within the established and globalised scope of this journal. We are grateful to the Editors for this opportunity. However, still, the main limitation of the special issue remains the insufficient set of countries covered. We wish to encourage future research and publishing by scholars from the CEE countries. Similarly, to other non-Western regions, there is a need from the established research environments to support colleagues and contributors only in critically reviewing but also by encouraging, actively supporting and suggesting solutions to publish in established journals. The call for the special issue was published before the Russia war on Ukraine started, thus the conflict, in which social media play a crucial communicational role, is not included in the proposed collection.

Table 1 .

 1 Democracy Index rankings for nations included in the research(EIU, 2023)

	Nation	Overa	Global	Region	Electoral	Functioning	Political	Political	Civil	Regime
		ll	Rank	Rank	Process &	of	participation	culture	liberties	type
		score			Pluralism	government
	CEE	5.39	n/a	n/a	7.97	6.74	6.36	6.84	7.77	n/a
	Czechia 7.97	25	1	9.58	6.43	7.22	7.50	9.12	Flawed
	Poland	7.04	46	7	9.17	6.07	6.67	6.25	7.06	Flawed
	Hungar	6.64	56	8	8.33	6.79	4.44	6.88	6.76	Flawed
	y
	Albania 6.41	64	13	7.00	6.43	5.00	6.25	7.35	Flawed

Table 2

 2

	. Changes in the overall Democracy Scores 2006-2022 (EIU, 2023)
	Nation	Overall score 2022	Overall Score 2006	Change
	Czechia	7.97	8.17	-0.20
	Poland	7.04	7.30	-0.26
	Hungary	6.64	7.53	-0.89
	Albania	6.41	5.91	+0.50

https://data.worldbank.org/indicator/IT.NET.BBND.P2


come

High return loss at the end face of fiber

Philippe Chanclou, Monique Thual, Jean

High return loss at the end face of fiber Philippe Chanclou Ecole Nationale Supérieure des Télécommunications de Bretagne, Departement d'Optique, BP226, 22303 Lannion, France

Monique Thual and Jean lostec France Telecom R&D, DTD/PIH, 2 avenue Pierre Marzin, 22307 Lannion, France A micro-optics has been developed to limit backreflection at the end face of single mode fibers. The measured return-loss at a wavelength of 1.55 µm is as high as 28 dB. This device allows preserving the geometric aspect of a normally cut end face fiber.

OCIS codes: (060.2340) Fiber optics components; (110.2760) Gradient-index lenses; (120.5700) Reflection; (120.3620) Lens design.

In transmission line in optical communication, when light is coupled into or out of an optical fiber through a flat cleaved or polished end face of the fiber, reflection may set up disturbing optical systems and devices. Consequently, to obtain the optimum performances of these systems and devices, it is effective to reduce the level of reflection. The primary cause of backreflection or return loss is a discontinuity in the index of refraction of the fiber. The typical Fresnel reflection of flat end face fiber is 3.3 10 -2 (-14.7 dB). To obtain lower optical backreflection, the end face of the fiber should have [START_REF] Ulrich | Beam-to-fiber coupling with low standing wave ratio[END_REF] either:

(1) anti-reflecting (AR) coating of the fiber end face. Dielectric AR multilayers can be deposited onto the end face of fiber. A power reflectivity of 10 -3 can be achieved. (2) lens shaping [START_REF] Purser | Low cost single mode optical collimators for use in air-spaced devices[END_REF]. The end fiber is heated to form a hemispherical shaping. The level of reflection of this termination has been measured as 10 -4 . (3) immersion cell. The immersed end face of the fiber in an index matching has a power of reflectivity of 10 -5 . (4) tilted end face. The end face of the fiber is cleaved or polished with an angle to separate in the angular dimension the reflected light flux from the incident mode.

Residual reflections of 10 -6 are obtained. In this paper, we report a solution to reduce backreflection using micro-optics at the end face of the fiber. The aim of this work is to reduce backreflection level without modifying the geometrical aspect of the fiber without any tilt or bubble lens. We have been looking for a process where the external diameter of 125 µm is maintained along the fiber. This solution using micro-optics may be used singly or in combination with the other methods to achieve high return loss. This micro-optics consists of an arrangement of graded index (GRIN) fiber and coreless silica fiber of calibrated lengths spliced at the end face of the SMF. The spot size is adjusted by changing the length of GRIN and silica sections. The fabrication process of micro-optics has already been published [START_REF] Chanclou | Collective microoptics on fiber ribbon for optical interconnecting devices[END_REF]. First techno-economical analysis shows a very attractive cost of this micro-optics. This is mainly due to the negligible cost of raw materials, the few number of fabrication steps, the collective treatment (by using fiber ribbon), the use of commercially available tools and the high yield of the whole process. We have named this micro-optics GRADISSIMO for GRADed Index, Silica, SIngle-MOde fiber. The principle of this low backreflection is schematically described in Fig. 1. The incident optical beam from the single mode fiber, is propagated through a free space section (silica) and a graded index medium before to be reflected on the end face. The dependence of the beam parameters can be described by the ray matrix transformation [START_REF] Chanclou | Collective microoptics on fiber ribbon for optical interconnecting devices[END_REF][START_REF] Emkey | Analysis and evaluation of graded-index fiber-lenses[END_REF]. The reflected beam is back propagated in these two sections before to be coupled in the single mode fiber. The level of backreflection describes only the amplitude fraction reflected back into the incident mode of the fiber. The coupling efficiency of the reflected beam with the fundamental mode LP01 of the fiber is function of the length of graded index and silica. This dependence with these sections has been calculated and measured. The level of reflection is a periodical function of the length of GRIN (Pitch/4 = 365 µm). Fig. 1. Schematic illustration of the high return loss scheme that utilizes the micro-optics.

To measure the dependence of the section lengths of the micro-optics on the level of back reflection, we have used a precision White-light INterferometry Reflectometer (WIN-R). This equipment measures the level and location of micro-reflections inside the fiber with a spatial resolution of 50 µm and a return-loss range of 0 to 100 dB. The measurements of the return loss have an accuracy 2 dB. WIN-R incorporates a fiber source at a wavelength 1550 nm. As an exemple, Fig. 2 shows a result of backreflection measure. Micro-optics characteristics are 400 µm of silica and 248 µm of GRIN. We measure backreflection level of -30.2 dB at the end face of the micro-optics. The two splicing interfaces of the SMF / silica and silica / GRIN are measured below -80 dB of reflection (Fig. 3). In the following, we outline measurements of micro-optics reflection. The results are reported in Fig. 4 (a) and (b) as function of GRIN and silica lengths. We show that the contribution of the GRIN length to the reflection is a periodical function. The reflected power from a SMF with a micro-optics without silica (Lsilica = 0 µm) is measured to be below a level line of the forward power oscillating between -14.7 dB and -18 dB (Fig. 4 (a)). For a length of silica of 400 µm, the level oscillates between -14.7 dB and -28 dB (Fig; 4 (b)). The theoretical curves (solid lines) are compared with the measured values. These curves are in good agreement with the upper limit of measured points. Since no polishing is applied in this work, the end face imperfection should modify the value of the return loss measurements. The larger the optical beam at the end face is, the more important are the influences of these end face imperfections on the level of reflection. So the dispersion of the results is all the more important as the spot size diameter at the end face is biggest. The spot size diameter of the micro-optics versus varying lengths of GRIN and silica sections has already been published [START_REF] Emkey | Analysis and evaluation of graded-index fiber-lenses[END_REF]. In this paper, we present a micro-optics at fiber end to reduce backreflection. The level of reflection can be reduced under -28 dB. The theory is in a good agreement with experimental results to define the upper limit of reflections. The fabrication process needs few simple and collective steps and is particularly suitable for mass production and low cost. The potential of this device to obtain very high return loss in combination with anti-reflecting coating is expected in the future. This solution allows preserving the geometric aspect of the fiber (no tilt) and simplifies the packaging process. Lsilica = 400 µm

Fig. 3 .Fig. 2 .

 32 Fig. 3. Schematic view of the measure. < -90 dB < -80 dB -30 dB

Fig. 4 .

 4 Fig. 4. Backreflection measures as a function of GRIN length for two lengths of silica Ls = 0 µm (a) and 400 µm (b). Solid lines: theoretical curves.