1Department of Clinical Pharmacology, The Royal College of Surgeons, Dublin 2, Ireland; and 2Cork Cancer Research Centre, Mercy University Hospital, University College Cork, Cork, Ireland
Submitted 25 March 2004 ; accepted in final form 28 June 2004
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ABSTRACT |
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platelet releasate; vitronectin; fibronectin
In the present investigation, we studied a shear-specific effect on metastatic cells and further characterized the interaction of tumor cells, platelets, and inflamed endothelium. Successful cell arrest in the circulation is dependent on the balance between adhesive and antiadhesive forces and on the rate at which adhesive interactions are broken (33). The initial interaction between adherent platelets and inflamed endothelium was recently described (10). Inflamed endothelium in cancer patients has been shown to enhance metastasis formation (1). Thus we hypothesized a dynamic interaction between inflamed endothelium and flowing metastatic cells.
Platelet tumor interactions are important in metastasis dissemination. Experimental evidence published in the 1960s demonstrated that a decrease in platelet count reduced the spread of cancer (11). When platelets are activated, they secrete mitogenic and angiogenic factors that have been implicated in tumor spread (25). The effect of this secreted component, i.e., the platelet releasate on enhancing bone cell migration and recruitment in vitro, has only recently been appreciated (26). We therefore examined the effect of preincubating tumor cells with platelet releasate under venous shear conditions. We further examined the involvement of the platelet-cyclooxygenase pathway in the platelet releasate by using the antiplatelet agent aspirin [acetylsalicylic acid (ASA)]. Platelet releasate potentiated the response of tumor cells to shear, while pretreating the tumor cells with platelet releasate from aspirinated blood abrogated this enhanced response.
Integrins support tumor cell arrest during blood flow (28). To identify the role of specific integrins in this process, we further characterized the effect of shear on tumor cell adhesion to matrices used by integrins. Our results show that the platelet releasate mediates its effects partially through the integrin v
3. Furthermore, depletion of vitronectin or fibronectin from the platelet releasate decreases the enhanced tumor response observed in the presence of platelet releasate.
In summary, we demonstrate a novel protein interaction between tumor cells expressing v
3 and platelet-secreted vitronectin and fibronectin under venous shear conditions. Our findings suggest that therapies targeted at specific integrins in a shear environment may prevent tumor cell spread from the circulation, thereby controlling metastatic spread.
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EXPERIMENTAL PROCEDURES |
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Antibodies and cells.
Monoclonal antibody against the integrin receptor v
3 (LM609) was obtained from Chemicon International (Temecula, CA). Monoclonal antibodies directed against Vn (clone VIT-2) and Fn (clone IST-4) were purchased from Sigma-Aldrich. Rib bone marrow micrometastases (RBM/E-3) were isolated from the rib bone marrow of a patient who underwent resection for primary esophageal cancer. Diagnostic and clinical investigations for staging of all patients included standards of care in accordance with guidelines of the human ethics committee for clinical research, National University of Ireland. Informed consent for surgery and bone aspiration was obtained in all cases. The cells were cultured from rib bone marrow cells (26). Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as previously described (14). MCF-7, Hs578T, and HuT cell lines were obtained from American Type Culture Collection (ATCC; Rockville, MD) and cultured as previously described (7, 12, 15).
Flow assay. The behavior of RBM/E-3 cells under physiologically relevant shear conditions was assayed using a modified parallel plate flow chamber assembly described by Lawrence et al. (22). The flow chamber was purchased from GlycoTech (Rockville, MD) and consisted of a flow deck and silicon rubber gasket, with the flow path defined precisely by gasket thickness and gasket flow path width. HUVECs were grown on 35-mm culture dishes (Corning, Corning, NY), which fit precisely within the flow chambers. Fifteen hours before performing the flow assay, HUVECs were pretreated with LPS (10 µg/ml) to upregulate the expression of adhesive proteins (30).
RBM/E-3 (1 x 106/ml) were suspended in PBS, prewarmed to 37°C, and kept at this temperature throughout the experiments. RBM/E-3 cells were injected via a side port into the flow path and allowed to settle on LPS-stimulated HUVECs in the parallel plate flow chamber for 5 min before being exposed to flow. The chamber was then perfused with PBS at a venous shear rate of 50 s1 for 1 min using a syringe pump (Harvard Apparatus, Holliston, MA). The cells were visualized in bright field with phase-contrast microscopy (x40 and x63 LD-Acroplan objectives) using a Zeiss Axiovert-200 epifluorescence microscope (Carl Zeiss, Gottingen, Germany).
As soon as flow was initiated, images were captured every 200 ms up to 1 min with a liquid-chilled Quantix-57 charge-coupled device camera (Photometrics, Tucson, AZ), resulting in 300 frames. Flow was then stopped for 5 min to analyze tumor adhesion. After the 5-min interval, the shear rate was increased incrementally to 100, 200, 400, 600, and 800 s1, with 5-min intervals of no flow. This resulted in six 1-min movies, each corresponding to an individual shear rate. Please refer to the Supplementary Material for this article to view renderings (Movies 13).1 The interaction of the RBM/E-3 cells and HUVECs was analyzed offline using the commercial software package MetaMorph (version 4.6.8; Universal Imaging, Downingtown, PA). Flow rates were calculated using the equation for laminar flow to give a venous shear rate between 50 and 800 s1. MCF-7, Hs578T, and HuT cells were also investigated as described above. In additional experiments, RBM/E-3 cells were incubated for 30 min with 70 µl of either platelet releasate or platelet releasate immunodepleted of Vn or Fn and then perfused over HUVECs as described above.
Isolation of platelet releasate. Gel-filtered platelets were prepared after being obtained from healthy volunteers who had not taken ASA for at least 10 days. Collection of blood was approved by The Royal College of Surgeons, Ireland, ethics committee, and informed consent was obtained from the volunteers. Blood was collected in acid-citrate-dextrose (ACD; in mM: 38 citric acid, 75 sodium citrate, 124 dextrose) as anticoagulant (0.15 vol/vol). The blood was centrifuged for 10 min at 150 g at room temperature. Platelet-rich plasma was then acidified to pH 6.5 with ACD. PGE1 (1 µM) was added to prevent platelet activation. Platelets were pelleted by centrifugation at 720 g for 10 min, and then the supernatant was removed and the pellet was resuspended in buffer A (in mM: 130 NaCl, 10 trisodium citrate, 9 NaHCO3, 6 dextrose, 0.9 MgCl2, 0.81 KH2PO4, and 10 Tris, pH 7.4). The platelets were gel filtered using Sepharose B resin and supplemented with 1.8 mM CaCl2. Gel-filtered platelets were stimulated with thrombin (0.1 U/ml) and collagen (0.19 mg/ml) and stirred for 15 min. The platelet aggregate was centrifuged at 720 g for 10 min, and the supernatant was aspirated and filtered through 0.22-µm-pore disks to remove platelet microparticles. The filtrate was then centrifuged at 6,000 g in 1-kDa cutoff filters for 12 h to concentrate the platelet releasate. For ASA-treated platelet releasate, whole blood was collected into ACD with ASA, and the releasate was collected.
Drug experiments. To assess the effects of ASA on the effects induced by platelet releasate, we added ASA (200 µM) directly to platelet releasate and preincubated this with RBM/E-3 cells or treated the tumor cells directly with ASA alone (200 µM). In separate experiments, we investigated the effects of adding a thromboxane antagonist (SQ-29548) to the platelet releasate with RBM/E-3 cells for 30 min or directly added a thromboxane mimetic (U-46619) to the tumor cells. The cells were then perfused over the HUVECs matrix as described above.
Tumor adhesion assays on matrix proteins.
Glass coverslips were coated overnight with 10 µg/ml of Vn, Fn, fibrinogen, and collagen type I or IV. RBM/E-3 cells were grown to 80% confluence and then suspended in prewarmed 37°C DMEM at a concentration of 1 x 106 cells/ml. The flow chamber containing the matrix-coated glass coverslip was assembled. RBM/E-3 cells were injected into the flow chamber, allowed to settle on the matrix for 5 min, and then exposed to a continuous shear rate of 100, 200, or 400 s1 for 1 h.
Blockade of v
3 in tumor cells.
To investigate the role of
v
3 in tumor adhesion under flow in the presence of platelet releasate, we pretreated RBM/E-3 cells with 10 µg/ml of the antibody LM609 for 30 min before addition of 70 µl of platelet releasate. LM609 inhibits the binding of
v
3 to matrix protein. The RBM/E-3 cells were then perfused over anti-
v
3-treated HUVECs at shear rates from 50 to 800 s1. The rates of adhesion and pseudopod formation were analyzed as described above.
Immunodepletion of vitronectin and fibronectin from platelet releasate.
Immunoprecipitation was performed with monoclonal anti-Vn, anti-Fn, or anti-IgG isotype antibodies. Immunoprecipitates were recovered with protein G-Sepharose beads. Immunodepleted platelet releasates and immunoprecipitates were separated by performing 7.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and probed with the above antibodies. The protein-antibody complexes were then visualized with a horseradish peroxidase-linked -mouse secondary antibody and SuperSignal ECL reagents.
Effect of shear on v
3 clustering in tumor cells.
Glass slides were coated with Vn (100 µg/ml) for 2 h at room temperature, blocked in 1% BSA in PBS for 1 h, and then washed three times with PBS before use. RBM/E-3 cells were suspended in serum-free DMEM at a concentration of 1 x 106 cells/ml. The tumor cell suspension (400 µl) was added to each slide and incubated for 1 h at room temperature. In separate experiments, the tumor cells were allowed to adhere to Vn for 30 min and then exposed to a continuous shear rate of 400 s1 for 30 min at 37°C. Both sheared and nonsheared adherent tumor cells were washed three times and fixed in 3.7% formaldehyde for 15 min at room temperature. Slides were incubated with 50 µl of LM609 (5 µg/ml) for 1 h at room temperature and then washed and incubated with a secondary goat anti-mouse IgG antibody for 10 min. The cells were then permeabilized with ice-cold acetone for 2 min. Phalloidin 488 was added at a concentration of 5 µg/ml, incubated at room temperature for 20 min to stain actin, washed, and incubated with a secondary antibody for 10 min. The cells were then washed and mounted using Dako fluorescent mounting medium. Slides were analyzed using a LSM510 Axioplan 2 upright confocal microscope (Carl Zeiss). Rhodamine fluorescence was detected at 546 nm, and FITC was detected at 488 nm.
Statistical analysis. Numerical values are means ± SD. Data were analyzed by unpaired t-tests or by one-way ANOVA.
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RESULTS |
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The patterns of adherence for shear rates of 50, 100, and 200 s1 were similar to those on the total chamber area, where 490 cells remained adherent. The number of adherent cells decreased as the shear rates were increased from 400 to 600 s1. A significant decrease in tumor cell adhesion occurred at 600 s1 (Fig. 1A), with only 12% of the initial adherent cell population remaining adherent (P = 0.001). Increasing the shear rate to 800 s1 did not affect this cell population. We observed some tumor cells clustering together at shear rates <400 s1, but at higher shear rates, only individual cells remained adherent to the endothelium.
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The tumor cells in the presence of the platelet releasate exhibited pseudopodia at a significantly lower shear rate of 50 s1 (n = 6) compared with the shear rate of 400 s1, which caused activation in the absence of the releasate. These results are shown in Fig. 1B. We also noted the formation of filopodia, i.e., cylindrical projections from the surface of the tumor cell. The percentage of adherent cells forming pseudopods is 1.2% at 0 s1, and this percentage progressively increased as shear rates strengthened, with a maximum response at 800 s1.
Platelet releasate from ASA-treated platelets abrogated the effects mediated by the platelet releasate in RBM/E-3 cells and was not due to thromboxane A2. High concentrations of thromboxane A2 (TxA2) and low concentrations of prostacyclin are observed in patients with cancer (16). ASA impairs TxA2 synthesis through acetylation of platelet cyclooxygenase. Because we had found that the platelet releasate potentiated the activation of RBM/E-3 cells, we investigated the effect of platelet releasate from platelets pretreated with ASA (200 µM).
The potentiation of shear-induced RBM/E-3 cell activation by the platelet releasate was completely abolished by ASA treatment. The interaction of tumor cells with HUVECs decreased and displayed a pattern similar to that observed in RBM/E-3 cells in the absence of the releasate (Fig. 1A).
To further characterize the effect of ASA in the platelet releasate, we pretreated RBM/E-3 cells directly with ASA alone or with ASA added to platelet releasate. The addition of ASA alone or ASA added directly to platelet releasate did not result in significant differences in tumor activation compared with platelet releasate derived from aspirinated blood (data not shown).
We investigated whether thromboxane caused increased tumor activation upon addition of the platelet releasate. We used a thromboxane receptor antagonist, SQ-29548, and a thromboxane mimetic, U-46619, as described in EXPERIMENTAL PROCEDURES. Both rates of adhesion and pseudopod formation did not significantly change when the tumor cells were exposed to shear rates from 50 to 800 s1, confirming that TxA2 was not involved in the process (data not shown).
Perfusion of MCF-7 and Hs578T cells over LPS-treated HUVECs resulted in shear-dependent pseudopod formation. We further characterized the behavior of other cell lines that frequently metastasize to bone. We tested the behavior of a metastatic breast cancer cell line (Hs578T) and a nonmetastatic breast cancer cell line (MCF-7) under venous shear conditions. The cells were injected at the same concentrations used for RBM/E-3 cells, allowed to settle on LPS-treated HUVECs for 5 min, and analyzed similarly for adhesion and pseudopod formation. Both MCF-7 and Hs578T cells were more adhesive than RBM/E-3 tumor cells at all shear rates; however, no significant differences were detected (data not shown).
Shear sensitivity in HuT cells.
To determine whether our shear response was a nonspecific response of metastatic cells, we examined the behavior of a cell line derived from circulating T cells (HuT cells). In these cells, we observed an entirely different response to shear. These cells formed a long-tailed pseudopodal structure at low venous shear rates of 50100 s1. RBM/E-3 cells demonstrated a different morphology at shear rates of 400 s1. Increasing the shear rate to >200 s1 resulted in rapid retraction of the tail in HuT cells (Fig. 3), demonstrating a shear-sensitive mechanism (Movie 3). As shear rates were increased to >400 s1, fewer HuT cells adhered and no tail formation was observed.
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Localization of v
3 in tumor cells exposed to shear.
Clustering of integrins is associated with actin rearrangement leading to cell motility (17). Because our results demonstrate that
v
3 played an important role in the adhesion to endothelium, we examined the clustering of this integrin in the absence and presence of shear. RBM/E-3 cells were stained with an anti-
v
3 antibody, shown in red in Fig. 8A. To assess the effect of shear on these cells, we perfused RBM/E-3 cells over Vn as described in EXPERIMENTAL PROCEDURES and stained the remaining adherent cells. Notable differences in the intensity of
v
3 staining were observed between sheared and nonsheared tumor cells, as shown in Fig. 8B. This suggests a possible role for shear in maximizing integrin clustering as well as tumor cell surface area, thus enhancing potential tumor cell arrest.
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DISCUSSION |
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Esophageal cancer is a relatively uncommon malignant condition with a low likelihood of cure. Once the cancer develops, it spreads rapidly via metastases through the bloodstream. A prerequisite for the progression of hematogenous metastasis is that circulating cancer cells lodge in the vascular bed of the target organ, a process that is opposed by flowing blood (3). Because leukocytes and platelets tether and roll in a shear-dependent manner, we hypothesized that cancer cells would respond similarly. In the present investigation, we used RBM/E-3 cells isolated from the rib bone marrow of a patient who underwent resection for primary esophageal cancer. Nonmetastatic MCF-7 and metastatic Hs578T breast cancer cells also were examined for their behavior under shear conditions. We have demonstrated that both metastatic and nonmetastatic cells activated at shear rates between 50 and 400 s1. In contrast, a cell line derived from circulating T cells behaved in an entirely different manner. RBM/E-3 adherence occurred in the continuous presence of shear stress, similar to leukocyte and platelet tethering and rolling on inflamed endothelium. Several researchers have reported the ability of cytokine-stimulated endothelium to cause E selectin-dependent rolling of cancer cells on the endothelial surface under conditions of fluid flow (13). Similarly, stimulating endothelial cells with endotoxin promotes the formation of mediators, which enhance tumor cell-endothelium interaction. To simulate the environment of circulating metastatic cells interacting with activated endothelium, we perfused RBM/E-3 cells over LPS-stimulated endothelium.
Investigators in numerous studies have examined the adhesion of cancer cells to endothelium, but they have used a static adhesion assay to quantify adhesive strength (18). More recently, the use of parallel plate flow chambers has enabled researchers to quantify the adhesion of cancer cells to endothelial monolayers under controlled flow conditions, which exposes both tumor and endothelial cells to shear stress. Shear stress affects both endothelial cell function and tumor cells (8). Exposing endothelial cells to shear stress is known to alter the surface expression of adhesion molecules (31), an effect that could explain why RBM/E-3 tumor cells adherent to inflamed HUVECs form pseudopodia at specific shear rates. We were unable to demonstrate a shear-induced activation response when RBM/E-3 cells were perfused over matrices such as fibrinogen, collagen I, and collagen IV. This suggests a specific response of metastatic cells that adhere to the endothelium. Previous studies have subjected tumor cells to shear conditions for periods of hours (24), thus maximizing the shear response in the tumor cells. In contrast, we investigated the initial response of tumor cells under hydrodynamic conditions.
Tumor cell activation is characterized by the protrusion of pseudopods (5), which ultimately leads to extravasation of the vascular wall. Under shear conditions, we did not observe pseudopod protrusion in the RBM/E-3 cells until a shear rate of 400 s1 was reached, which corresponded to 20-min adherence on endothelium. At shear rates >400 s1, there was no increase in the formation and retraction of pseudopods, suggesting that once activated, the formation of pseudopods becomes independent of shear. In the absence of shear, pseudopod formation did not occur until 40 min had elapsed.
We also investigated the shear-induced response in MCF-7 and Hs578T tumor cells. These cells also activated at a precise shear rate. The results of the present investigation suggest that a critical shear stress is necessary for tumor cells to activate and remain adherent. This may also reflect the point in the vasculature where these cells metastasize. Our results demonstrate that different tumor cells exhibit different activation states under the influence of venous flow. It is well known that malignant tumors preferentially metastasize to particular distant organ sites (19). Our results suggest that metastatic spread may reflect the shear forces encountered in that region.
Although it is well accepted that platelets play an important role during hematogenous metastasis, the direct involvement of the platelet releasate in tumor cell behavior under shear conditions has not yet been investigated. We examined the effects of fluid shear stresses on RBM/E-3 cells adherent to HUVECs in the presence of platelet releasate and releasate from ASA-treated platelets. We observed a dramatic response in tumor cells preincubated with the platelet releasate. First, the number of cells adherent to the HUVECs surface increased. Second, the minimum shear rate required to induce pseudopod formation was reduced to 50 s1, and in some cases, the cells became activated before shear was initiated. Thus one or more components present in the platelet releasate had an effect on the behavior of RBM/E-3 tumor cells under flow conditions. ASA-treated platelet releasate did not potentiate the activation response of the tumor cells. The effects of platelet releasate from ASA-treated platelets on tumor activation warrants further study, but in an initial evaluation, we have found that the effects of ASA platelet releasate are not due to ASA alone and that a thromboxane antagonist and mimetic do not cause significant differences in tumor activation under flow.
Characterization of the specific proteins responsible for increased tumor adhesion in the presence of platelet releasate was further investigated. Because integrins are the main mediators of tumor cell arrest in the vasculature (28), we examined the behavior of RBM/E-3 cells under flow on different matrix components that are ligands for specific integrins. Maximal tumor cell arrest and activation occurred on the matrix proteins Vn and Fn, suggesting a potential role for the integrin v
3. In addition, we found that blockade of
v
3 in RBM/E-3 cells followed by addition of platelet releasate resulted in less adhesion and activation at high venous shear rates of 600 and 800 s1. Examination of
v
3 further via confocal microscopy in pre- and postsheared tumor cells resulted in distinct differences in the intensity of
v
3 staining, with greater staining observed in sheared cells. The differences may be due to clustering of integrins, which has been intimately linked to actin rearrangement leading to cytoskeletal movement (23), or they may be due simply to the mechanical force of shear exposing maximal cell surface area to interacting proteins.
In summary, we have shown that the mechanical stimulus of shear evokes specific changes in the morphology of tumor cells at defined shear rates. The activation state of tumor cells was enhanced in the presence of the platelet releasate through secreted Vn and Fn, while platelets pretreated with ASA abrogated the effects mediated by the platelet releasate. This may in part explain the potential benefit of ASA therapy in the treatment of cancer and suggests that therapies targeted to defined regions of shear and specific integrins may be of potential benefit.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supplemental data for this article may be found at http://ajpcell.physiology.org/cgi/content/full/00159.2004/DC1/.
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