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Experimental Therapeutics |
vß3 Integrins in Tumor Growth, Angiogenesis, and Metastasis
Department of Biology Research, Centocor, Inc., Malvern, Pennsylvania 19355 [M. T., Z. Z., M. K., E. E., M. T. N.], and Department of Tumor Progression, National Institute of Oncology, Budapest, Hungary H-1122 [J. T., E. R.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3. We used chimeric (c) 7E3 Fab (ReoPro) and murine (m) 7E3 F(ab')2 to elucidate the role of these integrins in angiogenesis, tumor growth, and metastasis. These antibodies are potent inhibitors of GPIIb/IIIa and vß3. c7E3 Fab inhibited vß3-mediated human umbilical vein endothelial (HUVEC) and melanoma cell adhesion, migration, invasion, and basic fibroblast growth factor stimulated proliferation of HUVECs (IC50 values range from 0.15 to 5 µg/ml for different assays). In an in vitro angiogenesis assay, c7E3 Fab inhibited basic fibroblast growth factor and platelet-stimulated capillary formation of HUVECs (IC50 = 10 µg/ml and 15 µg/ml, respectively), demonstrating that endothelial vß3 is important for sprouting, and platelet-stimulated sprouting is mediated by GPIIb/IIIa. In an experimental metastasis assay, a single pretreatment of human melanoma cells with c7E3 Fab (2.5 µg/ml) inhibited lung colonization of the tumor cells in severe combined immunodeficient mice. In vivo, m7E3 F(ab')2 partially inhibited growth of human melanoma tumors in nude mice compared with control-treated animals. These data suggest that tumor cell-expressed integrins are important but not the only component involved in tumor growth. Because c7E3 Fab and m7E3 F(ab')2 do not cross-react with murine integrins, this inhibition of metastasis and tumor growth is attributable to direct blockade of human tumor vß3 integrins. m7E3 F(ab')2 completely blocked tumor formation and growth of human melanoma tumors growing in nude rats. In this xenograft model, m7E3 F(ab')2 simultaneously binds to both human tumor and host platelet GPIIb/IIIa and endothelial vß3 integrins, thus participating as an antiangiogenic and an antitumor agent. Collectively, these results indicate that combined blockade of GPIIb/IIIa and vß3 affords significant antiangiogenic and antitumor benefit. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IIbß3) receptor on platelets, which is the major receptor involved in platelet aggregation. c7E3 Fab also binds with equivalent affinity to the vitronectin receptor vß3, and it can redistribute between GPIIb/IIIa and vß3 receptors in vitro (2)
. We asked whether c7E3 Fab could be used to determine the contribution of platelet GPIIb/IIIa and vß3 integrins in tumor growth, angiogenesis, and metastasis.
There is now considerable evidence that progressive tumor growth is dependent on angiogenesis. The formation of new blood vessels provides tumors with nutrients and oxygen, allows the removal of waste products, and acts as conduits for the spread of tumor cells to distant sites (3)
. Several studies have defined the role of integrins in the angiogenic process (4, 5, 6)
. During the angiogenic process, vß3 is up-regulated on the surface of activated endothelial cells, which in turn helps these cells to migrate, proliferate, and invade the tumor (4, 5, 6)
. An antagonist of vß3, LM609, suppressed angiogenesis and blocked growth of human tumors that did not express this receptor (7)
. LM609 was used in a SCID mouse human chimeric angiogenesis model. In this system, vß3-negative human melanoma cells were injected into full thickness human skin grafted onto SCID mice. The resulting tumors induced an angiogenic response that enhanced the growth of tumor cells in an orthotopic microenvironment (7)
. Regular administration of LM609 significantly inhibited growth of vß3-negative tumors by blocking the growth of human blood vessels. Because LM609 does not cross-react with mouse integrins, its antiangiogenic activity was attributed to blockade of human vß3 receptors in the vasculature of the human skin. A subsequent study using the murine IgG equivalent of c7E3 Fab (m7E3 IgG) in the same model achieved similar results as LM609 (8)
. Similar to LM609, 7E3 does not cross-react with mouse integrins; therefore, it inhibited growth of human tumors by blocking human vß3 receptors in the vasculature of the human skin. In these studies, a partial inhibition of tumor growth was observed, and the combined effect of blocking tumor cell-expressed vß3 and endothelial cell-expressed vß3 was not evaluated. One limitation of this model is that tumors can grow even in the absence of human vasculature, because the mouse vasculature can sustain tumor growth. To the best of our knowledge, a relevant model examining simultaneous blockade of both host and tumor cell-expressed integrin has not yet been evaluated. One purpose of our study was to evaluate whether combined blockade of host and tumor cell-expressed integrins was superior to blockade of tumor cell-expressed integrins in vivo.
The clinical significance of ß3 integrin expression in human melanoma was determined in a prospective study that examined the expression of this integrin in patients who were followed for a mean of 98 months after diagnosis with intermediate thickness melanoma (9 , 10) . This study concluded that tumors in 64% of the patients expressed ß3 integrin, and a higher proportion (45%) of patients with ß3 positive melanomas were more likely to die of their disease when compared with those with ß3 negative tumors (8%).
Angiogenesis can also stimulate the metastatic cascade by providing conduits for the spread of tumor cells to distant sites (6
, 11) . Some have postulated that platelets are involved in tumor cell extravasation, adherence, or trapping of tumor cell-platelet aggregates to capillary walls, and protection of circulating tumor cells from the antitumor response of the host (reviewed in Refs. 1
, 11
, 12
). The role of platelets in facilitating hematogenous metastasis is well accepted, but little is known about their role in contributing to growth of the primary and/or metastatic tumor. Platelet granules contain a variety of angiogenic factors such as VEGF, platelet-derived growth factor, TGF-ß, and fibrinogen, and these modulators are immediately secreted after platelet activation (13)
. Tumor vasculature is leaky, and extravasated fibrin(ogen) that is deposited on the tumor surface can provide an ideal substrate for platelet binding and activation. In addition, tumor cells can activate platelet aggregation (14)
and cause the release of VEGF from platelets (15
, 16)
, which in turn can stimulate angiogenesis. c7E3 Fab can block GPIIb/IIIa-mediated platelet aggregation, degranulation, and adhesion to fibrinogen (1)
. One goal of this study was to determine whether blockade of platelets could inhibit tumor growth in vivo. Recently, Verheul et al. (17)
have demonstrated that platelets stimulate endothelial cell proliferation in vitro. Clinically thrombocytosis, an increase in platelet count, is directly correlated with survival of patients of lung and ovarian carcinoma (18, 19, 20)
, supporting the notion that platelets may play a role in tumor growth, angiogenesis, and metastasis. The central hypothesis for our study was that combined blockade of platelet GPIIb/IIIa, endothelial, and tumor cell-expressed vß3 could have an enhanced inhibitory effect compared with blockade of tumor cell-expressed vß3 alone. c7E3 Fab is one such agent that can antagonize GPIIb/IIIa and vß3, and it is widely used in the clinic as an antithrombotic agent. Therefore, we wanted to determine whether c7E3 Fab has anticancer properties. Results from this study indicate that c7E3 Fab and m7E3 F(ab')2, in addition to providing antithrombotic effect, also possess antiangiogenic and antitumor properties.
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MATERIALS AND METHODS |
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vß3, and MAB1961 (PIF6), a mAb against integrin vß5, were purchased from Chemicon (Temecula, CA). Biocoat cell culture inserts (pore size 8 µm) were purchased from Becton Dickinson (Bedford, MA). Vybrant cell adhesion assay kit (V-13181) was purchased from Molecular Probes (Eugene, OR). Human plasminogen-free fibrinogen (von Willebrand/fibronectin depleted) was purchased from Enzyme Research Labs (South Bend, IN). Bovine skin gelatin was purchased from Sigma (St. Louis, MO). Human vitronectin was purchased from Promega (Madison, WI), and type I collagen from Life Technologies, Inc. (Gaithersburg, MD). c7E3 Fab, m7E3 F(ab')2, and 10E5 were generated at Centocor. For animal experiments m7E3 F(ab')2, instead of the intact IgG, was used to eliminate platelet clearance and any other Fc receptor-mediated events.
Cell Lines.
HUVECs were purchased from Clonetics (Walkersville, MA), and cultured in EBM complete medium (Clonetics) containing 10% fetal bovine serum, long R insulin-like growth factor-1, ascorbic acid, hydrocortisone, human epidermal growth factor, human VEGF, gentamicin sulfate, and amphotericin-B. Cells were grown at 37°C and 5% CO2, and medium was changed every 2–3 days. Cells were passaged when they reached 80% confluence. Passages 3–8 were used in all of the experiments. The A375S2 human melanoma cell line was obtained from American Type Culture Collection (Rockville, MD), and deemed free of Mycoplasma and bacterial contaminants. The cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids. HT168M1 melanoma cells were isolated from a patient as described (21)
and were cultured in 10% FBS and RPMI 1640. Human colon carcinoma HT29 cells were obtained from American Type Culture Collection, and deemed free of Mycoplasma and bacterial contaminants. The cells were cultured in -MEM supplemented with 10% FBS, 2 mM L-glutamine, 1mM sodium pyruvate, and 0.1 mM nonessential amino acids.
Flow Cytometry.
To stain surface integrins, cells were harvested, rinsed, suspended in unsupplemented RPMI 1640, and sequentially incubated for 60 min at room temperature with anti-integrin mAbs (10 µg/ml) and FITC-labeled goat antimouse antibody (1:200). In some instances, cells were directly labeled with FITC-labeled anti-integrin mAbs (10 µg/ml). Absence of primary antibody or substitution of primary antibody with isotype-matched irrelevant antibody served as negative controls. Cells were immediately analyzed with a fluorescence-activated cell sorter Scan II flow cytometer (Becton Dickinson, Mountain View, CA).
Adhesion Assay.
Microtiter plates (Linbro-Titertek; ICN Biomedicals, Inc.) were coated at 4°C overnight with vitronectin (1 µg/ml), gelatin (0.1%), fibrinogen (100 µg/ml), type I collagen (10 µg/ml), or fibronectin (10 µg/ml). Fibrin-coated Microtiter wells were formed by thrombin treatment (1 units/ml) of fibrinogen. These concentrations of proteins supported optimal cell adhesion. Immediately before use plates were rinsed with PBS and blocked for 1 h with 1% BSA/PBS (pH 7.4). Adherent cells were labeled with Calcein a.m. fluorescent dye (Molecular Probes) according to the manufacturer’s instructions, harvested, washed twice, and suspended in 0.1% BSA in DMEM. After cell density was adjusted to 5 x 105/ml, cells were incubated with various concentrations of antibodies for 15 min at 37°C. The cell-antibody mixture was added to wells (100 µl/well) and incubated for 1 h at 37°C. Plates were rinsed twice with PBS to remove unbound cells, and adhesion was measured in a fluorescence plate reader (Fluoroskan; Tecan, Research Triangle Park, NC) at 485–538 nm. Cell adhesion to BSA-coated wells served as a negative control. Isotype-matched antibodies served as a negative control.
Cell Migration Assay.
Cell migration assays were performed in 24-transwell chambers with a polystyrene membrane (6.5-mm diameter, 10-µm thickness, and 8-µm pore size) as described previously (22)
. Briefly, the underside of the membrane was coated with vitronectin (2 µg/ml) for 60 min at room temperature and then blocked with a solution of 1% BSA/PBS at room temperature for 60 min. Next, membranes were washed with PBS and dried. Serum-free medium (750 µl) containing 0.1% BSA and bFGF (20 ng/ml) or medium containing 10% FBS was added to the lower chambers. Subconfluent 24-h cultures were harvested with trypsin-EDTA, washed twice, and resuspended in serum-free medium. Cells (100,000/500 µl) were added to the upper chambers in the presence or absence of antibodies. The chambers were placed in a tissue culture incubator, and migration was allowed to proceed for 4–6 h. Migration was terminated by removing the cells on the top with a cotton swab, and the filters were fixed with 3% paraformaldehyde and stained with Crystal Violet. The extent of cell migration was determined by light microscopy, and images were analyzed using the Phase 3 image analysis software (Glen Mills, PA). The software analyzes the total area occupied by the stained cells on the bottom side of the filter, and this is directly proportional to the extent of cell migration.
Invasion Assay.
The cell invasion assays were performed as described (23)
. Briefly, fibrinogen (plasminogen-free 100 µl of 10 mg/ml) and 100 µl of 1 unit/ml thrombin was mixed, and immediately added to the top chamber of 24-well transwell plates (6.5-mm diameter, 10-µm thickness, and 8-µm pore size). The plates were incubated at 37°C for 30 min to form a fibrin gel. Confluent tumor cells (A375S2) were trypsinized, centrifuged, resuspended in basal medium supplemented with 0.1% BSA and 10 µg/ml plasminogen (Enzyme Research Labs) with various concentrations of antibodies, and incubated for 15 min at room temperature. Cells (100,000/500 µl) were added to the upper chamber in the presence or absence of antibodies. The lower compartment of the invasion chamber was filled with 0.75 ml of 10% FBS-DMEM, which served as a chemoattractant, and the plate was transferred to a tissue culture incubator. After 24 h, invasion was terminated by removing the cells on the top with a cotton swab, and the filters were fixed with 3% paraformaldehyde and stained with Crystal Violet. The extent of cell migration was analyzed using the Phase 3 image analysis software as described above.
Endothelial MC-based Sprouting Assay.
A modification of the methods of Nehls and Drenckhahn (24)
was used to measure capillary tube formation in three-dimensional fibrin-based matrix. Gelatin-coated cytodex-3 MCs (Sigma) were prepared according to recommendations of the supplier. Freshly autoclaved MCs were suspended in EBM-2 + 20% FBS, and endothelial cells were added to a final concentration of 40 cells/MC. The cells were allowed to attach to the MCs during a 4-h incubation at 37°C. The MCs were then suspended in a large volume of medium and cultured for 2–4 days at 37°C in 5% CO2 atmosphere. MCs were occasionally agitated to prevent aggregation of cell coated beads. MCs were embedded in a fibrin gel that was prepared as follows: human fibrinogen (2 mg/ml) was dissolved in plain medium containing antibodies and/or bFGF, PRP containing 250,000 platelets/µl, PPP, or serum containing EBM-2 medium. PRP, PPP, and gel-filtered platelets were prepared from citrated whole blood obtained from healthy volunteers as described (2)
. To prevent excess fibrinolysis by fibrin-embedded cells, aprotinin was added to the fibrinogen solution and to growth medium at 200 units/ml. Cell-coated MCs were added to the fibrinogen solution at a density of 100–200 MCs/ml (50–100 beads/per 48-well plate), and clotting was induced by addition of thrombin (0.5 units/ml). After clotting was complete, 0.5 ml of solution (containing all of the components described above except fibrinogen and thrombin) was added to the fibrin matrices. The plates were incubated at 37°C and 5% CO2 for 1–3 days. After 1–3 days, gels were fixed with a solution of 3% paraformaldehyde in PBS, and the number of capillary sprouts with length exceeding the diameter of the MC bead (150 µm) was quantified by using the Phase 3 image analysis.
Endothelial Cell Proliferation and Apoptosis Assays.
Subconfluent HUVECs were trypsinized, washed, and resuspended in complete medium. Cells (5000) were added to each well of 96-well plates. To test whether the plates themselves may influence the assay, endothelial cells were plated on normal tissue culture plates, high protein-binding plates that were precoated with vitronectin (1 µg/ml), gelatin (0.1%), or type I collagen (2 µg/ml). Cells were allowed to attach for 2 h, medium was aspirated, wells were washed once with PBS, and 100 µl of medium (0.1% serum-M199 or 2% serum-M199) containing bovine bFGF-2 (R&D systems), human recombinant VEGF165 (r + D Systems), and/or various antibodies was added to each well. The plates were incubated at 37°C for 48 h. Extent of cell proliferation was determined by the Celltiter 96 Aqueous kit (Promega), ATP kit (Packard, Meridian, CT), or BrdUrd kit (Oncogene Research Products). For the MTS and the BrdUrd assay, absorbance was measured at 490 nm and 540/450 nm, respectively. Luminescence intensity was measured for the ATP assay in a TopCount reader (Packard). To quantify apoptosis, cells were treated as above with antibodies or positive control etoposide for 18 h, and extent of DNA fragments were measured by using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics GmbH, Mannheim, Germany).
Matrigel-based Angiogenesis Assay in Nude Rats.
The Matrigel plug-based angiogenesis assay was performed as described earlier (25)
with slight modifications. Briefly, cold Matrigel (Becton Dickinson) was mixed with bFGF (5 µg/ml) and m7E3 F(ab')2 (
300 µg/ml) or an equal volume of PBS. The next day, 2 ml of Matrigel solution was injected s.c. into nude rats (Taconic, Germantown, NY), and animals were dosed i.p. with 6 mg/kg of m7E3 F(ab')2. This dose of m7E F(ab')2 completely inhibits rat platelet aggregation ex vivo (26)
. Animals were dosed every day for 6 days, and plugs were removed on day 7. The extent of angiogenesis was quantified by using the Drabkins kit (Sigma) as described (25)
.
Lung Metastasis Assay.
The lung metastasis assay was performed as described previously (27)
. Human melanoma HT168M1 cells were pretreated with 2.5 µg/ml of c7E3 Fab or control antibody for 15 min at room temperature, washed, and 1 x 106 cells were tail vein injected into female SCID mice. After 1 month, animals were euthanized, lungs were removed and fixed in paraformaldehyde, and the number of lung colonies were counted.
Growth of Human Melanoma Tumors in Nude Mice and Nude Rats.
To determine whether m7E3 F(ab')2 could inhibit tumor growth in vivo, we used a human melanoma xenograft model in nude mice and nude rats. Briefly, A375S2 cells (3 x 106/animal) were s.c. injected into female nude mice (Charles River, Raleigh, NC) or nude rats (Taconic). Tumor cells were pretreated with antibody (100 µg/ml for 5 min) before injection or therapy was initiated after animals had developed measurable tumors. Antibody was injected i.p. at a dose of 200 µg/animal or at an animal body weight-adjusted dose of 3–10 mg/kg. Control groups were injected with equivalent volume of diluent (PBS). Tumor volume (mm3) was calculated based on the formula: (length x width x width)/2 and tumor wet weight (mg) was obtained at termination of the study.
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RESULTS |
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vß3 and vß5 integrins, whereas HT29 cells expressed vß5 but not vß3 (Fig. 1)
. A375S2 and HUVECs but not HT29 cells stained with c7E3 Fab (Fig. 1)
. Therefore, we used A375S2 and HUVECs to determine the effects of vß3 blockade in tumor growth and angiogenesis.
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vß3 binds gelatin, fibrinogen, fibrin, and vitronectin (28
, 29)
, we questioned whether c7E3 Fab could block cell adhesion to these matrix proteins. c7E3 Fab completely inhibited adhesion of HUVECs and A375S2 cells to fibrinogen and gelatin, and it partially inhibited cell adhesion to vitronectin (Fig. 2A
; Table 1
). c7E3 Fab completely inhibited tumor cell adhesion to fibrin, whereas it partially blocked endothelial cell adhesion to fibrin (Fig. 2
; Table 1
), suggesting that endothelial cells use more than the vß3 receptor to adhere to fibrin. Because HT-29 cells do not express vß3-integrin, c7E3 Fab did not block cell adhesion (data not shown). Collectively, the data indicate that vß3 mediates cell adhesion (Figs. 2
and 3
; Table 1
).
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vß3-mediated cell adhesion, therefore we determined whether c7E3 Fab could block cell migration. c7E3 Fab dose dependently inhibited bFGF-stimulated endothelial cell migration (Figure 3)
. Interestingly, c7E3 Fab also inhibited migration of A375S2 when serum was used as a chemoattractant (Figure 4)
. Although c7E3 Fab only partially inhibited cell adhesion to vitronectin (Figure 2)
, it completely blocked cell migration towards this matrix protein. Similar results were obtained with LM609, P1F6 and the combination of both antibodies (Table 1)
. 10E5 did not block migration of A375S2 cells, suggesting that GPIIb/IIIa is not functionally expressed in this tumor cell line. These findings suggest that endothelial and melanoma cells primarily use vß3 integrin to migrate towards vitronectin, and c7E3 Fab can inhibit both bFGF and serum stimulated cell migration.
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, suggesting the involvement of at least vß3 in this process. Similarly, c7E3 Fab dose-dependently inhibited tumor cell invasion through fibrin. P1F6 was only partially effective at inhibiting tumor cell invasion, and no enhanced inhibition was observed when it was combined with LM609, suggesting that vß5 is involved to a lesser degree than vß3 in tumor cell invasion. Irrelevant IgG and a mAb directed to platelet GPIIb/IIIa (10E5) served as negative controls. Collectively, these data suggest that blockade of vß3 by c7E3 Fab can effectively block invasion of human melanoma cells.
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. c7E3 Fab inhibited cell proliferation by inducing apoptosis of proliferating endothelial cells (Fig. 6B)
. No effect of the drug was observed on quiescent endothelial cells, suggesting that vß3 function is only essential for proliferating endothelial cells. These findings indicate that c7E3 Fab inhibits endothelial cell proliferation in response to serum and bFGF, suggesting that vß3 plays a central role in mediating endothelial cell proliferation.
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. Isotype-matched irrelevant mouse and human antibodies served as negative controls.
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indicates that PRP stimulated endothelial sprouting to a greater extent than PPP, suggesting that platelets can stimulate angiogenesis. c7E3 Fab completely inhibited PRP-stimulated endothelial cell sprouting; however, because c7E3 Fab also blocks vß3, it was difficult to interpret whether platelet GPIIb/IIIa was involved in platelet-stimulated endothelial sprouting. The involvement of GPIIb/IIIa was demonstrated by the observation that 10E5, a mAb to GPIIb/IIIa, completely blocked gel-filtered platelet-stimulated endothelial cell sprouting (Fig. 7D)
. Inhibition of both endothelial vß3 and platelet GPIIb/IIIa receptor inhibited endothelial sprouting that was stimulated by either platelets or by angiogenic factors contained in plasma such as bFGF.
c7E3 Fab Inhibits Experimental Metastasis of Human Melanoma Tumors.
Earlier studies indicated that m7E3 F(ab')2 recognizes rat integrins but not murine integrins, and it blocks experimental metastasis of mouse tumor cells in a rat (15)
. The proposed antimetastatic mechanism that explains these results is that the antibody blocks the host (rat) platelet GPIIb/IIIa and vß3 integrins, thereby preventing seeding of the murine tumor cells in the lung endothelium. To test if blockade of tumor cell expressed vß3 without inhibiting host integrins could inhibit lung metastasis, we chose a lung colonization model of human melanoma metastasis in SCID mice. In this model, c7E3 Fab only binds to the human tumor cell expressed integrin but not to the host (mouse) integrin. A single pretreatment of human melanoma HT168M1 cells with c7E3 Fab (2.5 µg/ml) significantly decreased the number and size of tumor colonies in the mouse lung (Fig. 8)
. These results collectively suggest that blockade of tumor cell vß3 can provide antimetastatic benefit by blocking tumor cell-platelet, tumor cell-endothelium, and platelet-endothelium interactions.
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vß3 and platelet GPIIb/IIIa integrins were involved in angiogenesis in vivo. c7E3 Fab does not cross-react with mouse or rat integrins; however, m7E3 F(ab')2 cross-reacts with rat integrins (26)
; therefore, we evaluated its antiangiogenic activity in vivo using a bFGF-stimulated Matrigel plug angiogenesis model. m7E3 F(ab')2 at 6 mg/kg completely inhibited ex vivo rat platelet aggregation (26)
; therefore, this dose of the antibody was used to determine whether m7E3 F(ab')2 could inhibit bFGF-stimulated angiogenesis. The antibody was administered daily for 1 week, Matrigel plugs were removed, and hemoglobin content indicated that m7E3 F(ab')2 significantly inhibited angiogenesis in nude rats (Fig. 9)
. These data demonstrate that blockade of rat vß3 and GPIIb/IIIa can inhibit angiogenesis in rats.
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indicates that m7E3 F(ab')2 partially inhibited growth of human melanoma tumors in nude mice (P = 0.0002). These results provide direct evidence that blockade of human melanoma cell-expressed vß3 integrin, without inhibiting mouse ß3 integrins, can partially inhibit tumor growth in vivo.
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vß3 and the host ß3 integrins (platelet GPIIb/IIIa and endothelial vß3) may result in enhanced inhibition of tumor growth in vivo. To test this hypothesis, the same human melanoma A375S2 cells used in the mouse studies were used in a rat model where m7E3 F(ab')2 blocks multiple integrins: platelet GPIIb/IIIa, endothelial vß3, and tumor cell vß3. This model mimics the clinical situation where the relevant integrins are expressed by the tumor and the host. A dose of 6 mg/kg of m7E3 F(ab')2 was used, because at this concentration the antibody completely inhibited ex vivo rat platelet aggregation (26)
and inhibited angiogenesis in nude rats (Fig. 9)
. Two series of experiments were performed in nude rats. In the first experiment, antibody and tumor cells were coinoculated into nude rats, and antibody therapy was administered either 3x/wk or 5x/wk for the duration of the study. Tumor formation and growth were dramatically inhibited in both antibody treated groups (Fig. 11)
. Animals treated with m7E3 F(ab')2 3x/wk developed measurable tumors 10 days later compared with the control group, and animals treated with the antibody 5x/wk did not develop tumors throughout the course of the study. Only 50% of the animals in the 3x/wk antibody treatment group developed tumors, and these tumors were significantly smaller compared with the control group (P < 0.001; Fig. 11
).
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150 mm3, and animals were randomized and then treated with m7E3 F(ab')2 (3 mg/kg, daily i.p. administration for the duration of the study) or vehicle control. Assays performed on terminal blood samples demonstrated that m7E3 F(ab')2 inhibited ex vivo platelet aggregation and did not cause thrombocytopenia in any of the animals (data not shown; Ref. 26
). m7E3 F(ab')2 was administered more frequently in rats compared with the mice, because it has a much shorter circulating half-life in rats. Approximately 150 µg/ml of m7E3 F(ab')2 was detected in the mouse serum the day after the last dose, whereas 3 µg/ml of circulating antibody was measured in the rat serum the day after the last dose (data not shown), suggesting that the antibody has a shorter circulating half-life in rats compared with mice. Yet, m7E3 F(ab')2 completely prevented growth of preformed tumors in the rat model (Fig. 11)
but only had a partial effect in the mouse model (Fig. 10)
. Collectively, these data provide evidence that combined antitumor and antiangiogenic targeting by m7E3 F(ab')2 is superior than antitumor targeting alone. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 participate in angiogenesis, tumor growth, and metastasis. Combined blockade of these receptors on three cell types was more effective at inhibiting tumor growth when compared with blockade of a single integrin receptor. c7E3 Fab, which binds with equivalent affinity to platelet GPIIb/IIIa and vß3, inhibited human melanoma and endothelial cell adhesion, migration, invasion, and lung colonization of human melanoma cells in nude mice. In addition, m7E3 F(ab')2 inhibited angiogenesis and growth of human melanoma tumors in vivo. Collectively, our results suggest that c7E3 Fab and m7E3 F(ab')2 with their multireceptor activity possess antiangiogenic and antimetastatic properties.
The requirement of platelets in hematogeneous spread of tumor cells was recognized almost 30 years ago and is reviewed in detail elsewhere (1
, 31
, 32)
. When metastatic tumor cells are shed into the blood circulation, they rapidly recruit platelets to form tumor cell-platelet aggregates, which results in a transient decrease in circulating platelet count (15
, 33)
. Several preclinical animal models have demonstrated that blockade of platelet GPIIb/IIIa integrin inhibits lung colonization of tumor cells (15
, 34)
. By blocking tumor cell-expressed vß3 integrin without inhibiting platelet function, c7E3 Fab, in this study, dramatically inhibited the metastatic ability of human melanoma cells in SCID mice. In this animal model, c7E3 Fab did not cross-react with mouse platelets; therefore, the results demonstrate that human melanoma cell-expressed vß3 integrin participates in lung metastasis.
In addition to facilitating metastasis, platelets can also stimulate tumor-induced angiogenesis. Platelet granules contain a variety of angiogenic factors such as VEGF that are rapidly secreted on platelet activation. Previous studies have revealed that an increase in platelet count is an indicator of poor prognosis in patients with lung and ovarian carcinoma (18, 19, 20) , and platelet-secreted VEGF is inversely correlated with survival of patients with cancer (35) . Pinedo et al. (12) have postulated that a true antiangiogenic agent must target platelets, but direct evidence to support this hypothesis is lacking. Our data provided novel evidence to support this hypothesis and demonstrate that platelets stimulated endothelial sprouting in vitro, and c7E3 Fab inhibited this sprouting. Earlier studies demonstrated that c7E3 Fab inhibited secretion of VEGF from platelets (15 , 16) ; therefore, it is conceivable that VEGF could be contributing to platelet-stimulated endothelial cell sprouting. Platelet-secreted VEGF is probably not the only factor that stimulates angiogenesis, because platelets also contain other growth factors such as TGF-ß and thrombin (13) that can stimulate endothelial cell sprouting. c7E3 Fab inhibits platelet-endothelial binding (36) and secretion of platelet granules containing growth factors (13) , which may explain why c7E3 Fab completely blocked platelet-stimulated endothelial cell sprouting. This is an important finding, because it demonstrates that not just tumor cells, but host cells can contribute to tumor angiogenesis.
In addition to blocking platelet GPIIb/IIIa, abciximab also inhibits vß3 function. Because vß3 is an essential receptor for angiogenesis, c7E3 Fab can inhibit endothelial cell proliferation, adhesion, migration, invasion, and induce apoptosis of proliferating cells. Human melanoma cell-expressed vß3 participates in cell adhesion, migration, and invasion, and increase in ß3 integrin inversely correlates with survival of melanoma patients (9
, 10)
. c7E3 Fab completely inhibited vß3-mediated human melanoma cell adhesion, spreading, and invasion. More importantly, m7E3 F(ab')2 has direct antitumor activity in vivo. Blockade of human melanoma cell-expressed vß3 by m7E3 F(ab')2, without blocking host cell integrin, resulted in a partial inhibition of tumor growth in nude mice. Interestingly, combined blockade of host integrins (platelet GPIIb/IIIa and endothelial vß3) and tumor cell-expressed vß3 completely prevented tumor formation and growth in nude rats. In this rat xenograft model, which mimics the clinical situation, combined antiangiogenic and antitumor activity of m7E3 F(ab')2 was superior at inhibiting tumor growth when compared with its antitumor activity in the mouse xenograft model.
Tumor growth and angiogenesis involves multiple integrin receptors; therefore, monospecific vß3 antagonists may not be effective at inhibiting tumor progression. Agents that block multiple integrin receptors may be more effective at inhibiting tumor growth and angiogenesis. This study provides novel evidence that combined inhibition of vß3 and GPIIb/IIIa may be an effective approach to inhibiting tumor growth, angiogenesis, and metastasis.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Centocor, Inc., 200 Great Valley Parkway, Malvern, PA 19355. Phone: (610) 651-6809; Fax: (610) 651-7363; E-mail: trikham{at}cntus.jnj.com 
2 The abbreviations used are: c, chimeric; m, murine; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; FBS, fetal bovine serum; mAb, monoclonal antibody; HUVEC, human umbilical vein endothelial cell; bFGF, bovine basic fibroblast growth factor; MC, microcarrier; PRP, platelet-rich plasma; PPP, platelet-poor plasma; 3x/wk, three times per week; 5x/week, five times per week; SCID, severe combined immuno-deficient.
Received 11/ 1/01. Accepted 3/18/02.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 integrins. Circulation, 98: 1085-1091, 1998.v integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J. Clin. Investig., 103: 1227-1241, 1999.[Medline]
v integrins. Science (Wash. DC), 270: 1500-1502, 1995.vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell, 79: 1157-1164, 1994.[Medline]
IIbß3 integrin is involved in invasion of human melanoma cells. Cancer Res., 57: 2522-2528, 1997.IIbß3 integrin. Cancer Res., : 5071-5078, 1996.
vß3, blocks in vivo thrombus formation in in vitro angiogenesis. Thromb. Haemostasis, 85: 896-902, 2001.[Medline]
IIbß3 integrin in prostate cancer metastasis. Prostate, 35: 185-192, 1998.[Medline]
(v) ß3) mediates platelet adhesion to the luminal aspect of endothelial cells: implications for reperfusion in acute myocardial infarction. Circulation, 96: 1809-1818, 1997.This article has been cited by other articles:
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  H. Laubli, K.-S. Spanaus, and L. Borsig Selectin-mediated activation of endothelial cells induces expression of CCL5 and promotes metastasis through recruitment of monocytes Blood, November 12, 2009; 114(20): 4583 - 4591. [Abstract] [Full Text] [PDF]
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 D. F. Meoli and R. J. White Thrombin induces fibronectin-specific migration of pulmonary microvascular endothelial cells: requirement of calcium/calmodulin-dependent protein kinase II Am J Physiol Lung Cell Mol Physiol, October 1, 2009; 297(4): L706 - L714. [Abstract] [Full Text] [PDF]
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 O. ENGEBRAATEN, M. TRIKHA, S. JUELL, S. GARMAN-VIK, and O. FODSTAD Inhibition of In Vivo Tumour Growth by the Blocking of Host {alpha}v{beta}3 and {alpha}IIb{beta}3 Integrins Anticancer Res, January 1, 2009; 29(1): 131 - 137. [Abstract] [Full Text] [PDF]
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 N. Li Platelet-lymphocyte cross-talk J. Leukoc. Biol., May 1, 2008; 83(5): 1069 - 1078. [Abstract] [Full Text] [PDF]
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 T. J. MacDonald, C. F. Stewart, M. Kocak, S. Goldman, R. G. Ellenbogen, P. Phillips, D. Lafond, T. Y. Poussaint, M. W. Kieran, J. M. Boyett, et al. Phase I Clinical Trial of Cilengitide in Children With Refractory Brain Tumors: Pediatric Brain Tumor Consortium Study PBTC-012 J. Clin. Oncol., February 20, 2008; 26(6): 919 - 924. [Abstract] [Full Text] [PDF]
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 A. Cumashi, N. A. Ushakova, M. E. Preobrazhenskaya, A. D'Incecco, A. Piccoli, L. Totani, N. Tinari, G. E. Morozevich, A. E. Berman, M. I. Bilan, et al. A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds Glycobiology, May 1, 2007; 17(5): 541 - 552. [Abstract] [Full Text] [PDF]
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 Y. Yu, E. Davicioni, T. J. Triche, and G. Merlino The Homeoprotein Six1 Transcriptionally Activates Multiple Protumorigenic Genes but Requires Ezrin to Promote Metastasis Cancer Res., February 15, 2006; 66(4): 1982 - 1989. [Abstract] [Full Text] [PDF]
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 A. Abdollahi, D. W. Griggs, H. Zieher, A. Roth, K. E. Lipson, R. Saffrich, H.-J. Grone, D. E. Hallahan, R. A. Reisfeld, J. Debus, et al. Inhibition of {alpha}v{beta}3 Integrin Survival Signaling Enhances Antiangiogenic and Antitumor Effects of Radiotherapy Clin. Cancer Res., September 1, 2005; 11(17): 6270 - 6279. [Abstract] [Full Text] [PDF]
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 J. J. Bergh, H.-Y. Lin, L. Lansing, S. N. Mohamed, F. B. Davis, S. Mousa, and P. J. Davis Integrin {alpha}V{beta}3 Contains a Cell Surface Receptor Site for Thyroid Hormone that Is Linked to Activation of Mitogen-Activated Protein Kinase and Induction of Angiogenesis Endocrinology, July 1, 2005; 146(7): 2864 - 2871. [Abstract] [Full Text] [PDF]
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J. S. Palumbo, K. E. Talmage, J. V. Massari, C. M. La Jeunesse, M. J. Flick, K. W. Kombrinck, M. Jirouskova, and J. L. Degen Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells Blood, January 1, 2005; 105(1): 178 - 185. [Abstract] [Full Text] [PDF]
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 N Cordes and V Meineke Modification of the cellular radiation survival and proliferation response by cell-matrix interactions: implications for integrin targeting in therapeutic approaches for radiation accident patients Br. J. Radiol., January 1, 2005; Supplement_27(1): 152 - 156. [Abstract] [Full Text] [PDF]
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 H. E. Turner, A. L. Harris, S. Melmed, and J. A. H. Wass Angiogenesis in Endocrine Tumors Endocr. Rev., October 1, 2003; 24(5): 600 - 632. [Abstract] [Full Text] [PDF]
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 S. E. Bojesen, A. Tybjaerg-Hansen, and B. G. Nordestgaard Integrin {beta}3 Leu33Pro Homozygosity and Risk of Cancer J Natl Cancer Inst, August 6, 2003; 95(15): 1150 - 1157. [Abstract] [Full Text] [PDF]
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M. M. Fuster, J. R. Brown, L. Wang, and J. D. Esko A Disaccharide Precursor of Sialyl Lewis X Inhibits Metastatic Potential of Tumor Cells Cancer Res., June 1, 2003; 63(11): 2775 - 2781. [Abstract] [Full Text] [PDF]
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