Malignant transformation in melanocytes is associated with increased production of procoagulant microvesicles Luize G. Lima1; Andreia S. Oliveira1; Luiza C. Campos2; Martin Bonamino2; Roger Chammas3; Claudio C. Werneck4; Cristina P. Vicente5; Marcello A. Barcinski6; Lars C. Petersen7; Robson Q. Monteiro1
1Institute of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 2Division of Experimental Medicine, National Cancer Institute, Rio de Janeiro, Brazil; 3Laboratory of Experimental Oncology, School of Medicine, University of São Paulo, São Paulo, Brazil; 4Department of Anatomy, Cell Biology and Physiology and Biophysics, Institute of Biology, University of Campinas, São Paulo, Brazil; 5Department of Biochemistry, Institute of Biology, University of Campinas, São Paulo, Brazil; 6Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil; 7Biopharmaceuticals Research Unit, Novo Nordisk A/S, Måløv, Denmark
Summary Shedding of microvesicles (MVs) by cancer cells is implicated in a var-iety of biological effects, including the establishment of cancer-associ-ated hypercoagulable states. However, the mechanisms underlying ma-lignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initi-ator protein tissue factor (TF). On the other hand, tumour- and mel-anocyte-derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial throm-
Correspondence to: Robson Q. Monteiro Instituto de Bioquímica Médica/CCS/UFRJ Avenida Bauhínea 400 (Bloco H, segundo andar, sala 08) Cidade Universitária, Ilha do Fundão Rio de Janeiro, 21941–590, Brazil Tel.: +55 21 2562 6782, Fax: +55 21 2270 8647 E-mail: [email protected]
bosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plas-ma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs pro-duced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagu-lation-related protumoural responses.
Financial support: This research was supported by the Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro Carlos Chagas Filho (FAPERJ), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP – 2007/01112–6 and 2009/00950–3), and Fundação do Câncer. Received: March 2, 2011 Accepted after major revision: June 30, 2011 Prepublished online: July 28, 2011
An important link between malignancy and hypercoagulant states has long been established (1, 2). In fact, the occurrence of cancer is commonly associated with a variety of clinical thrombotic syn-dromes including local and systemic venous and arterial throm-boses (3). Also, thrombosis is often diagnosed as the first clinical manifestation of a tumour and different authors demonstrated a significant correlation between the incidence of thromboembolic events and a worse prognosis of the neoplastic disease (4, 5), which supports the idea that the activation of blood coagulation system contributes to tumour aggressiveness and vice versa. It is note-worthy, however, that changes in the haemostatic balance during cancer progression transcends thrombosis occurrence, as evidenc-
ed by abnormalities on in vitro coagulation tests found in more than 90% of oncologic patients, irrespective to their thrombotic status (6).
Microvesicles (MVs) are membrane fragments that can be shed from the surface of various cell types under conditions such as cell activation and apoptosis (7) being this phenomenon closely as-sociated with phosphatidylserine (PS) exposure (8). The release of MVs from surface membranes generates circular structures with relative large size (0.1–1 μm diameter) and heterogeneous com-position, which reflects their cellular origin. Although found in the blood and other biological fluids of normal healthy individuals, MVs are thought to be implicated in the pathogenesis of a number of disorders. It includes cancer (9, 10), since elevated MV levels are observed in these patients. In this context, a wide variety of biologi-
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2 Lima et al. Release of procoagulant microvesicles by melanoma cells
cal effects related to MVs have been described, including diverse aspects of tumour biology, such as angiogenesis, metastasis and modulation of immune responses (11, 12).
A number of reports have suggested a potential role for circu-lating MVs in establishing a thrombophilic state in cancer patients (13–16). Procoagulant properties of MVs include surface exposure of tissue factor (TF), the central trigger of the coagulation cascade, (17) and PS (18), which provides a negatively charged surface required for the assembly of catalytic active coagulation complex-es (tenase and prothrombinase). TF has been long implicated in the in vitro procoagulant activity of malignant cell lines, being its expression frequently correlated with cell aggressiveness (19, 20). In addition, TF has been shown to be overexpressed in samples of patients with various types of neoplasias, including melanoma and most epithelial carcinomas (21–23), and its expression has often been found to correlate with tumour grade, increased angiogenesis and decreased survival (24, 25). Therefore, it has been suggested that TF may play a causative role in prothrombotic complications in cancer patients.
Although it was recently demonstrated that tumour-derived MVs are prothrombotic in vivo (26), there are few studies that cor-relate acquisition of the malignant phenotype with changes in the procoagulant and prothrombotic pattern of MVs. To further ad-dress this question, we investigated the in vitro procoagulant and the in vivo prothrombotic properties of MVs produced by a non-tumourigenic melanocyte-derived cell line as compared to its tu-mourigenic melanoma counterpart. Thrombotic events are not as common in melanoma patients as in other cancer types such as pancreas adenocarcinoma or glioblastoma (5). However, it was previously demonstrated that the procoagulant status of mel-anoma cells is determinant for tumour aggressiveness (19). In this context, a paired model in which the malignant cell line derives from the non-malignant one could be an excellent tool to evaluate a possible correlation between malignant transformation and the production of procoagulant MVs.
Our results suggest that release of MVs may be a key feature as-sociated with tumour progression. In addition, it offers new in-sight into the possible involvement of tumour-derived MVs in the establishment of cancer-associated prothrombotic states, indicat-ing an important role for TF in this process.
Material and methods
Reagents
Human factor Xa (FXa) was purchased from Calbiochem (San Diego, CA, USA). Human factor Va (FVa) and murine factor X (FX) were purchased from Haematological Technologies Inc. (Essex Junction, VT, USA). Murine factors VIIa (FVIIa) and active site-blocked VIIa (FFR-FVIIa) were expressed and purified as pre-viously described (27). Chromogenic substrates for thrombin (S-2238, H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroani-line dihydrochloride) and FXa (S-2765, N-α-benzyloxycar-
bonyl-D-Arg-Gly-Arg-p-nitroanilide) were purchased from Chromogenix AB (Molndal, Sweden). Ethylenediaminetetraacetic acid (EDTA), polyethyleneglycol and human placental annexin-V were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Cell culture
The murine melanocyte cell line melan-a (28) was grown at 37ºC in a humidified, 5% CO2 atmosphere in culture flasks in Dulbec-co´s Modified Eagle Medium (DMEM; GibcoBRL), pH 6.9, con-taining foetal bovine serum (FBS) 10% (v/v) and supplemented with 2.4 g/l HEPES, 3.7 g/l sodium bicarbonate, 125 mg/l sodium dihydrogen phosphate, 110 mg/l sodium pyruvate, 100,000 U/l penicillin, 100 mg/l streptomycin, 2 mM L-glutamine, 55 μM β-mercaptoethanol, and 200 nM 12-o-tetradecanoyl PMA (Sigma). The melanoma cell line derived from melan-a, Tm1 (29), and the murine melanoma B16F10 were cultured in the same con-ditions, except for PMA. After separation of cell culture supernat-ant for posterior MVs purification, cells were detached with Hank’s solution containing 10 mM HEPES and 0.2 mM EDTA, spun at 350 x g for 7 minutes (min), resuspended in DMEM con-taining 10% FBS (supplemented as previously described) and transferred to another culture flask (or maintained at 4oC until utilisation).
MVs purification from cell culture supernatants
Cell culture supernatants were consecutively centrifuged at 800 x g for 10 min and at 20,000 x g for 20 min, always at 4ºC. The final pel-let was then washed once, resuspended in phosphate-buffered sa-line (PBS) and stored at – 80ºC until utilisation. MVs were quanti-fied by counting in a BD FACScalibur™ Flow Cytometer (Becton Dickinson, San Jose, CA, USA). Although the limited efficacy of flow-cytometric analysis for detecting low-size MV populations has been clearly demonstrated, it remains the most used technique to detect and enumerate MVs, as indicated by a previous Vascular Biology Subcomittee survey of the International Society on Thrombosis and Haemostasis (30).
Flow-cytometric analyses
For surface PS detection, MVs were resuspended in PBS containing 10% adult bovine serum (v/v) and incubated for 15 min at room temperature with 3G4 murine monoclonal antibody against phos-phatidylserine (previously described by Ran et al. (31), and kindly donated by Dr. Philip E. Thorpe, Department of Pharmacology and Simmons and Hamon Cancer Centers, University of Texas Southwestern Medical Center at Dallas, TX, USA), diluted 1:200. Samples were extensively washed by repeated cycles of centrifu-
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gation and resuspension in PBS and then analysed using a BD FACScalibur™ Flow Cytometer (Becton Dickinson). Although 3G4 was shown to specifically bind anionic phospholipids in the presence of serum or β2-glycoprotein I (31–35), and PS is con-sidered its primary target, it is noteworthy that analysis of PS expo-sure by this antibody was confirmed by staining with annexin-V, which also binds PS. MVs were incubated for 15 min at room tem-perature with fluorescein-conjugated annexin-V (Santa Cruz Bio-technology Inc., Santa Cruz, CA, USA) diluted 1:100 in binding buffer (10 mM HEPES, 150 mM NaCl, 2.5 mM CaCl2).
Cells and MVs were also resuspended in PBS containing 1% bovine serum albumin (BSA) and incubated for 30 min at 4ºC with a rabbit polyclonal antibody against murine tissue factor (200 μg/ml; American Diagnostica, Stamford, CT, USA) or normal rabbit IgG (200 μg/ml; Santa Cruz Biotechnology Inc.). Samples were ex-tensively washed by repeated cycles of centrifugation and resus-pension in PBS to remove unbound antibody. After post-fixation with 1% paraformaldehyde, samples were incubated for 30 min at 4ºC with 1 μg/ml of a phycoerythrin-conjugated anti-rabbit IgG (Santa Cruz Biotechnology Inc.). Cells or MVs were then washed again and analysed using a BD FACScaliburTM Flow Cytometer (Becton Dickinson).
In all cases, data were analysed by the BD CellQuest Pro™ soft-ware (Becton Dickinson).
In vitro activation of plasma coagulation
MVs procoagulant activity was measured by a clotting assay em-ploying normal pooled murine platelet-poor plasma (PPP). Fifty microliters of cell or MVs ressuspended in PBS at different concen-trations were added to 50 μl of PPP containing 3.8% sodium ci-trate diluted 1:9 (v/v). After 1 min incubation at 37 ºC, 100 μl of 6.25 mM CaCl2 was added and the clotting times were recorded on a KC-4 coagulometer (Amelung Ltd, Diagnostic Grifols, Barce-lona, Spain).
FX activation as followed by hydrolysis of chromogenic substrate
Activation of FX by FVIIa was performed in 50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 1 mg/ml BSA, pH 7.5 (HEPES-BSA buffer), as follows: FVIIa (1 nM, final concentration) was incu-bated with cells (5 x 104/ml) or MVs (5 x 103/ml) for 10 min at 37°C in HEPES-BSA buffer. Reaction was initiated by addition of FX (135 nM, final concentration) and aliquots of 25 μl were removed after 30 min into microplate wells containing 25 μl of Tris-EDTA buffer (50 mM Tris-HCl, 150 mM NaCl, 20 mM EDTA, 1 mg/ml polyethyleneglycol 6000, pH 7.5). After the addition of 50 μl of 200 μM S-2765 prepared in Tris-EDTA buffer, absorbance at 405 nm was recorded at 37°C for 30 min at 6-second (s) intervals using a Thermomax Microplate Reader (Molecular Devices, Menlo Park,
CA, USA). Velocities (mOD/min) obtained in the first minutes of reaction were used to calculate the amount of FXa formed, as com-pared to a standard curve using known enzyme concentrations. Appropriate controls performed in the absence of FVIIa or MVs showed no significant formation of FXa.
The effect of the active site-blocked TF antagonist FFR-FVIIa on FXa formation was also tested by incubating cells or MVs with a mixture of FVIIa (1 nM, final concentration) and FFR-FVIIa (10 nM, final concentration) for 10 min prior to incubation with FX.
Prothrombin activation as followed by hydrolysis of chromogenic substrate
Activation of prothrombin by the prothrombinase complex (FXa/FVa) was performed in HEPES-BSA buffer, using a discontinuous assay, as previously described (36). FXa (10 pM, final concen-tration) was incubated with FVa (1 nM, final concentration) in the presence of MVs (3.6 x 104/ml) for 2 min at 37ºC. Reaction was initiated by addition of prothrombin (500 nM, final concen-tration) and aliquots of 10 μl were removed every 1 min into microplate wells containing 40 μl of Tris-EDTA buffer. After addi-tion of 50 μl of 200 μM S-2238 prepared in Tris-EDTA buffer, ab-sorbance at 405 nm was recorded at 37ºC for 20 min at 6-s intervals using a Thermomax Microplate Reader (Molecular Devices). Vel-ocities (mOD/min) obtained in the first minutes of reaction were used to calculate the amount of thrombin formed, as compared to a standard curve using known enzyme concentrations. Appropri-ate controls performed in the absence of FXa/FVa or MVs showed no significant formation of thrombin.
The effect of annexin-V on thrombin formation was also tested as follows: MVs (3.6 x 104/ml) were incubated with annexin-V (10 nM) for 5 min at 37oC in HEPES-BSA buffer. Then, MVs were in-cubated with FXa (10 pM)/FVa (1 nM) for 2 min, at 37oC, followed by addition of prothrombin (500 nM). Aliquots of 10 μl were re-moved after 5 min, and delivered to microplate wells containing 40 μl of Tris-EDTA buffer. The amount of thrombin formed was evaluated as described above using its chromogenic substrate S-2238.
RNA isolation and reverse transcriptase polymerase chain reaction
RNA was isolated from Tm1 or melan-a cells (2.5 x 105) using the Trizol reagent (Invitrogen) following the manufacturer’s instruc-tions. Total RNA (1 μg) was reversely transcribed into cDNA using cDNA Archive (Applied Biosystems, Carlsbad, CA, USA) accord-ing to the manufacturer’s protocol. An aliquot of the reaction mix-ture was further used for polymerase chain reaction (PCR) am-plification. Primers used were: TF (forward: 5’-CCTCGGA-CAGCCAGTAATTC-3’ and reverse: 5’-GTGTGAGCGTTAGCGGCTTC-3’) and β-actin (forward:
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4 Lima et al. Release of procoagulant microvesicles by melanoma cells
5’-ACACCCGCCACCAGTTCGCC-3’ and reverse: 5’-GCA-CAGTGTGGGTGACCCCGTCTC-3’). Thermocycling conditions were as follows: 35 cycles of 95°C for 30 s (denaturation), 55°C for 45 s (annealing), and 72°C for 45 s (extension). PCR products were separated by 1% agarose gel electrophoresis and further stained with ethidium bromide.
Generation of the GFP-B16F10 cell line
Lentiviral vectors were produced as previously described (35), using the same methodology but the 3rd generation lentivirus sys-tem containing the following plasmids: pMD-G (expressing the G protein of Vesicular Stomatitis Virus- 6 μg/plate), pRRLSIN.cPPT.EF1a-GFP.WPRE (carrying the enhanced green fluorescent protein under the control of the Elongation Factor 1 alpha promoter – 20 μg/plate), pMDLg/pRRE (expressing HIV’s gag and pol proteins – 10 μg/plate) and pRSV.REV (expressing HIV’s rev protein – 5 μg/plate). Supernatants containing viral vec-tors were filtered (0.22 μm filter), concentrated by centrifugation at 33,000 x g for 30 min and frozen at –80ºC. The vector titer was determined by transducing 293T cells and evaluating GFP ex-pression by flow cytometry (BD FACScalibur™ Flow Cytometer; Becton Dickinson). For transduction, B16F10 cells were plated and transduced 18 hours (h) later by spinning the cells for 45 min at 400 x g twice with vector-containing medium at a multiplicity of infection (MOI) of 100, with a 10 h interval. Cells were kept in cul-ture for two aditional weeks without viral vectors to certify that GFP expression was stable and cells were virtually 100% GFP-posi-tive.
Photochemically induced carotid artery thrombosis
Carotid artery thrombosis was induced as previously described (38). Adult mice (8–12 weeks of age; 22–25 g body weight) were an-esthetised with intramuscular injection of ketamine 100 mg/kg and xylasine 16 mg/kg, secured in the supine position, and placed under a dissecting microscope. The right common carotid artery was isolated through a midline cervical incision, and an ultrasonic
flow probe (Model 0.5 PSB; Transonic Systems, Ithaca, NY, USA) was applied. A 1.5-mW, 540-nm laser beam (Melles Griot, Carls-bad, CA, USA) was applied to the artery from a distance of 6 cm. Five minutes before induction of thrombosis, animals were in-jected in the tail vein with MVs (2.5 x 104/ml for Tm1 or melan-a MVs; 1.0 x 105/ml for B16F10 or GFP+B16F10 MVs) or saline (control). Rose bengal dye (3’,4’,5’,6’-tetrachloro-2,4,5,7-tetraio-dofluorescein; Fisher Scientific, Fair Lawn, NJ, USA) at a dose of 50 mg/kg body weight was then injected into the lateral tail vein, and blood flow was monitored continuously. The occlusion time was taken as the interval between injection of rose bengal dye and com-plete and stable (> 5 min) cessation of flow.
Fluorescence microscopy
Frozen sections of the carotid artery were fixed with acetone for 20 min at 4°C and examined with an Olympus BX60 microscope equipped with 10x ocular and 10 or 20x objective lenses. Images were acquired with a Olympus 32–0044C-228, Q-color 3 cooled, RTV (made in Canada for Olympus America by QImaging) digital camera and QCapture 2.81.0 (2005) software.
MVs isolation from cell-free plasmas of melanoma or melanocyte-injected mice
All animal experiments were performed under approved protocols of the institutional animal care and use committee. Melan-a or Tm1 cells (5 x 105) resuspended in 100 μl DMEM were injected subcutaneously into the dorsal area of six- to eight-week-old C57BL/6 isogenic mice. After 17 days, every animal injected with melanoma cells presented a tumour mass > 100 mm3, while mel-anocyte cells were non-tumourigenic. Mice were then anesthe-tised, and blood (1 ml) was obtained by cardiac puncture in the presence of 3.8% sodium citrate diluted 1:9 (v/v). Cell-free plas-mas were separated by centrifugation of blood at 3,500 rpm for 15 min, and samples were processed according to the same protocol of MVs purification from cell culture supernatants (20,000 x g for 20 min at 4ºC). MVs present in the final pellet was then analysed and quantified by counting in a BD FACScalibur™ Flow Cytometer (Becton Dickinson), and stored at – 80ºC until utilisation.
For melanoma-associated antigen (MAA) detection, MVs were resuspended in PBS containing 1% BSA and incubated for 1 h at 4°C with a murine monoclonal antibody against MAA (MM29B6 (39), kindly donated by Dr. Elieser Gorelik, University of Pitts-burgh Cancer Institute, Hillman Cancer Center), hybridoma supernatant diluted 1:1. After extensive washing by repeated cycles of centrifugation and resuspension in PBS to remove unbound antibody, MVs were incubated with a fluorescein-conjugated anti-mouse IgG (2 μg/ml, Santa Cruz Biotechnology Inc.), for 1 h at 4°C. MVs were then washed again and analysed using a BD FACScalibur™ Flow Cytometer (Becton Dickinson).
Table 1: Comparison between Tm1 melanoma and melan-a mel-anocyte cells of some phenotypic characteristics.
Chacteristics* melan-a Tm1
Tumourigenic when injected into syngeneic mice No Yes
Contact-inhibited / anchorage-dependent growth Yes / Yes No / No
Dependence on phorbol ester as growth factor Yes No
Morphology Epithelioid Smaller and bipolar cells
*Summarised from Correa et al., 2005 (40) and Oba-Shinjo et al., 2006 (29).
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5 Lima et al. Release of procoagulant microvesicles by melanoma cells
Statistical analysis
One-way ANOVA or unpaired t-test were performed using the In-Stat software (GraphPad, La Jolla, CA, USA).
Results
Malignant transformation increases the shedding of MVs in vitro
In order to investigate the procoagulant properties of tumour cell-derived MVs, we applied the melanoma cell line Tm1 and com-pared it to the parental melanocyte-derived cell line, melan-a. The Tm1 lineage exerts several phenotypic characteristics associated with malignant transformation in contrast to what has been ob-served for melan-a cells (�Table 1).
In this context, we first analysed the spontaneous shedding of MVs by both cell lines in vitro. By differential centrifugation we were able to isolate MVs from the supernatants of both Tm1 and melan-a cell cultures. Cells were cultivated for 24 h (when both cell lines still presented the same growth rate [40]) in the same condi-tions, including the presence of PMA. Flow-cytometric analyses showed a similar forward vs. side scattering (FSC x SSC) profile for both MVs (�Fig. 1A). However, the rate of MVs production was significantly higher in the tumourigenic cell line Tm1 (�Fig. 1B), resulting in a two-fold greater accumulation of MVs in melanoma cultures during the 24 h assay period. It is important to note that all flow-cytometric stainings presented in this study, for MVs ob-tained in vitro, have been gated as indicated on FSC x SSC plot shown in �Figure 1A.
Procoagulant activity of MVs is elevated upon malignant transformation
Using one stage clotting-assay, we observed that melanoma-de-rived MVs shortened the coagulation time of murine plasma at a lower concentration range than did melan-a MVs (�Fig. 2A). Since TF significantly contributes to activation of plasma coagu-lation, and previous reports have shown that a number of mel-anoma cell lines express TF (19, 41) in a cell aggressiveness corre-lated manner (42), its expression in Tm1 and melan-a MVs was examined. Flow-cytometric analyses showed that Tm1 melanoma MVs express higher levels of TF antigen on their surface, whereas a less pronounced TF expression was detected on melanocyte-de-rived MVs under identical assay conditions (�Fig. 2B). Tm1 MVs present an approximately two-fold increase in global geometric mean of fluorescence intensity compared to melan-a (ΔMIF repre-sents the difference between stained and unstained samples). Ac-cumulation of melanoma MVs in a highly expressing TF subpopu-lation (indicated as S2 in �Fig. 2B) was also observed. Accord-ingly, a higher level of TF antigen was exposed on the surface of melanoma than on melanocyte cells (�Fig. 2B). Tm1 melanoma cells also showed a markedly increased TF gene expression when compared to melan-a cells as analysed by semi-quantitative reverse transcriptase-PCR analysis (�Fig. 2C).
Next, we investigated TF activity in both Tm1 and melan-a MVs. Specific enzymatic assays – FXa generation in the presence of FVIIa – showed that Tm1 MVs supported FX activation in a more efficient way than melan-a MVs. FX activation was strictly FVIIa-dependent (�Fig. 2D), indicating assembly of the extrinsic tenase complex (FVIIa/TF/FX). No significant FX activation was ob-served in the absence of MVs or in the absence of FVIIa (data not shown). In the same way, Tm1 cells supported FX activation more efficiently than melan-a ones (�Fig. 2E).
Taken together these results demonstrate that, compared to non-tumourigenic melanocyte cells, Tm1 melanoma cells shed a higher number of MVs with a generally higher level of TF exposure.
Figure 1: Shedding of MVs by melan-a and Tm1 cells. A) Example of Tm1– and melan-a-derived MVs analysed by flow cytometry in a dot plot of forward (FSC-H) vs. side (SSC-H) light scattering. Plots shown indicate one representative experiment. B) MVs were isolated from 24 h culture superna-
tants of melan-a and Tm1 cells, resuspended in equal PBS volumes and quantified by flow-cytometric counting. Bars represent mean ± SD of five in-dependent experiments.
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6 Lima et al. Release of procoagulant microvesicles by melanoma cells
Exposure of phosphatidylserine on MVs is not affected by malignant transformation
Previous studies have demonstrated that PS exposure on cell-de-rived MVs is obligatory for the assembly of membrane-dependent procoagulant complexes (18). We therefore investigated whether differential PS exposure could contribute for the differences in procoagulant properties of melanoma and melanocyte-derived MVs. Flow-cytometric analyses employing a monoclonal antibody against PS or annexin-V (see Materials and methods) indicated that PS exposure was essentially identical on MVs from melan-a and
Tm1 cells (see �Suppl. Fig. 1 available online at www.thrombosis-online.com). A more quantitative analysis was obtained by calcu-lating the mean fluorescence intensity for anti-PS labelling in each sample (�Fig. 3A). We further investigated the assembly of the prothrombinase complex (FVa/FXa/prothrombin), a process that is critically dependent on the presence of PS-rich anionic mem-branes. Consistently, both types of MVs supported prothrombin activation at similar rates (�Fig. 3B). Controls performed in the absence of MVs or in the absence of FXa and FVa showed no effi-cient thrombin formation. Finally, PS-dependency was confirmed by preincubation of MVs with annexin-V, a protein that blocks
Figure 2: Characterisation of procoagulant properties of Tm1 and melan-a MVs. A) Procoagulant activity of Tm1 and melan-a MVs. Black circle represents coagulation time of murine plasma alone. Each point represents mean ± SD of three independent experiments. *p < 0.001 relative to control plasma (Student’s t-test). B) Flow-cytometric analysis of TF expression in Tm1 and melan-a MVs as compared to their parental cells. Black lines represent labelling with anti-murine TF polyclonal antibody followed by phycoerythrin-conjugated secondary antibody. Gray regions represent cells labeled with normal rabbit IgG. ΔMIF represents geometric mean of fluorescence inten-sity difference between stained and unstained samples. C) Reverse transcrip-
tase polymerase chain reaction (RT-PCR) analysis of TF gene expression in Tm1 and melan-a cells. Assay was performed as described in Materials and methods. D and E) Assembly of extrinsic tenase complex on Tm1 and melan-a MVs (D) or cells (E). Activation of murine FX by murine FVIIa was examined in the presence of MVs or cells. Inhibitory effect of an active site-blocked FVIIa derivative (FFR-FVIIa) on FX activation was tested by preincubating MVs or cells with a mixture of FVIIa/FFR-FVIIa (1:10). The amount of FXa formed was measured after 30 min of reaction. Bars represent mean ± SD of three independent experiments. * p < 0.05 or **p < 0.001 relative to Tm1 MVs or cells (Student’s t-test).
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7 Lima et al. Release of procoagulant microvesicles by melanoma cells
binding to PS sites. As seen in �Figure 3C, annexin-V markedly decreased prothrombin activation by the FXa/FVa complex on MVs.
We conclude that the high procoagulant activitiy of Tm1 MVs relative to melan-a MVs is primarily due to a high level of TF ex-pression rather than a difference in PS exposure.
Melanoma- but not melanocyte-derived MVs accelerate thrombus formation in vivo
It was recently demonstrated by Thomas et al. that mice bearing experimental tumours display increased arterial thrombosis ten-dency, and tumour-derived MVs accumulate in the thrombus growth (26). Besides the higher prevalence of venous than arterial thrombosis in cancer patients, it seems clear therefore that the ar-terial model is a valuable tool for studying the prothrombotic properties of MVs. In order to evaluate the impact of melanocyte transformation on these features, the same laser-induced in vivo model of arterial thrombosis was employed. Mice were injected in-travenously with Tm1- or melan-a-derived MVs. Control animals were given an equal volume of saline. After a 5-min equilibration period, endothelial injury was triggered by photochemical acti-vation of rose bengal dye in the common carotid artery, and the in-terval between the onset of injury and complete thrombotic occlu-sion of the artery was then determined. As seen in �Figure 4A, time to occlusion was not affected by melan-a MVs, as compared to
the control group (mean time to occlusion 64 ± 5 min for melan-a-derived MVs vs. 58 ± 7 min in control group). On the other hand, melanoma MVs were highly thrombogenic (mean time to occlu-sion 31 ± 8 min). Strikingly, mice injected with higher numbers of melanoma MVs died shortly after injection and exhibited com-mon signs of pulmonary embolism, for instance unconsciousness and shallow breathing (data not shown).
In order to gain insight into the mechanisms underlying throm-bus formation by tumour-derived MVs, MVs obtained from a well known TF-expressing murine melanoma cell line, B16F10 (12, 41), were also used. As seen in �Figure 4B, B16F10-derived MVs dis-played high prothrombotic ativity (mean time to occlusion 37 ± 9 min) as seen for Tm1 MVs (�Fig. 4A). The thrombogenecity of melanoma MVs was completely abolished when these MVs were pre-incubated with a TF antagonist, active site-inhibited FVIIa (FFR-FVIIa), prior to injection into the mice (�Fig. 4B).
To determine if tumour-derived MVs accumulate at the site of vascular injury, MVs carrying the enhanced green fluorescent pro-tein (eGFP) were used in the same model of arterial thrombosis, and sections of the thrombi were further processed for inspection by fluorescence microscopy. Briefly, B16F10 melanoma cell line was transduced with a lentiviral vector containing the gene for eGFP, and MVs were then isolated from the culture supernatants of the GFP-positive (GFP+) B16F10 cell line. Upon induction of a photochemical injury to the common right carotid artery, we ob-served a complete thrombotic occlusion in 32 ± 11 min (p < 0.05, compared to control group) after injection of GFP+B16F10-de-rived MVs into mice (�Fig. 4B). Moreover, a fluorescent signal
Figure 3: PS exposure and assembly of prothrombinase complex on Tm1 and melan-a MVs. A) Detection of PS exposure by flow cytometry. Relative specific labelling of Tm1– and melan-a-derived MVs by Alexa 488-conjugated anti-PS mAb is shown (ΔMFI represents geometric mean of fluorescence intensity difference between stained and unstained samples). Bars represent mean ± SD of four independent experiments. B) Kinetics for the activation of prothrombin by FXa and FVa, in the absence (white dia-monds) or in the presence (white square/black square) of MVs. A negative
control was included by incubating prothrombin with MVs in the absence of FXa and FVa (white triangle/white circle). Each point represents mean ± SD of four independent experiments. C) Inhibitory effect of annexin-V on pro-thrombin activation by MVs. MVs were incubated with annexin-V prior to in-cubation with FXa/FVa/prothrombin, and the amount of thrombin formed was measured after 5 min of reaction. Results are expressed as the percen-tage of prothrombin activation relative to control. Bars represent mean ± SD of three independent experiments.
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8 Lima et al. Release of procoagulant microvesicles by melanoma cells
Figure 4: Effect of Tm1-, melan-a-, B16F10- or GFP+B16F10-derived MVs on arterial thrombus formation in mice. Mice were injected intra-venously with saline (control) or MVs 5 min before injection of rose bengal dye. B16F10 derived-MVs preincubated with active site-inhibited FVIIa (FFR-FVIIa) were also tested. A and B) Bars represent mean ± SD of thrombotic oc-clusion times after photochemical injury of the carotid artery (n ≥ 3). *p <
0.05 or **p < 0.01 relative to control (Student’s t-test). C) Imaging of GFP+B16F10-derived MVs using fluorescence microscopy. GFP+B16F10-de-rived MVs were detected accumulating at the site of photochemical injury of the right carotid artery after infusion into the blood circulation of a living mouse (left panel). No fluorescent signal was detected in the common left ca-rotid from the same animal (right panel).
corresponding to GFP+MVs was detected at the injured vascula-ture, whereas no signal was identified in the common left carotid from the same animal (�Fig. 4C).
By demonstrating a direct participation in acceleration of thrombus formation in vivo, our results indicate that TF derived from melanoma MVs may play a direct role in cancer-associated thrombosis.
Melanoma-bearing C57BL/6 mice produce procoagulant MVs in vivo
Finally, to elucidate the in vivo relevance of MVs production by Tm1 cells, we inoculated C57BL/6 mice subcutaneously with these cells and examined their plasma for the presence of MVs after 17 days, when every animal presented a tumour mass > 100 mm3. Once more applying a protocol of successive centrifugations, we were able to isolate a population of MVs displaying a forward vs. side scattering profile (FSC x SSC) similar to that previously deter-mined by flow-cytometric analysis for Tm1 MVs produced in vitro (�Fig. 5A). In contrast, MVs population (identified by the same gate of FSC x SSC plot shown in �Fig. 5A) was less frequent in the plasma from tumour-free animals, i.e. which have been inoculated subcutaneously with melan-a cells or vehicle alone (�Fig. 5B). Using the one-stage plasma coagulation assay, we observed that ex vivo MVs significantly shortened the clotting time, similarly to MVs purified in vitro from Tm1 culture supernatants (�Fig. 5C).
We finally investigated the cellular origin of MVs obtained from the plasma of the melanoma-bearing mice. By employing an anti-body against the melanoma-associated MAA antigen, specific for melanomas originating in C57BL/6 mice (39), we observed that
about 67% of MVs population is tumour-derived (�Fig. 5D). As expected, no staining for MAA antigen was observed in a pool of MVs obtained from plasmas of control mice inoculated with ve-hicle (data not shown). Most strikingly, staining with anti-murine TF revealed that about 94% of tumour-derived MVs (63% of total MVs) are TF-positive, demonstrating that tumour-bearing mice exhibit high levels of circulating procoagulant MVs.
Discussion
Cellular shedding of MVs was first described by Wolf in 1967 (43), but since then little is known about the exact mechanisms under-lying MVs production. Although formation of MVs has been shown for a number of different cell types, increasingly attention has been directed to cellular vesiculation in the context of cancer, since various tumour-related biological processes have been as-sociated to MVs (44). Furthermore, accumulation of MVs is often observed in tumour cell cultures, as well as in biological fluids of oncologic patients. So, it is reasonable to speculate that malignant transformation could be closely associated to changes in the bio-genesis of MVs, with important impact in their composition and function.
To further address this question, we analysed MVs shed by tu-mourigenic melanoma cell lines as compared to normal mel-anocytes. Our results showed that melanoma cells produce more elevated amounts of MVs, corroborating the existence of a close correlation between shedding of MVs and the acquisition of a ma-lignant phenotype, which was suggested by others using different types of tumour models (45, 46). Actually, the presence of mutant oncogenes, such as K-ras (20) and the epidermal growth factor
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9 Lima et al. Release of procoagulant microvesicles by melanoma cells
variant III (EGFRvIII) (47), seems to stimulate MVs production. Similarly, loss of tumour suppressors such as p53 (20) appears to regulate tumour vesiculation, which agrees with the idea that the oncogenic status of tumour cells could dictate MVs release.
MVs have long been studied in the context of the coagulation system (9, 10). More recently, different reports have correlated the presence of high levels of circulating MVs with thrombotic events in cancer patients (13–16) and MVs specifically derived from tu-mour cells were shown to support thrombus formation in vivo. In this regard, we investigated changing patterns in the procoagulant and prothrombotic activities of MVs possibly associated with the malignant transformation by melanocytes. Notably, tumour- but not melanocyte-derived MVs significantly reduced the plasma clotting time in vitro (�Fig. 2) and accelerated thrombus formation in vivo (�Fig. 4). Melanoma MVs also accumulated at sites of vascular injury, whereas no cancer cell-derived MVs could be found at intact vasculatures. This suggests that neoplastic con-version could stimulate the production of prothrombotic MVs.
Once in the circulation, tumour-derived MVs could mediate sys-temic effects associated with the thromboembolic events in cancer patients, whose incidence seems to be directly related to tumour progression (4, 5).
Procoagulant activity of MVs appears to be particularly associ-ated with their content of the primary clotting initiator TF and the phospholipid PS. The former one is overexpressed in a variety of malignancies (23) and its presence on tumour cell-derived MVs has been implicated in cancer-associated prothrombotic states (48). In this study, we observed that melanoma MVs carry higher levels of functionally active TF on their surface (�Fig. 2), indicat-ing a qualitative change of MVs associated with melanocyte trans-formation. Accordingly, Yu et al. showed that oncogenic pathways such as the expression of mutants K-ras and p53 gene upregulates both TF expression and the emission of TF-containing MVs by co-lorectal cancer cells (20). Indeed, previous reports have shown that TF expression in different melanoma cell lines correlates with their aggressiveness (42). Since MVs were shown to expose a molecular
Figure 5: Procoagulant activity of MVs obtained from plasma of mel-anoma-bearing C57BL/6 mice. A) MVs isolated from plasma of melanoma-bearing mice were analysed by flow cytometry in a dot plot of forward vs. side light scattering. B) Animals were inoculated subcutaneously with Tm1 or melan-a cells or vehicle alone (control). Lines represent the mean number of MVs isolated from the plasmas of each group on day 17 post-inoculum. *p < 0.05 (Student’s t-test). C) MVs purified from plasma of melanoma-bearing mice (black squares) or isolated from Tm1 culture supernatant (black
triangles) were incubated with murine plasma prior to recalcification with CaCl2. Black circle represents coagulation time of murine plasma alone. Each point represents mean ± SD of three independent experiments. D) MVs iso-lated from plasma of melanoma-bearing mice were analysed by flow cyto-metry in a dot plot of TF vs. MAA double labelling. Quadrant regions were de-fined from a negative control in which double staining with normal rabbit IgG and mouse control isotype IgG was applied.
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repertoire that reflected the type and the developmental state of the cells from which they originate (7), it was not surprising that Tm1– and melan-a-derived MVs displayed a TF expression pattern similar to that of their respective parental melanoma- and normal melanocyte cells (�Fig. 2). Nevertheless, the parental pattern ap-peared not to be exactly recapitulated on MVs, especially regarding TF activity. Current evidence suggests that formation of MVs is not entirely an ad random process (49), but involves shedding of spe-cific membrane domains, allowing the cell to “mold off” a specific profile of MVs antigens. This was supported by recent studies indi-cating that the density of active TF on MVs was higher than the density on their parental cells (26).
Participation of TF on thrombus formation induced by tu-mour-derived MVs was demonstrated by results showing that pre-incubation of melanoma MVs with an active site-blocked FVIIa derivative completely abolished their thrombogenecity (�Fig. 4). Melanoma MVs also expose the anionic phospholipid PS on their surface, which may contribute along with TF to propagate the co-agulation cascade (18). However, an appreciable difference in PS exposure between the two MVs derived from either melan-a or Tm1 cells was not observed (�Fig. 3). This supports the idea that TF plays a key role in the prothrombotic activity of tumour-de-rived MVs.
In vivo assays showed that melanoma-bearing mice exhibit a dramatic increase in circulating MVs in contrast to control ani-mals subcutaneously injected with vehicle or melan-a cells (�Fig. 5). MVs isolated from plasma of tumour-bearing mice displayed a procoagulant pattern similar to melanoma MVs obtained from Tm1 cell line supernatants. This observation is in accordance with a previous study which demonstrated that TF antigen and activity were readily detected in the circulation of mice bearing orthotopi-cally grown human pancreatic cancer (50). Studies employing samples of cancer patients or plasma samples collected from tu-
mour-bearing mice have also shown that circulating MVs may originate from platelets, monocytes, endothelium, as well as from the cancer cells themselves (13, 15, 26, 50). Herein, we studied the co-expression of the TF antigen and a specific marker associated with melanoma cells (MAA) and showed that specific cancer cell-derived TF-bearing MVs were present in the peripheral blood of melanoma-bearing mice at levels corresponded to more than 60% of the total amounts of circulating MVs (�Fig. 5). It definitively demonstrated that endogenous TF-exposing MVs arising from a growing tumour appeared in the circulation ready to participate in thrombus formation in vivo. In a similar way, Zwicker et al. (16) observed that approximately 50% of the circulating TF-bearing MVs obtained from three patients with pancreatic cancer were positive for the epithelial malignancy marker MUC-1 antigen. On the other hand, it does not exclude a possible contribution of vas-cular-derived MVs to the maintenance of a thrombotic state as-sociated with cancer, since TF and/or PS-exposing MVs derived from activated platelets, monocytes and endothelial cells may pro-vide additional procoagulant surfaces (51). In any case, it seems clear that targeting TF would diminish systemic procoagulant changes in the host as well as tumour growth/metastasis-related properties (52, 53).
It has long been established that idiopathic prothrombotic events are closely associated with further cancer diagnosis (1). In addition, circulating MVs seems to be directly involved in the pa-thogenesis of cancer-associated hypercoagulability. In this context, our data provide direct evidence for the principle that malignant transformation can dictate quantitative and qualitative changes in the release of MVs by tumour cells, in particular a remarkable in-crease in their procoagulant and prothrombotic properties. There-fore, therapeutic strategies against production of procoagulant MVs, such as oncogene-directed treatments (54), would attenuate their prothrombotic effects in cancer, which could certainly have a great impact on the morbidity and survival of oncologic patients.
Acknowledgments We thank Dr. I.M.B. Francischetti (National Institutes of Health, Bethesda, MD, USA) for careful revision of the manuscript and Thais M. Gameiro for technical assistance.
Conflict of interest None declared.
What is known about this topic? ● Shedding of microvesicles by neoplastic cells has been implicated
in several pro-tumoural responses. ● Tissue factor-bearing microvesicles are highy procoagulant
in vitro. ● Thrombotic manifestations have been correlated with high levels
of tissue factor-bearing microvesicles in plasma of cancer patients. ● Acquisition of microvesicle procoagulant properties upon malig-
nant transformation is poorly understood.
What does this paper add? ● Malignant transformation in melanocytes increases microvesicle
production. ● Microvesicle-associated tissue factor is elevated upon malignant
transformation. ● Melanocyte- and melanoma-derived microvesicles display ident-
ical levels of phosphatidylserine exposure. ● Melanoma- but not melanocyte-derived microvesicles are pro-
thrombotic in vivo.
Abbreviations FFR-FVIIa, active site blocked factor VIIa; MVs, microvesicles; PS, phos-phatidylserine; TF, tissue factor.
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