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Abstract
Background While malignant pleural effusion (MPE) is a common and significant cause of morbidity in patients with cancer, current treatment options are limited. Human heparanase, involved in angiogenesis and metastasis, cleaves heparan sulfate (HS) side chains on the cell surface.
Aims To explore the coagulation milieu in MPE and infectious pleural effusion (IPE) focusing on the involvement of heparanase.
Methods Samples of 30 patients with MPE and 44 patients with IPE were evaluated in comparison to those of 33 patients with transudate pleural effusions, using heparanase ELISA, heparanase procoagulant activity assay, thrombin and factor Xa chromogenic assays and thromboelastography. A cell proliferation assay was performed. EMT-6 breast cancer cells were injected to the pleural cavity of mice. A peptide inhibiting heparanase activity was administered subcutaneously.
Results Levels of heparanase, factor Xa and thrombin were significantly higher in exudate than transudate. Thromboelastography detected almost no thrombus formation in the whole blood, mainly on MPE addition. This effect was completely reversed by bacterial heparinase. Direct measurement revealed high levels of HS chains in pleural effusions. Higher proliferation was observed in tumour cell lines incubated with exudate than with transudate and it was reduced when bacterial heparinase was added. The tumour size in the pleural cavity of mice treated with the heparanase inhibitor were significantly smaller compared with control (p=0.005).
Conclusions HS chains released by heparanase form an anticoagulant milieu in MPE, preventing local thrombosis and enabling tumour cell proliferation. Inhibition of heparanase might provide a therapeutic option for patients with recurrent MPE.
- heparan sulfate
- pleural effusion
- tumor
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Key messages
What is the key question?
Why malignant pleural effusion does not clot?
What is the bottom line?
Heparan sulfate (HS) chains released by heparanase form an anticoagulant milieu in malignant pleural effusion (MPE).
MPE enhances tumour cell proliferation and the effect is modulated by cell surface HS chains.
Why read on?
Inhibition of heparanase might provide a therapeutic option for patients with recurrent MPE.
Background
The pleura comprises a layer of mesothelial cells and the underlying connective tissue.1 The entire surface area of the visceral pleura is vast, amounting to 4000 cm2 in a person with a body weight of 70 kg. Unlike in the past, when mesothelial cells had been thought to purely provide an inactive wrapping to serosal cavities, they have presently been established as reactive entities, responding to inflammatory stimuli by expression of additional enzymes, release of cytokines, growth factors and chemotactic peptides. The submesothelial connective tissue encompasses blood vessels and lymphatics.1 2 Normally, approximately 15 mL/day of fluid move into this potential space, primarily from the capillaries of the parietal pleura and the fluid is removed through the lymphatics in the parietal pleura.3 A pleural effusion is an anomalous accumulation of fluid in the pleural space subsequent to excessive fluid production, reduced absorption or both. Malignant pleural effusion (MPE) presents a common and major clinical problem. Its pathogenesis is poorly understood and therapeutic choices are restricted. As the effects of pleurodesis that obliterates the pleural space resulting in extensive adhesion of the visceral and parietal pleura or of an indwelling pleural catheter (IPC) are not optimal, mainly due to inadequate prevention of recurrent fluid accumulation, other potentially effective measures need to be explored.
The haemostatic system is a significant contributing factor to tumour growth and angiogenesis. Activated coagulation factors (eg, tissue factor (TF), thrombin, fibrin) possess angiogenic along with metastatic properties.4 5 In addition, cytokines produced during tumour growth (eg, interleukin (IL)-1, IL-6) can induce cell surface expression of coagulation factors in endothelial cells, further activating the coagulation system6 and enforcing a positive feedback loop between these processes. Heparanase protein has emerged as an enzyme capable of degrading heparan sulfate (HS) side chains in the extracellular matrix (ECM) and on the cell surface.7 8 Platelets and tumour cells have long been known as ample sources of heparanase.9 10 Our earlier studies have demonstrated that heparanase may also enhance the coagulation system in a non-enzymatic manner. Heparanase has been shown to upregulate the expression of the blood haemostasis initiator—TF11 and interact with tissue factor pathway inhibitor (TFPI) on the cell surface, leading to release of TFPI from the cell membrane of endothelial and tumour cells, followed by increased cell surface coagulation activity.12 Additionally, our previous findings have indicated that heparanase directly augments the TF activity, resulting in enhanced factor Xa generation and activation of the haemostatic system.13 14 We have lately defined new peptides originating from TFPI-2 first Kunitz domain that impede the heparanase procoagulant activity by interfering with the heparanase-TF complex. In vivo, the newly identified peptides have reduced activation of the haemostatic system and attenuated sepsis severity, without predisposing to substantial bleeding tendency.15 Furthermore, the peptides inhibiting heparanase procoagulant activity have been demonstrated to substantially decrease tumour growth, vascularisation and relapse.16
The presence of activated coagulation factors in the pleural effusion was previously demonstrated. Levels of thrombin-antithrombin complex, fibrinopeptide 1+2 and fibrin degradation products were shown to be highest in IPE, lower in MPE and lowest in transudate samples.17 18 Given the evidence on coagulation system activation in the pleural effusion, while clinically, transudate and MPE do not usually form a clot, the current study has aimed to investigate the haemostatic balance in different kinds of pleural effusions, focusing on the involvement of the heparanase protein. The results obtained could widen our understanding of the pleural effusion mechanisms to be targeted by novel therapeutic modalities.
Materials and methods
Study group
Patients were enrolled over a 6-month period, between December 2016 and May 2017. Thoracocentesis was done at the discretion of the treating physician in patients aged above 18 years. Details were obtained from patient medical charts. According to the criteria by Porcel and Light,19 an effusion was considered an exudate if it met any of the following three requirements1: the ratio of pleural fluid protein to serum protein was >0.5,2 the pleural fluid lactate dehydrogenase (LDH) to serum LDH ratio was >0.6,3 pleural fluid LDH was greater than two-thirds of the upper limit of normal for serum LDH. If a malignancy was a concern, a cytological examination was done. All MPE samples were positive in cytological examination. All cases of infectious pleural effusions (IPE) were related to clinically treated pneumonia. Twenty-eight out of 33 patients with transudative fluid had congestive heart failure. In the remaining five cases, the aetiology of the effusion was not clear and the fluids were evaluated with cytological and bacterial tests. Of the 107 evaluated patient samples, diagnostic small-volume aspiration of pleural fluid (50–60 mL) was performed in 12 cases; in the rest, large-volume aspiration was carried out using chest tube placement. Test samples were collected to tubes with no additive and immediately subjected to centrifugation at 1500 g for 10 min at room temperature. Fluids without sediment were immediately stored in aliquots of 250 µL at −20°C and all samples were frozen and thawed once. None of the recruited patients had been previously treated with either IPC or pleurodesis (talc). Exclusion criteria were: pregnancy, use of oral contraceptives or therapeutic anticoagulant therapy. If prophylactic enoxaparin was used, thoracocentesis was done just prior to the next dose administration. Samples of pleural fluid were collected in siliconised tubes.
The mouse model
Mouse breast cancer 104 cells (EMT-6) were injected to the intrapleural cavity of BALB/c mice, as previously described.20 Heparanase inhibitory peptide 615 was injected subcutaneously on alternate days, in the area opposite to the tumour side, starting on the first day after the tumour cell injection. Control mice were injected with phosphate-buffered saline (PBS in a manner similar to that used in the treatment group. After 14 days the experiment was ended. EMT-6 cells were chosen as a model of rapidly growing tumour and were not injected as an orthotopic procedure (ie, to the breast tissue). These anaplastic cells derived from BALB/c mice are devoid of oestrogen or progesterone receptors.21 Heparanase expression was previously demonstrated to be upregulated by oestrogen.22 23 Hence, all the studies evaluating heparanase were performed in male mice aged 7–8 weeks in order to avoid age and hormonal effects.
Reagents and antibodies
A single chain GS3 heparanase gene construct, comprising the 8 and 50 kDa heparanase subunits (8+50) was purified from the conditioned medium of baculovirus-infected cells. GS3 heparanase was assayed for the presence of bacterial endotoxin (Biological Industries, Beit Haemek, Israel), using the gel-clot technique (limulus amebocyte lysate test) and was found to contain <10 pg/mL of the endotoxin.11 Polyclonal antibody 1453 was raised in rabbits against the entire 65 kDa heparanase precursor isolated from the conditioned medium of heparanase-transfected HEK-293 cells. The antibody was affinity-purified on immobilised bacterially expressed 50 kDa heparanase glutathione-S-transferase fusion protein.24 Monoclonal antiheparanase antibody 1E1 was generated by immunising BALB/c mice with the entire 65 kDa heparanase protein. Antibody 733 was raised in rabbits against a 15 amino acid peptide that maps at the N-terminus of the 50 kDa heparanase subunit.24 Recombinant human factor VIIa and plasma-derived human factor X were purchased from Sekisui Diagnostics (Stamford, Connecticut, USA). All coagulation factors were dissolved in double-distilled water. Chromogenic substrate to factor Xa (I.D. 222, solubility: Tris buffer, pH −8.4) and chromogenic substrate to thrombin (I.D. 238, solubility: Tris buffer, pH −8.4) were purchased from Sekisui Diagnostics. Bovine factor Xa and bacterial heparinase II were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Polyclonal antihuman TF and polyclonal antihuman TFPI antibodies were purchased from Santa Cruz (Santa Cruz, California, USA). Polyclonal anti-TFPI-2 was purchased from Bioss (Woburn, Massachusetts, USA).
Cell culture
EMT-6 mouse breast carcinoma, T47D human breast carcinoma, MCF-7 human breast carcinoma, Chinese hamster ovary (CHO) cells CHO-745 (deficient in the synthesis of proteoglycans due to the lack of xylosyl transferase activity) and CHO-K1 (wild type) were purchased from the American Type Culture Collection. Cells were grown in the Dulbecco’s modified Eagle’s medium (Biological Industries) supplemented with 10% fetal calf serum and antibiotics.
Heparanase ELISA
Wells of microtitre plates were coated (18 hours, 4°C) with 2 µg/mL of antiheparanase monoclonal antibody 1E1 in 50 µL of coating buffer (0.05 M Na2CO3, 0.05 M NaHCO3, pH 9.6) and were then blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour at 37°C. Samples (200 µL) were loaded in triplicates and incubated for 2 hours at room temperature, followed by the addition of 100 µL of antibody 1453 (1 µL/mL) for 2 hours at room temperature. Horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (1:20 000) in blocking buffer was added (1 hour, room temperature) and the reaction was visualised by the addition of 50 µL of chromogenic substrate (tetramethylbenzidine (TMB); MP Biomedicals, Germany) for 30 min. The reaction was stopped with 100 µL of H2SO4 and absorbance at 450 nm was measured using an heparanase ELISA plate reader (PowerWave XS, BioTek, Winooski, Vermont, USA). Plates were washed five times with a washing buffer (PBS, pH 7.4 containing 0.1% (w/v) Tween 20) after each step. As a reference for quantification, a standard curve was established by serial dilutions of recombinant 8+50 GS3 heparanase (390 pg/mL–25 ng/mL).25
Heparanase procoagulant activity assay
As previously reported,26 we performed a basic experiment of factor Xa generation in the following manner. The mentioned concentrations were final. Twenty-five µL of pleural effusion, recombinant human factor VIIa (0.04 µM) and plasma-derived human factor X (1.4 µM) were incubated in 50 µL of the assay buffer (0.06 M Tris, 0.04 M NaCl, 2 mM CaCl2, 0.04% w/v BSA, pH 8.4) at a total volume of 125 µl in a 96-well sterile plate. After 15 min at 37°C, chromogenic substrate to factor Xa was added (1 mM). Following 20 min, the reaction was stopped with 50 µL of glacial acetic acid and the level of Xa generation was determined using an ELISA plate reader (PowerWave XS, BioTek). Heparins had been shown to abrogate the TF/heparanase complex,13 so, in parallel, the same assay was performed but with the addition of fondaparinux (2.5 µg/mL) to the assay buffer. Bovine factor Xa diluted in the assay buffer was used to generate a standard curve. The subtraction of the first assay result from the second assay result determined heparanase procoagulant activity. Thus, the assay provided three results: heparanase procoagulant activity, TF activity and TF+heparanase procoagulant activities.
Factor Xa chromogenic assay
Fifty μL of pleural effusion were added to 50 µL of Tris buffer (0.06 M, pH 8.4) and 25 µL of chromogenic substrate to factor Xa (5 mM). After 30 min, the reaction was stopped with glacial acetic and the absorbance at 405–490 nm was measured using an ELISA plate reader (PowerWave XS, BioTek). Bovine factor Xa diluted in the assay buffer was used to generate a standard curve.
Thrombin chromogenic assay
Fifty μL of pleural effusion were added to 50 µL of Tris buffer (0.06 M, pH 8.4) and 25 µL of chromogenic substrate to thrombin (5 mM). After 30 min, the reaction was stopped with glacial acetic and the absorbance at 405490 nm was measured using an ELISA plate reader (Power Wave XS, BioTek). Human thrombin diluted in the assay buffer was used to generate a standard curve.
Thromboelastography
This assay was performed according to the manufacturer's recommendations. Briefly, blood was collected in a bottle containing 3.2% (0.12M) sodium citrate (9:1) and stored at room temperature. Recalcification and thromboelastography (TEG) measurements at 37°C were performed in disposable cups of the Thrombelastograph coagulation analyser (Haemoscope, Skokie, Illinois, USA). Apart from calcium no other activators were added.
Anti-XA enoxaparin level
Pleural effusion samples were tested for the chromogenic anti-Xa activity with HemosIL Liquid anti-Xa (Instrumentation Laboratory, Bedford, USA) reagent, using the analyser system ACL TOP 550 CTS (Instrumentation Laboratory). In each test, 50 µL of effusion were added to 100 µL of pooled normal plasma from three volunteers. The anti-factor Xa activity measurement was expressed in IU/mL.
Heparan sulfate chromogenic assay
Levels of HS were measured in the following way: 16 mg of dimethylmethylene blue (DMMB, Sigma-Aldrich) were dissolved in 1 L of double distilled water containing 3.04 g glycine, 1.6 g NaCl and 95 mL of 0.1 M acetic acid. The solution was filtered using Whattman 3 MM. Standard curve was prepared using HS derived from bovine kidney (Sigma-Aldrich, H7640). To 20 µL of a sample, 5 µL of protamine sulfate (10 mg/mL, Fresenius Kabi, Toronto, Canada) were added followed by addition of 20 µL of a sample to 200 µL of DMMB solution. Absorbance was immediately read using a plate reader at 525 nm. Each sample was evaluated with and without protamine sulfate and the subtraction of the first assay result from the second assay result determined the HS level in a sample.
Proliferation assay
The XTT Cell Proliferation Kit (Biological Industries) was used according to the manufacturer’s instructions. Briefly, 1×105 cells were cultivated in a 96-well plate. One hundred μL of growth media were added to each well and incubated for 24 hours. A blank containing complete medium without cells was used as negative control. The reaction solution included 0.1 mL of activation solution added to 5 mL of XTT reagent. Fifty μL of the reaction solution were added to each well and the plate was incubated for 4 hours; then absorbance was measured by ELISA reader at a wavelength of 450 nm. Non-specific readings were measured at a wavelength of 630 nm and subtracted from the 450 nm measurement.
Immunohistochemistry
Staining of formalin-fixed, paraffin-embedded 5-micron sections was performed. Slides were deparaffinised with xylene, rehydrated and endogenous peroxidase activity was quenched for 30 min by 3% hydrogen peroxide in methanol. Slides were then subjected to antigen retrieval by boiling (20 min) in 10 mM citrate buffer, pH 6. Slides were further incubated with 10% normal goat serum in PBS for 60 min to block non-specific binding followed by incubation (20 hours, 4°C) with anti-TF, anti-TFPI, anti-TFPI-2 or antiheparanase 733 antibodies, diluted 1:100 in blocking solution. Slides were then extensively washed with PBS containing 0.01% Triton X-100 and incubated with a secondary reagent (Envision kit; Dako, Glostrup, Denmark) or secondary fluorescent reagent (Jackson ImmunoResearch, West Grove, Pennsylvania, USA) according to the manufacturer’s instructions. Following additional washes, a colour was developed with the AEC reagent (Sigma-Aldrich). The slides were blindly assessed by two experts not informed about the allocated groups. Representative intensity of protein immunostaining was shown.
Statistical analysis
Data were evaluated using SPSS software for Windows V.13.0 (SPSS, Chicago, Illinois, USA). For variables with normal distribution, the parametric t-test was used and variables were summarised as mean±SD. For variables with non-normal distribution, the non-parametric Mann-Whitney U test was used and variables were reported as median and IQR. The significance level was set at p<0.01. Table 1 presents results of patient characteristics analysis, where the t-test was used to assess statistical significance of age and the Mann-Whitney U test was applied to evaluate statistical significance of male gender enoxaparin prophylaxis. The t-test was used to compare data of different assays employed to evaluate patient samples of pleural effusion (figures 1 and 2). The effects of pleural effusion and cell surface HS chains on tumour cell proliferation were analysed using the Mann-Whitney U test (figures 3 and 4). This test was also applied to assess mouse model data (figure 5).
Demographic characteristics of patients with pleural effusion
Activation of coagulation system is higher in infectious pleural effusion (IPE) than in malignant pleural effusion (MPE). Transudate pleural effusion (n=33), IPE (n=44) and MPE (n=30) were evaluated. Although the heparanase level was about twofold higher in the IPE and MPE compared with the transudate samples (A), heparanase procoagulant activity was only slightly increased in the IPE compared with the transudate and was similar in the MPE and transudate (B). Factor Xa and thrombin levels demonstrated a significant increase in IPE compared with transudate and a less prominent increase in the MPE compared with transudate (C, D). T-test was used in A–D. Results represent mean±SD; *p<0.01, **p<0.001.
Pleural effusions contain heparan sulfate (HS) chains. Thromboelastography (TEG) with the addition of 5 µL transudate (B), infection pleural effusion (D) and malignant pleural effusion (F) to 335 µL of pooled whole blood from three healthy volunteers was significantly pathologic compared with baseline TEG (A) in terms of the time to clot formation (R) and the thrombus strength (MA). Malignant effusion exerted the most prominent effect. Bacterial heparinase addition to the assays (0.6 unit/mL) prior to the effusion completely reversed the anticoagulant effect of the effusion, indicating that the effect is related to HS chains in the effusion. The evaluations were repeated five times using five different samples from each group. Representative images are presented herein (C, E, G). Transudate pleural effusion (n=33), infectious pleural effusion (IPE) (n=44) and malignant pleural effusion (MPE) (n=30) were evaluated to assess HS chain levels. The highest level was found in the MPE samples (H). T-test was used in H. Results represent mean±SD; ***p<0.0001.
Pleural effusions increase tumour cell proliferation. T47D cells were incubated with pleural effusion (pool derived from three patients randomly allocated) for 48 hours (10% pleural effusion in serum-free medium). Control cells were incubated with serum free medium. The most prominent increase in proliferation was observed when infectious pleural effusion (IPE) was added. Mann-Whitney U test was used. Results represent median and IQR of three different experiments; *p<0.01, **p<0.001. MPE, malignant pleural effusion.
Cell surface heparan sulfate (HS) modulates tumour cell proliferation. MCF-7 cells were incubated with malignant pleural effusion (MPE) (pool derived from three patients) for 48 hours (10% pleural effusion in serum-free medium). MPE significantly increased cell proliferation (A). The effect was attenuated when bacterial heparinase (0.6 unit/mL) was added to the assay for 48 hours. Notably, bacterial heparinase also decreased proliferation in the control cells by half (A). In order to answer the question if the effect on proliferation is related to the free HS chains or to the release from the cells surface, we repeated the assay using two cell lines, CHO; CHO-745, that have a very low level of cell surface HS (B) and the wild type CHO-K1 cells (C). In CHO-745, the effect of the MPE on proliferation was minor, similar to the addition of heparinase, indicating that the cell surface HS chains are the dominant parameter affecting the proliferation and free HS chains have a less prominent role. Mann-Whitney U test was used. Results in A–C represent median and IQR of three different experiments; *p<0.01, **p<0.001. (D) CHO-745 or CHO-K1 2×104 cells/well were plated in triplicates on top of the basement membrane extract (Cultrex, Trevigen, Germany) diluted in phosphate-buffered saline (1/2) for 48 hours in full medium. Notably, CHO-745 cells proliferated less and formed a decreased number of cell islands and remained separated from each other, compared with CHO-K1 cells. Representative images (x50 magnification) of the triplicate experiment, captured with a Nikon E995 digital camera (Nikon, Tokyo, Japan).
Heparanase inhibition attenuates tumour growth in the pleural cavity. Mouse breast cancer 104 cells (EMT-6) were injected to the intrapleural cavity of BALB/c mice. Heparanase inhibitory peptide 6 (3 mg/kg) was injected subcutaneously, on alternate days, in the area opposite to the tumour side, starting on the first day after the tumour cell injection. Control mice were injected with phosphate-buffered saline in a manner similar to that used in the treatment group. After 14 days, the experiment was ended. A significant reduction in the number of mice who developed tumours was observed in the treatment group. Mann-Whitney U test was used. (A). Representative images (x50 magnification) of parietal pleura (arrows) of control mice with tumour (T) and treated mice that were stained for heparanase, tissue factor (TF), tissue factor pathway inhibitor (TFPI) and TFPI-2 (B). Images were captured with a Nikon E995 digital camera (Nikon, Tokyo, Japan).
Results
Activation of coagulation system is higher in IPE than in MPE
Samples of transudate pleural effusion (n=33), IPE (n=44) and MPE (n=30) were evaluated. Demographic characteristics of the patients are presented in table 1. There was no difference between the groups in terms of age or gender. In the IPE group, eight cases of positive effusion culture were identified. The 36 other cases were those of parapneumonic effusion. The most common malignancy in the MPE group was adenocarcinoma. About one-third of the patients from all the groups received prophylactic enoxaparin. Heparanase levels, heparanase procoagulant activity, factor Xa level and thrombin level were analysed in all the samples (figure 1). Interestingly, although the heparanase level was about twofold higher in the IPE and MPE than in the transudate samples (figure 1A, p<0.001), heparanase procoagulant activity was only slightly increased in the IPE compared with the transudate, while being similar in MPE and transudate (figure 1B). Factor Xa (figure 1C) and thrombin (figure 1D) levels demonstrated a significant increase in IPE compared with transudate (p<0.001) and a less prominent increase in the MPE compared with transudate (p<0.01). Results represent mean±SD.
Pleural effusions contain heparan sulfate chains
To evaluate the net effect of the pleural effusion on coagulation, the TEG assay was used. Five µL of transudate (figure 2B), infection pleural effusion (figure 2D) or malignant pleural effusion (figure 2F) were added to 335 µL of pooled whole blood from three healthy volunteers and analysed by TEG. Results were significantly pathologic compared with baseline TEG (figure 2A), in terms of the time to clot formation (R) and the thrombus strength (MA). Malignant effusion exerted the most prominent effect. Notably, the addition of bacterial heparinase to the assays (0.6 unit/mL) prior to the effusion completely reversed the anticoagulant effect of the effusion, indicating that this effect was related to effusion HS chains. The experiments were repeated five times using five different samples from each group. Representative images are presented (figure 2C,E,G). Transudate pleural effusion (n=33), IPE (n=44) and MPE (n=30) were evaluated to assess HS chain levels. The highest level was found in the MPE samples (2 hours, p<0.0001). The finding of high HS chain levels in the MPE and IPE gives explanation to the discrepancy in the heparanase assays results (figure 1A,B). While the ELISA for heparanase levels does not depend on the presence of HS chains in the sample, the procaoagulant activity assay is inhibited in the presence of HS chains. In order to exclude the contribution of enoxaparin to the effusion anticoagulant effect, we studied 32 samples of patients treated with enoxaparin from the study group (10 transudates, 12 IPE, 10 MPE) and 6 new 2-week samples of (2 transudates, 2 IPE, 2 MPE) to the assay of anti-Xa level of enoxaparin. The mean (±SD) anti-Xa enoxaparin level was 0.01 IU/mL (±0.01), indicating negligible levels.
Tumour cells release heparan sulfate chains to the medium
We hypothesised that part of the HS chains in the effusion are released from the tumour cells. The effects of the tumour cell medium were assessed in the TEG assay. Fifty µL of 48 hours serum-free medium of T47D cells (80% confluence) were added to 290 µL of pooled whole blood obtained from three healthy volunteers, whereas 50 µL of normal serum-free medium were added to the control. The malignant medium (figure 6) was found to prolong the time to clot formation (R) and reduce the thrombus strength (MA). The effect was reversed when bacterial heparinase was incorporated in the assay (figure 6C), suggesting that the anticoagulant effect could result from HS chains.
Tumour cells release heparan sulfate (HS) chains to the medium. Fifty µL of 48 hours serum-free medium of T47D cells (80% confluence) were added to 290 µL of pooled whole blood from three healthy volunteers. Fifty µL of normal serum-free medium were added to the control (A). The malignant medium (B) prolonged the time to clot formation (R) and reduced the thrombus strength (MA). The effect was reversed when bacterial heparinase was added to the assay (C), indicating that the anticoagulant effect results from HS chains. Representative images of three separate experiments.
Pleural effusions increase tumour cell proliferation
To evaluate the contribution of the effusions to tumour cell proliferation, T47D cells were incubated with pleural effusion (pool derived from three patients randomly allocated) for 48 hours (10% pleural effusion in serum-free medium). The most prominent increase in proliferation was observed on IPE addition (p<0.001). Results represent median and IQR of three different experiments (figure 3).
Cell surface heparan sulfate modulates tumour cell proliferation
MCF-7 cells were incubated with MPE (pool derived from three patients) for 48 hours (10% pleural effusion in serum-free medium). MPE significantly increased cell proliferation (figure 4A). The effect was attenuated when bacterial heparinase (0.6 unit/mL) was incorporated in the assay for 48 hours (p<0.001). Notably, bacterial heparinase also decreased by half the proliferation rate in the control cells (figure 4A, p<0.001). To clarify if the effect on proliferation is associated with free HS chains or with the release of cell surface HS chains, induced by heparanase, present in the effusion, we have repeated the assay using two CHO cell lines. The first line, CHO-745, is deficient in the synthesis of proteoglycans due to the lack of xylosyl transferase activity, resulting in a very low level of cell surface HS chains, and the second line, CHO-K1, is the wild type. The effects of MPE and the addition of bacterial heparinase on proliferation were found to be minor in CHO-745 cells (figure 4B) compared with CHO-K1 cells (figure 4C, p<0.001). This result indicates that the cell surface HS chains are the dominant parameter affecting the proliferation and free HS chains have a minor role. Results represent median and IQR of three different experiments. CHO-745 or CHO-K1 2×104 cells/well were plated in triplicates on top of the basement membrane extract (Cultrex, Trevigen, Germany) diluted in PBS (1/2) for 48 hours in full medium. Representative images of the triplicate experiment are presented in figure 4D. CHO-745 cells proliferate less and they formed a decreased number of cell islands and remained separated from each other, compared with CHO-K1 cells. Thus, it appears that the role of cell surface HS chains may not be limited to modulating proliferation, but may also potentially include the cell-to-cell adherence.
Heparanase inhibition attenuates tumour growth in the pleural cavity
As delineated in the ‘Materials and methods’ section, mouse breast cancer cells (EMT-6) were injected to the intrapleural cavity of BALB/c mice followed by heparanase inhibitory peptide injections. A significant reduction in the number of mice who developed tumours was observed in the treatment group (figure 5A, p=0.005). Representative images of parietal pleura (arrows) of control mice with tumour and treated mice that were stained for heparanase, TF, TFPI and TFPI-2 are presented in figure 5B.
Discussion
The present study has demonstrated that while activation of the coagulation system does occur in pleural effusion, the net effect appears to be anticoagulant, which could be attributed to HS chains. The significantly higher level of HS chains observed in the MPE compared with that in the transudate or IPE is likely to be explained by HS chain release from the tumour cells. The anticoagulant nature of the effusion enables its accumulation and tumour cell proliferation, as otherwise, it would have turned to a fibrin clot obstructing the pleural cavity. Heparanase, which is the only human protein capable of degrading HS chains, is responsible for the HS chain accumulation in the pleural effusion. We have further shown that tumour cell proliferation induced by the pleural effusion is regulated by the tumour cell surface HS chains and, to a lesser extent, by the free HS chains. Tumour cells in the MPE tend to be separated and not to form a solid tumour as occurs in the primary malignancy. We have demonstrated that the cell surface HS chains may also affect this feature. In our study, inhibition of heparanase by a peptide derived from TFPI-2 has significantly reduced tumour development in a mouse model of pleural cavity tumour. This peptide, preventing the interaction of heparanase with TF, has been demonstrated to inhibit the heparanase activity in the coagulation system and to attenuate tumour growth14 and impede further release of heparanase from the cells (Nadir et al, unpublished data, 2019). Thus, release of HS chains from the tumour cells by heparanase enables effusion accumulation without clotting, and increases tumour cell proliferation, while inhibition of heparanase almost completely prevents MPE occurrence.
Activation of the coagulation system in the pleural effusion was previously reported. Vaz et al studied 54 samples of pleural fluids, 15 transudates and 39 exudates, including 9 parapneumonic, 15 tuberculosis and 15 cancer samples. Levels of thrombin-antithrombin complex, fibrinopeptide 1+2 and fibrin degradation products were highest in the IPE, lower in the MPE and lowest in the transudate samples.17 Gieseler et al showed that in the MPE, despite the presence of both procoagulant and anticoagulant factors, activation of the coagulation system appeared to dominate. In the latter study, the TF level was similar to that in the normal plasma, while levels of prothrombin and antithrombin were about one-third of those in the plasma. Levels of D-dimer and prothrombin fragment 1+2 were about 20 times higher than in the plasma.18 We have also demonstrated activation of the coagulation system in the various effusions, but the finding of high levels of HS chains is innovative and can explain why the pleural effusion causes no clots locally.
Heparanase is an enzyme capable of cleaving HS side chains in a limited number of sites, yielding HS fragments of still appreciable size (˜5–7 kDa)7 8 that interact with antithrombin and heparanase. What is the estimated anticoagulant effect of HS chains in the MPE? In our study, the addition of 5 µL of MPE to 335 µL of blood (12.5 µL/mL) resulted in no clotting, as demonstrated by TEG. While 1 unit of unfractionated heparin (UFH)/1 mL of blood is an established therapeutic dose, >2–3 units of UFH/mL usually induce no clotting. Hence, 12.5 µL of MPE contain at least 2–3 equivalent units of UFH that is 160–240 equivalent units of UFH/mL. This level is about 200-fold higher than the therapeutic dose of UFH. The reason of HS chain accumulation in the pleural effusion is unclear. It is known that HS degradation is mainly related to the action of macrophages in the liver, spleen and bone marrow. MPE is reported to be rich in macrophages, although they have weak cytotoxic activity against tumour cells, possibly contributing to tumour cell resistance to apoptosis.27 It is possible that the function of macrophages against HS chains in the MPE fluid is weakened. This intriguing issue needs to be further investigated. A single human heparanase cDNA sequence was independently reported by several research groups.28–31 Thus, unlike the large number of proteases that can degrade polypeptides in the ECM, one major heparanase appears to be used by cells to degrade the HS side chains of HS proteoglycans. While previous studies demonstrated an association of several cancer types and clinical parameters with some single nucleotide polymorphism in the heparanase gene,32–35 no effect of the described polymorphisms was studied in terms of heparanase ability to degrade HS chains in cancer or infection. This issue warrants further investigation.
According to our results, in the CHO and MCF-7 cells incubated with MPE, the addition of bacterial heparinase has brought about reduction in cell proliferation. These experiments have demonstrated that a change in the cell surface HS milieu has an effect on tumour cell proliferation. It has been previously reported that cell surface HS chains are involved in tumour cell signalling, inflammation, angiogenesis and metastasis.36 Liu at al have reported that bacterial heparinase I increases tumour cell growth, while bacterial heparinase III decreases the growth,37 indicating that the length of the remaining HS on the cell surface imposes a biological effect. Hence, the use of bacterial heparinase II, which has the activity of heparinase I and III, can explain the proliferation reduction observed in our study.
In conclusion, the present study demonstrates that HS chains released by heparanase to the pleural effusion prevent clotting. In addition, the change in cell surface HS chains affects tumour cell proliferation. Obstruction of heparanase release from cells by a subcutaneously injected peptide significantly inhibited tumour growth. The findings of this study regarding the ability of an heparanase inhibitor to ameliorate malignant pleural effusion accumulation need to be further evaluated. Comparison of the heparanase inhibitory peptides derived from TFPI-2 to other currently evaluated heparanase inhibitors38–45 in terms of their effects on malignant pleural effusion is an extensive area of investigation. The involvement of heparanase and HS chains in other cavities, such as the peritoneal cavity or the subdural space, is an intriguing arena for further research.
Acknowledgments
The authors would like to thank Professor Israel Vlodavsky and Dr Neta Ilan (The Technion, Haifa, Israel) for providing the heparanase antibodies. The abstract of this article has previously been published in Research and Practice in Thombosis and Haemostasis: Abstracts of the XXVI Congress of the International Society on Thrombosis and Haemostasis, 8–13 July 2017.
References
Footnotes
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests European Patent Application No. IL201200027 filed on 18 January 2012 in the name of Nadir, Brenner, Vlodavsky. Entitled: Methods and kits for assessing heparanase procoagulant activity, compositions comprising heparanase and methods for the treatment of coagulation-related disorders.
Patient consent for publication Not required.
Ethics approval This study was approved by the Institutional Review Board of the Rambam Health Care Campus (Approval #0578–14-RMB). The study was approved by the Technion Ethics Committee for Animal Research, and the procedures were conducted in accordance with institutional guidelines.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on reasonable request.