Platelets promote breast cancer cell MCF-7 metastasis by direct interaction: surface integrin α2β1-contacting-mediated activation of Wnt-β-catenin pathway
- 39 Downloads
Integrin-mediated platelet-tumor cell contacting plays an important role in promoting epithelial-mesenchymal transition (EMT) transformation of tumor cells and cancer metastasis, but whether it occurs in breast cancer cells is not completely clear.
The purpose of this study was to investigate the role of integrin α2β1 in platelet contacting to human breast cancer cell line MCF-7 and its effect on the EMT and the invasion of MCF-7 cells.
Human platelets were activated by thrombin, and separated into pellets and releasates before the co-incubation with MCF-7 cells. Cell invasion was evaluated by transwell assay. The surface integrins on pellets and MCF-7 cells were inhibited by antibodies. The effect of integrin α2β1 on Wnt-β-catenin pathway was assessed by integrin α2β1-silencing and Wnt-β-catenin inhibitor XAV. The therapeutic effect of integrin α2β1-silencing was confirmed in the xenograft mouse model.
Pellets promote the invasion and EMT of MCF-7 cells via direct contacting of surface integrin α2β1. The integrin α2β1 contacting activates Wnt-β-catenin pathway and promotes the expression of EMT proteins in MCF-7 cells. The activated Wnt-β-catenin pathway also promotes the autocrine of TGF-β1 in MCF-7 cells. Both Wnt-β-catenin and TGF-β1/pSmad3 pathways promote the expression of EMT proteins. Integrin α2β1-silencing inhibits breast cancer metastasis in vivo.
The direct interaction between platelets and tumor cells exerts its pro-metastatic function via surface integrin α2β1 contacting and Wnt-β-catenin activation. Integrin α2β1-silencing has the potential effect of inhibiting breast cancer metastasis.
KeywordsEpithelial-mesenchymal transition Platelet Direct interaction MCF-7 cell Wnt-β-catenin TGF-β1
Chromatin Immunoprecipitation Assay
Quantitative Real-time PCR
Transforming growth factor-β1
Platelets, as small cell fragments, are not only important coagulation-related factors, but also play a vital role in cancer metastasis . The increased level of platelets was correlated with tumor progression, recurrence and distant metastasis in various cancer types [2, 3]. The high level of platelets also has a close correlation with poor recurrence-free survival rates and cancer-specific survival rates [4, 5]. The interaction between platelets and tumor cells activates signaling pathways, such as the Wnt-β-catenin pathway, and promotes the expression of epithelial-mesenchymal transition (EMT) proteins, thus making tumor cells change to a more aggressive phenotype . In breast cancer, Ishikawa et al. found that the accumulation of platelets in primary tumor cells was positively correlated with chemoresistance, and can induce EMT of breast cancer cells . Therefore, inducing EMT transformation of tumor cells is the major way of platelets promoting cancer metastasis.
Previous study mainly investigated the indirect interactions between platelets and tumor cells——secreting releasates (mainly α-granules) of platelets to promote EMT of tumor cells . It has been reported that platelet-derived α-granules containing transforming growth factor-β1 (TGF-β1) increased the growth of primary tumors in murine models of ovarian cancer [9, 10]. However, in recent years, the effect of direct interaction of platelet and tumor cells on promoting EMT has been attracting attentions in the field of cancer treatment. Pang et al. demonstrated that the direct interaction between tumor cells and platelet fractions, named as pellets (with the activation marker of P-selectin, but without releasates ), promotes the EMT and extracellular matrix (ECM) degradation, inducing a more aggressive phenotype in tumor cells . Labelle et al. demonstrated that direct platelet-tumor cell contacting and platelet-derived TGF-β1 synergistically activate the TGF-β1/Smad pathway in cancer cells, resulting in their EMT transformation and enhanced metastasis . Whereas, the mechanism underlying the direct interaction between platelets and breast cancer cells is largely unknown.
Integrins mediate cell-to-cell contacting, and are key mediators in delivering signals between cells. They were discovered by Hynes RO and his colleagues [14, 15]. Also, integrins are suggested to be involved in the tumor growth and progression , and were regarded as a therapeutic target for tumors. As reported, the combined blockade of both tumor integrin αvβ3 and platelet integrin αIIbβ3 inhibited platelet-cell contacting and suppressed angiogenesis and tumor growth of melanoma . α2β1, a kind of integrin, has been shown to exist in the surface of breast cancer cells, and is believed to mediate the direct interaction between tumor cells and platelets . However, how integrin α2β1 mediates the EMT by platelet-breast cancer cell contacting remains unknown.
Herein, this current study defined the role of integrin α2β1 in mediating the direct interaction between platelets and breast cancer cells, and explored its effect on the promotion of EMT by controlling the correlated signaling molecules, thus implying the important role of direct contacting of platelets and breast cancer cells in promoting breast cancer metastasis.
Materials and methods
Preparation of platelets and platelet fractions
Platelets and platelet fractions used in this study were collected from human blood as previously described . Briefly, healthy human blood was centrifuged at 250G for 10 min. The plasma and buffy coat were gently transferred to a fresh tube, and the centrifugation was repeated at 2000G for 10 min. The platelet-rich plasma in the bottom was then washed with the platelet washing solution for 3 times. To prepare platelet fractions, platelets were stimulated by thrombin 0.5 U/ml for 15 min at 37 °C. The pellets (platelet membranes) were separated from the releasates (supernatant containing active components) by centrifugation at 2800G for 7 min. To determine the extent of contamination of the thrombin-activate pellets with soluble platelet proteins, platelet factor 4 (PF4) was measured using an ELISA assay (Abcam, Cambridge, UK) and compared with the PF4 levels in the releasates and whole platelets solubilised by the addition of 0.1% Triton-X 100 .
P-selectin exposure analysis
A minimum of 10,000 gated events was analyzed on a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, US). The anti-CD41 conjugated with fluorescein (FITC, BD Pharmingen, San Jose, CA) and the anti-CD62P conjugated with phycoerythrin (PE, BD Pharmingen) were used as antibodies. Then they were added to the activated platelets with a final dilution of 1:20 (v/v). FITC and PE-conjugated isotype-matched antibodies were used to control nonspecific labeling of antibodies. The samples were fixed with 0.4% paraformaldehyde and analyzed by flow cytometry after antibody incubation for 15 min at 37 °C.
Transwell invasion assay
Human breast cancer cell lines, MCF-7, MDA-MB-231, and SK-BR-3 (all from American Type Culture Collection, ATCC, Manassas, VA, US), were maintained at 37 °C, 5% CO2, grown in Dulbecco’s Modified Eagle’s medium (DMEM; ThermoFisher, Pittsburgh, PA, US) containing 1% penicillin-streptomycin (Gibco, NY, USA) and 10% fetal bovine serum (FBS; ThermoFisher) at pH 7.2 overnight. The invasion assay was performed in 24-well BD Biocoat™ Matrigel™ Invasion Chambers (8 μm pore size; BD Biosciences) . Total 5 × 104 MCF-7 cells were plated in transwell inserts. Then the DMEM buffer (the buffer group), thrombin-activated platelets (the platelet group), releasates (the releasate group), pellets (the pellet group), and the supernatant of the co-incubation of pellets with MCF-7 cells for 48 h [the pellet + MCF-7 conditioned medium (CM) group] were added. Both upper and lower chambers contained DMEM. After 48 h, cells remaining in the upper part of the transwell were removed with a cotton swab. Migrated cells were then stained with Crystal Violet 0.5% and the total number of cells was counted with a Zeiss Axiovert 200 microscope (Zaventem, Belgium). The invasion assay was also performed in MDA-MB-231 cells and SK-BR-3 cells after adding platelets.
Direct interaction between tumor cells and platelets or platelet fractions
The medium of MCF-7 cells was changed for fresh DMEM immediately prior to the treatment. Then the DMEM buffer (the buffer group), thrombin-activated platelets (the platelet group), releasates (the releasate group), pellets (the pellet group), and the supernatant of the co-incubation of pellets with MCF-7 cells for 48 h (the pellet + MCF-7 CM group) were added, and incubated for 40 h.
Quantitative real-time PCR (qRT-PCR)
Primers and promoter sequences used in qRT-PCR and ChIP
qRT-PCR Primers (from 5′ to 3′)
ChIP Primers (from 5′ to 3′)
Total protein was extracted from the tumor cell line or tumor tissues using RIPA lysis buffer. The concentration of total protein was quantified using a BCA kit (ThermoFisher). The protein samples (20 μg/sample) were separated by SDS-PAGE and then transferred to poly-vinylidene difluoride (PVDF) membranes (Merck Millipore, Danvers, MA, US). After blocking with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h, the proteins were immunoblotted with primary antibodies at 4 °C overnight. After washing with TBST three times, the proteins were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After washing with TBST three times, protein signals were determined using a substrate chemiluminescence detection system (ThermoFisher) with the Image Lab Software (Bio-Rad, Hercules, CA, US). The primary antibodies used were anti-β-catenin (1:5000, Rabbit monoclonal antibody, Abcam, ab32572), anti-integrin α2 (1:10000, Rabbit monoclonal antibody, Abcam, ab133557), anti-β-actin (1:500, Mouse monoclonal antibody, Abcam, ab8226), anti-Lamin B1 (0.1 μg/ml, Rabbit polyclonal antibody, Abcam, ab16048), and anti-pSmad3 (1:2000, Rabbit monoclonal antibody, Abcam, ab52903).
Analysis of tumor cell contacting to platelets or pellets
The platelet adhesion assay was performed to evaluate the contacting between platelets/pellets and tumor cells as previously described . Briefly, 5 × 104 cells/ml breast cancer cells were seeded in a 96-well plate. Washed thrombin-activated platelets or pellets labeled with fluorescein (FITC, BD Pharmingen) were added to the cells for 30 min at 37 °C. The non-adherent platelets or pellets were discarded. Then the flow cytometry was used to perform the platelet/pellet adhesion assay.
Antibody inhibition of pellet or tumor cell membrane
The antibody inhibition of pellet and tumor cell membrane was performed as previously described . In brief, the 5 × 108 CFDA-SE-conjugated thrombin-activated pellet were pre-incubated with 50μg/ml anti-P-selectin, anti-AK7 (integrin α2β1), or anti-CRC64 (integrin αIIbβ3) on ice for 30 min. Then they were added to 3 × 105 MCF-7 cells in 100 μl of DMEM in a 96-well plate and incubated at 37 °C for 30 min. To prevent further binding before flow cytometry, 100 μl /well ice cold 0.1% BSA/PBS was added and the flow cytometry was performed immediately. The antibody inhibition of MCF-7 cell membrane was also performed as described above, and the antibodies used were anti-AK7 and anti-CRC64. The anti-AK7 was used to inhibit the cell surface integrin α2β1 of MDA-MB-231 cells and SK-BR-3 cells.
Integrin α2β1-silencing MCF-7 cell line
The shRNA specific for the integrin α2β1 subunit (Sigma, St. Louis, MO, US) was cloned to the lentiviral plasmid vector pLKO.1-puro, and the pLKO.1-puro lentiviral vector without shRNA (empty vector) was used as the control . Lentiviruses were produced in HEK293T cells by co-transfection of plasmid vector containing shRNA or control vector. Then the MCF-7 cells were infected with shRNA lentivirus or empty lentivirus in the presence of polybrene (8 mg/ml) and selected with puromycin (1–2 mg/ml) for 4–6 days.
TGF-β1 levels were detected in MCF-7 cells co-cultured with DMEM (the buffer group), platelets, releasates, or pellets for 40 h with the Quantikine TGF-β1 immunoassay kit (R&D Systems, Minneapolis, MN, US).
Chromatin Immunoprecipitation assay (ChIP)
The promoter of Snail and Slug binding by pSmad3 and/or β-catenin was determined by ChIP . Briefly, after the co-culture with activated platelets with or without XAV (Wnt-β-catenin inhibitor) or SB (TGF-β1/Smad3 inhibitor), the MCF-7 cells were fixed with 1% formaldehyde, sonicated on ice to shear the DNA into the fragments from 200 bp to 500 bp. The lysate was subjected to immunoprecipitations with anti-pSmad3, anti-β-catenin, or non-specific rabbit IgG. The immunoprecipitated DNA was subjected to PCR to amplify a fragment of Snail, Slug, or tgfb1 promoter. The PCR products were run electrophoretically on a 1% agarose gel and visualized by ethidium bromide staining. The ChIP primers used were shown in Table 1.
For pSmad3/β-catenin IP, three subconfluent 35-mm dishes of platelet-co-cultured MCF-7 cells were each extracted with 1 ml each of PBS containing 0.1% Triton X-100, 1:1000 CLAP, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, and 1 mM sodium vanadate. The dishes were rocked at 4 °C for 15 min, and the cell lysates were centrifuged for 10 min at 4 °C. Then the precipitate was reextracted in a volume of 3 ml radioimmunoprecipitation assay buffer for 15 min at 4 °C. The supernatants were pooled and divided into 500-μl aliquots after centrifugation for 10 min. Each aliquot was then incubated with appropriate combinations of immune and preimmune sera and protein A–Sepharose beads for 14 h at 4 °C. The beads were collected by brief centrifugation and washed three times in PBS containing 0.1% Triton X-100 and once in PBS alone. Then the beads were suspended in SDS-PAGE sample buffer, heated to 95 °C for 5 min, and analyzed by western blotting.
The promoter activity was determined by the luciferase reporter assay. In brief, the promoter sequences of tgfb1, Snail, and Slug were cloned into the pGL3 vector. The MCF-7 cells with different treatments were transiently transfected with the vectors carrying tgfb1, Snail, or Slug promoter sequences using Lipofectamine 2000 (Invitrogen, Pittsburgh, PA, US). After 48 h of transfection, luciferase activity was detected using a luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer’s protocol and was normalized to the renilla luciferase activity.
Pulmonary metastasis assays
MCF-7 cells were co-incubated with platelets (the MCF-7 + platelet group) or without platelets (the MCF-7 group) for 40 h. MCF-7 cells which were stably interfering integrin α2β1 (Si-MCF-7) were obtained using the lentivirus vectors. The Si-MCF-7 were co-incubated with platelets (the Si-MCF-7 + platelet group) or without platelets (the Si-MCF-7 group) for 40 h. On the other hand, following the treatment of anti-AK7 for 30 min, the platelets were then co-incubated with MCF-7 cells for 40 h (the MCF-7 + platelet/AK7 group). The co-incubation between anti-AK7-untreated platelets and MCF-7 cells was the control (the MCF-7 + platelet group). The tumor cell monolayer was washed three times to remove platelets and dead cells. Then the tumor cells (1.5 × 106 cells/100 μl PBS) were intravenously injected into the lateral tail vein of immunodeficient nude mice (6-week old, female). There were 5 mice in each group.
MDA-MB-231 cells were co-incubated with platelets for 40 h (the MDA-MB-231 + Platelet group). Following the simultaneous inhibition of surface integrin α2β1, MDA-MB-231/AK7 cells were co-incubated with platelet/AK7 for 40 h (the MDA-MB-231/AK7 + platelet/AK7 group). MDA-MB-231 cells cultured in the normal medium was the control (the MDA-MB-231 group). The tumor cell monolayer was washed three times to remove platelets and dead cells. Then the tumor cells (1 × 106 cells/100 μl PBS) were intravenously injected into the lateral tail vein of immunodeficient nude mice (6-week old, female). There were 5 mice in each group. The Balb/c nude mice used in this study were bought from Shanghai Lab Animal Research Center (Shanghai, China).
After 5 weeks, mice were sacrificed and the lungs were separated. For hematoxylin-eosin (HE) staining, the pulmonary nodules were fixed in 10% formaldehyde of NaCl/Pi buffer with a pH of 7.4. After being dehydrated in alcohol, they were embedded in paraffin. Paraffin blocks were sliced in 4 μm pieces and stained with HE.
Statistical analysis was performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, US) with a Student’s t-test or analysis of variance. The data were expressed as the mean ± standard deviation (SD). P < 0.05 was considered statistically significant. All experiments were performed in triplicate.
Activated platelets promote MCF-7 EMT via direct contacting
Platelets contact breast cancer cells via surface integrin α2β1
Integrin α2β1 contacting between platelets and breast cancer cells activates Wnt-β-catenin pathway
Activated Wnt-β-catenin pathway promotes tgfb1 transcription and TGF-β1 autocrine in MCF-7 cells
Both Wnt-β-catenin and TGF-β1/pSmad3 pathways promote MCF-7 EMT
Integrin α2β1-silencing inhibits breast cancer metastasis in vivo
Accumulating studies have indicated the pro-metastatic function of platelets in various types of tumors, including cholangiocarcinoma , multiple myeloma , and ovarian cancer . In breast cancer, the pro-metastatic function of platelets was initiated by the accumulation of platelets in primary tumor cells . The mechanism of platelet accumulation is mainly related to the “First Responder” property of platelets in circulation . Tumor cells with metastatic characteristics are often more aggressive, and easily enter the circulation, releasing direct signals that promote platelet aggregation . Meanwhile, the aggregated platelets release angiogenic factors that exaggerate tumor growth and angiogenesis . In the present work, we demonstrated the pro-metastatic function of platelets was initiated via coincubation of breast cancer cells with platelets and platelet fractions. Interestingly, our findings indicated that the direct interaction between tumor cells and pellets, or known as platelet membranes, plays the most important role in promoting MCF-7 cell invasion (Fig. 1), which is consistent with the former research . Previous study indicated that releasates promote MDA-MB-231 cell (human breast cancer cell line) invasion , however, the pro-invasive effect of releasates to MCF-7 cells was low in our findings. We believed that it may be the potential mechanism of the lower metastatic property of MCF-7 cells than that of the MDA-MB-231 cells, although further investigations are still needed.
The importance of integrins that affect tumor progression has made them an appealing target for cancer therapy. Integrins are heterodimeric cell surface receptors that formed by the combination of 18 α-subunits and 8 β-subunits, mediating the contacting to the ECM and other molecules . In breast cancer, integrin α6β4 is reported to increase the tumor size and grade, and decrease the survival rates [28, 29], while integrin αvβ3 is reported to promote bone metastasis of breast cancer patients [30, 31]. Meanwhile, tumor cell–platelet interactions mediated by integrins on the surfaces of both tumor cells and platelets are correlated with increased tumor metastasis. It is reported that the combined blockade of both tumor integrin αvβ3 and platelet integrin αIIbβ3 inhibited the angiogenesis and tumor growth of melanoma . In the current study, we confirmed the high proportion of surface integrin α2β1 on MCF-7 cells, and revealed the reduced MCF-7 cell-pellets contacting ability after the blockade of tumor integrin α2β1 or pellet integrin α2β1 (Fig. 2), suggesting that antagonists that target integrin α2β1 on platelets or tumor cells may inhibit tumor invasion. Hence, we introduced integrin α2β1-silencing MCF-7 cells into the mouse xenograft model, and found the anti-metastatic effect of integrin α2β1-silencing. Although previous studies and our current work suggest that integrin α2β1 promotes metastasis of breast cancer, a study conducted by Ramirez et al.  indicated that integrin α2β1 acts as a metastasis suppressor in a mouse model of breast cancer. In their study, the number and size of metastatic foci in the lungs were significantly increased in the integrin α2-deficient mice in comparison with the wild type mice. We believed that apart from the platelet-related metastatic way, there may be other metastatic ways that mediated by integrin α2β1, which will be investigated in our further studies.
As reported, integrin α2β1 is a collagen receptor which can bind to type I, II, III, IV, and XI collagens . Although little is known concerning the integrin α2β1-mediated cell contacting, a previous study revealed that the inhibition of the binding between integrin α2β1 and collagen suppressed platelet aggregation , suggesting collagen may mediate the binding between integrins. Plus, collagens are the essential component of ECM and provide both integrity and biological cues for cells . We supposed that the direct binding between integrins in platelet and breast cancer cells was mediated by collagens, although more evidence is needed.
The Wnt-β-catenin signaling pathway is activated by cell surface receptors, and plays an important role in promoting EMT of breast cancer cells [36, 37]. Integrins are important cell surface receptors on tumor cells, and regulate the cellular β-catenin level, thus affecting transcriptions of target molecules. Cheng et al. revealed that the integrin β3/Wnt signaling mediate the chemoresistance and stemness of breast cancer cells . Li et al. demonstrated that integrin α5/β-catenin signaling promotes the stemness and metastasis of triple negative breast cancer . Based on these previous studies, we assessed the cytoplasm and nucleus levels of β-catenin in MCF-7 cells incubated with thrombin-activated platelets and pellets, and it appeared a significant enhancement of β-catenin in both cytoplasm and nucleus after contacting platelets and pellets (Fig. 3a). By integrin α2β1-silencing and Wnt-β-catenin blockade, we confirmed that the integrin α2β1/β-catenin pathway promotes MCF-7 cell invasion via increasing the expression of EMT correlated proteins Snail, Slug, and E-cadherin. Notably, the direct interaction between platelets and MCF-7 cells exerted more apparent influence on EMT correlated proteins than the transwell invasion assay (Fig. 5d), and we surmised that it may be related to the low invasive property of MCF-7 cells.
Except for the Wnt-β-catenin signaling pathway, the activation of the TGF-β1/Smad pathway in cancer cells also contributes to the EMT and metastasis of breast cancer . Previous research demonstrated that the direct platelet-tumor cell contacting and platelet-derived TGF-β1 synergistically activate the TGF-β1/Smad pathway in cancer cells . In our work, we found that both supernatant TGF-β1 and tgfb1 mRNA levels were markedly enhanced after MCF-7 cell-platelet contacting, and the subsequently increased expression of pSmad3 was also confirmed. By integrin α2β1-silencing and Wnt-β-catenin blockade, we confirmed the activation of integrin α2β1/β-catenin/tgfb1 signaling cascade after MCF-7 cell-platelet/pellet contacting, indicating that the MCF-7 cells autocrine TGF-β1 after the contacting (Fig. 4). Moreover, we found that the TGF-β1/Smad pathway needs Wnt-β-catenin participation to regulate Snail and Slug transcriptions, as TGF-β1/pSmad3 blockade partly reduced transcription of Snail and Slug, while Wnt-β-catenin blockade almost cut off the transcription of Snail and Slug (Fig. 5). Combined with the IP results, we demonstrated that Wnt-β-catenin promotes EMT via dependent or independent of the TGF-β1/pSmad3 pathway.
In conclusion, our findings indicated that the direct interaction between platelets and breast cancer cells exerts its pro-metastatic function via integrin α2β1 contacting and Wnt-β-catenin activation. The activated Wnt-β-catenin promotes the autocrine of TGF-β1 in MCF-7 cells. Meanwhile, both Wnt-β-catenin and TGF-β1/pSmad3 pathways promote the transcription of EMT related genes. Additionally, the integrin α2β1-silencing has the potential effect of inhibiting breast cancer metastasis, providing a novel target of treating breast cancer.
X-xZ and Y-gS put forward the concept of the study, designed the study, prepared the manuscript and contributed to the statistical analysis. YY contributed to the data acquisition. YZ contributed to the quality control of data and algorithms. Z-gZ analyzed the data and interpretation. X-fW edited the manuscript. Y-gS revised the manuscript. All authors read and approved the final manuscript.
This study was supported by the National Natural Science Foundation of China (Grant No. 81702595).
Ethics approval and consent to participate
This study was approved by the Institute Research Medical Ethics Committee of the First Affiliated Hospital of Zhengzhou University. All the individuals were participating in this study with written informed consent. All animals-treatment operations were executed according to the Zhengzhou University Ethical Guidelines for Animal Experiment.
Consent for publication
The study was undertaken with Zhengzhou University.
The authors declare that they have no competing interests.
- 5.Pardo L, et al. The prognostic value of pretreatment platelet count in patients with head and neck squamous cell carcinoma. Auris Nasus Larynx. 2016;44(3):S0385814616301900.Google Scholar
- 21.Qijin L, Malinauskas RA. Comparison of two platelet activation markers using flow cytometry after in vitro shear stress exposure of whole human blood. Artif Organs. 2015;35(2):137–44.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.