The novel chemokine receptor CXCR7 regulates trans-endothelial migration of cancer cells
© Zabel et al; licensee BioMed Central Ltd. 2011
Received: 15 March 2011
Accepted: 14 June 2011
Published: 14 June 2011
Migration of metastatic tumor cells from the bloodstream into lymph nodes is thought to be facilitated by expression of the chemokine receptors CCR7, CXCR4 and, for B cell-derived tumors, CXCR5. Expression of their respective chemokine ligands (CCL19, CCL21, CXCL12 and CXCL13) by endothelial cells inside the lymph nodes facilitates the trans-endothelial migration (TEM) of these cells through high endothelial venules into the lymph node parenchyma. It is known that CXCR7, a second CXCL12 receptor, regulates TEM of CXCR4+CXCR7+ tumor cells towards a CXCL12 source. In this study, we set out to assess the potential stimulation by CXCL12 of tumor cell TEM towards other chemokines and whether CXCR7 might be able to regulate such effects.
The human Burkitt's lymphoma cell line NC-37, which expresses CXCR4, CXCR5, CXCR7 and CCR7, was selected as a model system. TEM of these cells through a human HUVEC endothelial cell monolayer was used as the main model system for these studies. Regulation of their TEM behavior by various concentrations of the various cognate chemokines for the above-mentioned receptors, placed in either the source or target wells of modified Boyden chamber migration plates, was assessed by quantifying the number of cells migrated under each experimental condition.
Exposure of CXCR4+CXCR7+ cancer cells to CXCL12 greatly potentiated their TEM towards the chemokines CCL19 and CXCL13. This CXCL12-potentiated TEM was inhibited by the second CXCR7 chemokine ligand, CXCL11, as well as CXCR7-specific small molecule antagonists and antibodies. In contrast, the CXCR4 antagonist AMD3100 was less effective at inhibiting CXCL12-potentiated TEM. Thus, CXCR7 antagonists may be effective therapeutic agents for blocking CXCL12-mediated migration of CXCR4+CXCR7+ tumor cells into lymph nodes, regardless of whether the cancer cells follow a CXCL12 gradient or whether serum CXCL12 stimulates their migration towards CCR7 and CXCR5 chemokines in the lymph nodes.
Trans-endothelial migration (TEM) is a critical step in the metastatic dissemination of malignant cells from a primary tumor to distant vital organs, which is the primary cause of morbidity and mortality in cancer patients (reviewed in ). During metastasis, cancer cells in the bloodstream cross the endothelial cell layer of the blood vessel to enter the parenchyma of the target organ, in a manner similar to the extravasation of leukocytes. Metastasis of tumor cells to lymph nodes, whether from blood or directly via the lymphatics, is likely mediated by the same processes used by lymphocytes when they enter lymph nodes . Like primary lymphocytes, tumor cells of hematopoietic and non-hematopoietic origin can express multiple chemokine receptors. CXCR4 is the most common chemokine receptor expressed by cancer cells, and has been thoroughly implicated in metastasis [3–6]. In model systems, CXCR4 regulates cancer metastasis to lymph node, bone, liver, and lung, the four most common metastatic destinations, which also express high levels of CXCL12, the only known chemokine ligand for CXCR4 [3–6]. High levels of CXCL12 are also present in the bloodstream [7–10].
CCR7, the most studied lymph node homing chemokine receptor, is expressed by certain cancer cells, in particular hematopoietic malignancies and lymph node metastases , as well as naïve T and B cells, while CCL19 and CCL21, the chemokine ligands for this receptor, are expressed in the T cell areas of lymph nodes . Similarly, the chemokine receptor CXCR5, which guides cells to the chemokine CXCL13 present in lymph node follicles , has been detected on leukemia and lymphoma cells and on naïve B cells [12–15].
A poorly understood but important area of chemokine biology is the synergistic and/or inhibitory effects produced by simultaneous activation or inhibition of multiple chemokine receptors. For example, CXCL12 has been shown to potentiate the chemotaxis of CXCR4+ cells towards CCL19, CCL21 or CXCL13. In one report, CXCL12 treatment increased T cell responsiveness to CCL19 and CCL21 in vitro and increased CCR7-dependent recruitment of T cells into lymph nodes in vivo. Moreover, Okada et al. showed that CXCL12-treated T cells homed more efficiently to lymph nodes and Peyer's patches than non-treated cells .
CXCR7 was recently identified as a second, high-affinity receptor for CXCL12 . This receptor is highly expressed by a variety of cancers, including breast , brain [20, 21], liver , pancreas , lung , prostate , melanoma [26, 27] and rhabdosarcomas . Like CXCR4, CXCR7 has also been implicated in tumor metastasis [24, 28, 29]. We recently showed that, although it does not directly mediate cell migration, CXCR7 can regulate TEM of CXCR4+CXCR7+ tumor cells towards CXCL12, an effect that can be blocked by CXCR7-specific antagonists and the second CXCR7 chemokine ligand, CXCL11 . We now describe that CXCL12 may enhance cell homing to lymph nodes by potentiating TEM towards CCL19, CCL21 and CXCL13, and show that CXCR7 can regulate this CXCL12-mediated potentiation of TEM. In the current study, we have studied the CXCL12-mediated TEM of Burkitt's lymphoma cells toward CCL19 and CXCL13. We then evaluated the involvement of CXCR7 by assessing the ability of CXCL11 and CXCR7-specific antagonists to interfere with such TEM. These studies illustrate a potential mechanism by which tumor cells metastasize to lymph nodes and other tissues, providing the rationale for antagonizing CXCR7 in vivo in order to block tumor metastasis.
Cells and reagents
The human Burkitt's lymphoma cell line NC-37 was obtained from the American Type Culture Collection (Manassas, VA). Human umbilical vein endothelial cells (HUVEC) were obtained from Lonza, Inc. (San Jose, CA), cultured according to the manufacturer's specifications, and used at passage 3. Chemokines CCL2, CCL19, CXCL9, CXCL11, CXCL12, and CXCL13 were purchased from R&D Systems (Minneapolis, MN). Anti-CCR7 (clone 150503), -CXCR3 (49801), -CXCR4 (12G5), -CXCR5 (clone RF8B2), and mouse IgG2a isotype control mAbs were also purchased from R&D Systems. Anti-CXCR7 (clone 8F11) and mouse IgG2b isotype control mAbs were purchased from BioLegend (San Diego, CA). PE-conjugated goat anti-mouse IgG was purchased from Jackson ImmunoResearch Labs, Inc. (West Grove, PA). Anti-CXCR7 (clone 11G8) mAb, mouse IgG1 isotype control mAb, CCX771 and CCX704  were generated at ChemoCentryx, Inc. AMD3100 was purchased from Sigma Aldrich (St. Louis, MO). Flow cytometry was performed on a FACScan (BD Biosciences, San Jose, CA).
Bare filter chemotaxis assay
Chemokine was diluted in chemotaxis buffer (HBSS containing 0.1% BSA), 29 μl/well was transferred to a 96-well microchamber plate, and the plate was covered with a 5 μm 96-well filter (NeuroProbe, Gaithersburg, MD). NC-37 cells were resuspended at 1 × 107cells/ml in chemotaxis buffer, mixed with chemokine, antibody or compound, and 20 μl/well was added on top of the filter. The plate was incubated for 2 h at 37°C in a humidified incubator, the filter was removed, and 5 μl/well of the DNA-intercalating reagent CyQuant (Invitrogen, Carlsbad, CA) was added to the wells. Fluorescence was measured using the SpectraFluor Plus plate reader (TECAN, San Jose, CA).
Trans-endothelial migration assay
The TEM assay was performed using 24-well plates with microporous (5 μm) transwell membrane inserts (Corning Costar Corp., Lowell, MA). HUVEC (passage 3) were suspended at 1 × 106/ml in HUVEC media (Lonza), 100 μl/well was placed in the top wells and 600 μl/well of HUVEC media was placed in the bottom wells. The plate was incubated overnight at 37°C, after which the HUVEC monolayers were washed with PBS lacking Ca2+ and Mg2+. NC-37 cells were suspended at 5 × 106cells/ml in assay medium (IMDM (Invitrogen, Carlsbad, CA) with 0.1% BSA), incubated with test reagents (AMD3100, CCX704, CCX771, CXCL9, CXCL11, 11G8, mIgG1, 8F11, mIgG2b, 12G5, or mIgG2a) at room temperature for 10 min, and then added (100 μl/well) to the upper wells containing the HUVEC monolayers. In potentiated TEM experiments, CXCL12 was also added to the NC-37 cells. Assay medium containing CCL2, CCL19, CXCL12, and/or CXCL13 was then added (600 μl/well) to the bottom wells. The plates were incubated overnight at 37°C, the top wells were removed, and the cells in the bottom wells were counted.
HUVEC (passage 1-2) were transfected with double-stranded RNAi oligonucleotides using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. Stealth RNAi duplex targeting CXCR7 (RNA sequence (GGCUAUGACACGCACUGCUACAUCU) and a scramble RNAi duplex control (GC content 48%) were purchased from Invitrogen. HUVEC were collected for the TEM assays three days after transfection.
Results and Discussion
Chemokine receptor expression on NC-37 human B lymphoma cells includes CCR7 and CXCR5
CXCL12 potentiates NC-37 cell TEM to CXCL13
A CXCR7-specific compound blocks CXCL12-potentiated NC-37 cell TEM to CXCL13
CXCR7-specific mAbs and CXCL11 block CXCL12-potentiated NC-37 cell TEM to CXCL13 or CCL19
To determine whether these agents could block CXCL12-potentiated NC-37 cell TEM to other chemokines, we performed the same assay with the CCR7 ligand CCL19. Although NC-37 cells migrated to CCL19 in the bare-filter migration assay (Figure 1), the cells did not migrate to CCL19 in the TEM assay (Figure 4B). However, NC-37 cells did migrate to CCL19 in the TEM assay (optimal concentration 1 μM) when CXCL12 (10 nM) was present in both wells. As seen in the TEM assay with CXCL13, CXCL12-potentiated TEM of NC-37 cells to CCL19 was inhibited by CXCL11, CCX771, and the CXCR7 and CXCR4-specific mAbs. CXCL9, AMD3100 and CCX704 did not block migration, supporting the fact that CXCL12 potentiation of CCL19-driven TEM occurred through CXCR7.
To confirm that CCX771, CXCL11 and the mAbs did not interfere with TEM by interacting directly with CXCR5, CXCL13, CCR7, or CCL19, we tested these agents in the bare filter chemotaxis assay. CCX771, CXCL11 and the mAbs had no effect on bare filter migration of NC-37 cells to CXCL13 (Figure 4C) or CCL19 (Figure 4D), ruling out the possibility of direct inhibitory effects of these agents on CXCR5 or CCR7 or their ligands.
CXCR7 expression by HUVEC is dispensable for CCX771 blockade of NC-37 cell TEM
In this report we demonstrate that CXCL12 can work in concert with CCL19 or CXCL13 to promote efficient TEM of CXCR7+ Burkitt's lymphoma NC-37 cells. While CCL19 or CXCL13 alone did not induce TEM of these cells, which also express CCR7, CXCR4, and CXCR5, the presence of CXCL12 on the apical side or both sides of the endothelial cell layer resulted in TEM of NC-37 cells towards CCL19 or CXCL13. These observations may relate to metastasis in vivo, since CXCL12 is implicated in metastasis but is present both in the bloodstream and in the parenchyma of many organs (reviewed in [3–6]). Importantly, the sensitization of NC-37 cells by CXCL12 was blocked by a CXCR7-specific antagonist. Since the antagonist also blocks TEM of the NC-37 cells towards CXCL12 alone , we conclude that CXCR7 can regulate both CXCL12-directed TEM and CXCL12-potentiated TEM of CXCR4+CXCR7+ lymphoma cells.
In addition to potentiating TEM towards CCL19 and CXCL13, CXCL12 alone induced TEM. That is, a subset of NC-37 cells migrated across the endothelial cell layer when CXCL12 was present at equal concentrations on both sides of the layer. This non-directed TEM was also blocked by the CXCR7 antagonist. The CXCR4-specific antagonist AMD3100 was unable to block either the non-directed TEM or the CXCL12-potentiated TEM, supporting the notion that these processes occur through CXCR7, not CXCR4. In addition, the inhibitory effects of the CXCR7 antagonist were observed regardless of whether or not the endothelial monolayer expressed CXCR7, suggesting that, in vivo, inhibition of TEM might require pharmacologic antagonism of CXCR7 only on the tumor cell.
Agents that target CXCR4, a chemokine receptor expressed by at least 23 different types of cancer , can block tumor cell TEM in mouse models in vivo. Treatment of mice with AMD3100 transiently reduced the seeding of i.v.-injected CXCR4+ syngeneic 4T1 mammary tumor cells in the lung . In a xenograft model in nude mice, treatment with AMD3100 reduced the dissemination of CXCR4+ human epithelial ovarian carcinoma cells onto mesothelial cells lining the peritoneal cavity . In both animal models, however, the effect on tumor cell dissemination was partial. We previously showed that AMD3100 was an inefficient inhibitor of NC-37 cell TEM to CXCL12  and here we show that AMD3100 is unable to block CXCL12 potentiation of NC-37 cell TEM to CCL19 or CXCL13. Although our studies have been performed only with a lymphoma cell line and should be expanded to include other tumor types, it is possible that, for tumor cells that express both CXCR4 and CXCR7, small molecules that target CXCR7 may therefore prove to be superior agents to inhibit metastasis in vivo. An additional limitation of our study is the use of HUVEC cells, which might not recapitulate the behavior of specialized endothelial cells such as those found in the high endothelial venules of lymph nodes.
To the extent that the chemokine CXCL12, which is found in blood and in organs commonly colonized by metastatic tumors, participates in promoting the migration of tumor cells across endothelium into those organs, our data suggest that the newly discovered CXCR7 receptor for CXCL12 may be a more effective therapeutic point of intervention than the previously studied CXCR4 receptor, which also recognizes CXCL12.
List of Abbreviations
human umbilical vein endothelial cell
- Duffy MJ, McGowan PM, Gallagher WM: Cancer invasion and metastasis: changing views. J Pathol. 2008, 214: 283-293. 10.1002/path.2282View ArticlePubMed
- Zlotnik A: Chemokines in neoplastic progression. Semin Cancer Biol. 2004, 14: 181-185. 10.1016/j.semcancer.2003.10.004View ArticlePubMed
- Ben-Baruch A: Organ selectivity in metastasis: regulation by chemokines and their receptors. Clin Exp Metastasis. 2008, 25: 345-356. 10.1007/s10585-007-9097-3View ArticlePubMed
- Kryczek I, Wei S, Keller E, Liu R, Zou W: Stroma-derived factor (SDF-1/CXCL12) and human tumor pathogenesis. Am J Physiol Cell Physiol. 2007, 292: C987-995.View ArticlePubMed
- Sun X, Cheng G, Hao M, Zheng J, Zhou X, Zhang J, Taichman RS, Pienta KJ, Wang J: CXCL12 / CXCR4 / CXCR7 chemokine axis and cancer progression. Cancer Metastasis Rev. 2010, 29: 709-722. 10.1007/s10555-010-9256-xPubMed CentralView ArticlePubMed
- Zlotnik A: Involvement of chemokine receptors in organ-specific metastasis. Contrib Microbiol. 2006, 13: 191-199.View ArticlePubMed
- Costa V, Sommese L, Casamassimi A, Colicchio R, Angelini C, Marchesano V, Milone L, Farzati B, Giovane A, Fiorito C, Rienzo M, Picardi M, Avallone B, Marco Corsi M, Sarubbi B, Calabrò R, Salvatore P, Ciccodicola A, Napoli C: Impairment of circulating endothelial progenitors in Down syndrome. BMC Med Genomics. 2010, 3: 40-52. 10.1186/1755-8794-3-40PubMed CentralView ArticlePubMed
- Hassan S, Baccarelli A, Salvucci O, Basik M: Plasma stromal cell-derived factor-1: host derived marker predictive of distant metastasis in breast cancer. Clin Cancer Res. 2008, 14: 446-454. 10.1158/1078-0432.CCR-07-1189View ArticlePubMed
- Kirkpatrick B, Nguyen L, Kondrikova G, Herberg S, Hill WD: Stability of human stromal-derived factor-1alpha (CXCL12alpha) after blood sampling. Ann Clin Lab Sci. 2010, 40: 257-260.PubMed
- Roodhart JM, Langenberg MH, Vermaat JS, Lolkema MP, Baars A, Giles RH, Witteveen EO, Voest EE: Late release of circulating endothelial cells and endothelial progenitor cells after chemotherapy predicts response and survival in cancer patients. Neoplasia. 2010, 12: 87-94.PubMed CentralView ArticlePubMed
- Luther SA, Bidgol A, Hargreaves DC, Schmidt A, Xu Y, Paniyadi J, Matloubian M, Cyster JG: Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis. J Immunol. 2002, 169: 424-433.View ArticlePubMed
- Airoldi I, Cocco C, Morandi F, Prigione I, Pistoia V: CXCR5 may be involved in the attraction of human metastatic neuroblastoma cells to the bone marrow. Cancer Immunol Immunother. 2008, 57: 541-548. 10.1007/s00262-007-0392-2View ArticlePubMed
- Burkle A, Niedermeier M, Schmitt-Graff A, Wierda WG, Keating MJ, Burger JA: Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia. Blood. 2007, 110: 3316-3325. 10.1182/blood-2007-05-089409View ArticlePubMed
- Capriotti E, Vonderheid EC, Thoburn CJ, Bright EC, Hess AD: Chemokine receptor expression by leukemic T cells of cutaneous T-cell lymphoma: clinical and histopathological correlations. J Invest Dermatol. 2007, 127: 2882-2892. 10.1038/sj.jid.5700916View ArticlePubMed
- López-Giral S, Quintana NE, Cabrerizo M, Alfonso-Pérez M, Sala-Valdés M, De Soria VG, Fernández-Rañada JM, Fernández-Ruiz E, Muñoz C: Chemokine receptors that mediate B cell homing to secondary lymphoid tissues are highly expressed in B cell chronic lymphocytic leukemia and non-Hodgkin lymphomas with widespread nodular dissemination. J Leukoc Biol. 2004, 76: 462-471. 10.1189/jlb.1203652View ArticlePubMed
- Bai Z, Hayasaka H, Kobayashi M, Li W, Guo Z, Jang MH, Kondo A, Choi BI, Iwakura Y, Miyasaka M: CXC chemokine ligand 12 promotes CCR7-dependent naive T cell trafficking to lymph nodes and Peyer's patches. J Immunol. 2009, 182: 1287-1295.View ArticlePubMed
- Okada T, Ngo VN, Ekland EH, Förster R, Lipp M, Littman DR, Cyster JG: Chemokine requirements for B cell entry to lymph nodes and Peyer's patches. J Exp Med. 2002, 196: 65-75. 10.1084/jem.20020201PubMed CentralView ArticlePubMed
- Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME, Sunshine MJ, Littman DR, Kuo CJ, Wei K, McMaster BE, Wright K, Howard MC, Schall TJ: A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med. 2006, 203: 2201-2213. 10.1084/jem.20052144PubMed CentralView ArticlePubMed
- Miao Z, Luker KE, Summers BC, Berahovich R, Bhojani MS, Rehemtulla A, Kleer CG, Essner JJ, Nasevicius A, Luker GD, Howard MC, Schall TJ: CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature. Proc Natl Acad Sci USA. 2007, 104: 15735-15740. 10.1073/pnas.0610444104PubMed CentralView ArticlePubMed
- Gross N, Meier R: Chemokines in neuroectodermal cancers: the crucial growth signal from the soil. Semin Cancer Biol. 2009, 19: 103-110.View ArticlePubMed
- Hattermann K, Held-Feindt J, Lucius R, Müerköster SS, Penfold ME, Schall TJ, Mentlein R: The Chemokine Receptor CXCR7 Is Highly Expressed in Human Glioma Cells and Mediates Antiapoptotic Effects. Cancer Res. 2010, 70: 3299-3308. 10.1158/0008-5472.CAN-09-3642View ArticlePubMed
- Zheng K, Li HY, Su XL, Wang XY, Tian T, Li F, Ren GS: Chemokine receptor CXCR7 regulates the invasion, angiogenesis and tumor growth of human hepatocellular carcinoma cells. J Exp Clin Cancer Res. 2010, 29: 31-44. 10.1186/1756-9966-29-31PubMed CentralView ArticlePubMed
- Maréchal R, Demetter P, Nagy N, Berton A, Decaestecker C, Polus M, Closset J, Devière J, Salmon I, Van Laethem JL: High expression of CXCR4 may predict poor survival in resected pancreatic adenocarcinoma. Br J Cancer. 2009, 100: 1444-1451. 10.1038/sj.bjc.6605020PubMed CentralView ArticlePubMed
- Iwakiri S, Mino N, Takahashi T, Sonobe M, Nagai S, Okubo K, Wada H, Date H, Miyahara R: Higher expression of chemokine receptor CXCR7 is linked to early and metastatic recurrence in pathological stage I nonsmall cell lung cancer. Cancer. 2009, 115: 2580-2593. 10.1002/cncr.24281View ArticlePubMed
- Wang J, Shiozawa Y, Wang J, Wang Y, Jung Y, Pienta KJ, Mehra R, Loberg R, Taichman RS: The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer. J Biol Chem. 2008, 283: 4283-4294.View ArticlePubMed
- Schutyser E, Su Y, Yu Y, Gouwy M, Zaja-Milatovic S, Van Damme J, Richmond A: Hypoxia enhances CXCR4 expression in human microvascular endothelial cells and human melanoma cells. Eur Cytokine Netw. 2007, 18: 59-70.PubMed CentralPubMed
- Izraely S, Klein A, Sagi-Assif O, Meshel T, Tsarfaty G, Hoon DS, Witz IP: Chemokine-chemokine receptor axes in melanoma brain metastasis. Immunol Lett. 2010, 130: 107-114. 10.1016/j.imlet.2009.12.003View ArticlePubMed
- Grymula K, Tarnowski M, Wysoczynski M, Drukala J, Barr FG, Ratajczak J, Kucia M, Ratajczak MZ: Overlapping and distinct role of CXCR7-SDF-1/ITAC and CXCR4-SDF-1 axes in regulating metastatic behavior of human rhabdomyosarcomas. Int J Cancer. 2010, 127: 2554-2268. 10.1002/ijc.25245PubMed CentralView ArticlePubMed
- Salmaggi A, Maderna E, Calatozzolo C, Gaviani P, Canazza A, Milanesi I, Silvani A, DiMeco F, Carbone A, Pollo B: CXCL12, CXCR4 and CXCR7 expression in brain metastases. Cancer Biol Ther. 2009, 8: 1608-1614. 10.4161/cbt.8.17.9202View ArticlePubMed
- Zabel BA, Wang Y, Lewén S, Berahovich RD, Penfold ME, Zhang P, Powers J, Summers BC, Miao Z, Zhao B, Jalili A, Janowska-Wieczorek A, Jaen JC, Schall TJ: Elucidation of CXCR7-mediated signaling events and inhibition of CXCR4-mediated tumor cell trans-endothelial migration by CXCR7 ligands. J Immunol. 2009, 183: 3204-3211. 10.4049/jimmunol.0900269View ArticlePubMed
- Balkwill F: The significance of cancer cell expression of the chemokine receptor CXCR4. Semin Cancer Biol. 2004, 14: 171-179. 10.1016/j.semcancer.2003.10.003View ArticlePubMed
- Smith MC, Luker KE, Garbow JR, Prior JL, Jackson E, Piwnica-Worms D, Luker GD: CXCR4 regulates growth of both primary and metastatic breast cancer. Cancer Res. 2004, 64: 8604-8612. 10.1158/0008-5472.CAN-04-1844View ArticlePubMed
- Kajiyama H, Shibata K, Terauchi M, Ino K, Nawa A, Kikkawa F: Involvement of SDF-1alpha/CXCR4 axis in the enhanced peritoneal metastasis of epithelial ovarian carcinoma. Int J Cancer. 2008, 122: 91-99. 10.1002/ijc.23083View ArticlePubMed
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