Oncogene (2015), 1–10 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc
ORIGINAL ARTICLE
Inhibition of TGFBIp expression reduces lymphangiogenesis and tumor metastasis Y-S Maeng1, B Aguilar2, S-I Choi1 and EK Kim1,3 Transforming growth factor-β-induced protein (TGFBIp) is an extracellular matrix protein that has a role in a wide range of pathological conditions. However, the role of TGFBIp signaling in lymphangiogenesis is poorly understood. The purpose of this study was therefore to analyze the effects of TGFBIp on lymphangiogenesis and determine whether TGFBIp-related lymphangiogenesis is important for the metastasis of tumor cells. TGFBIp increased adhesion, migration, and morphologic differentiation of human lymphatic endothelial cells (LECs), consistent with an increase in lymphatic vessel sprouting in a three-dimensional lymphatic ring assay. TGFBIp also induced phosphorylation of intracellular signaling molecules SRC, FAK, AKT, JNK and ERK. TGFBIp-induced lymphatic vessel sprouting was inhibited by addition of anti-integrin β3 antibody and pharmacologic inhibitors of FAK, AKT, JNK or ERK. TGFBIp increased both CCL21 expression in LECs, a chemokine that actively recruits tumor cells expressing the cognate chemokine receptors to lymphatic vessels and LEC permeability by inducing the dissociation of VE-cadherin junctions between LECs via the activation of SRC signaling. In vivo, inhibition of TGFBIp expression in SW620 cancer cells dramatically reduced tumor lymphangiogenesis and metastasis. Collectively, our findings demonstrate that TGFBIp is a lymphangiogenic factor contributing to tumor dissemination and represents a potential target to inhibit metastasis. Oncogene advance online publication, 16 March 2015; doi:10.1038/onc.2015.73
INTRODUCTION Metastatic spread of tumor cells to distant organs is the leading cause of mortality from cancer.1,2 Although metastatic tumor spread can occur via a variety of mechanisms, including direct local invasion of tissue or the seeding of body cavities, most metastases arise following the invasion of and dissemination via the circulatory systems. Although both blood and lymphatic vascular systems have been implicated, preclinical experimental systems suggest the most common pathway of initial metastasis is via the lymphatic system.3,4 Indeed, in many human cancers, the detection of tumor metastases in the tumor-draining lymph node (LN) is the first step in tumor dissemination and is one of the most important markers of both patient prognosis and therapeutic strategy decisions. Historically, lymphatic vessels were considered as passive participants in tumor metastasis by simply providing channels through which tumor cells could transit. However, the discovery of several key lymphatic-specific molecular markers and the increased availability of in vitro and in vivo experimental systems to study lymphatic biology have highlighted a much more complex, active role for the lymphatic vasculature in metastatic tumor spread. A vast number of lymphangiogenic factors, some previously identified as regulators of blood vascular endothelium,5 have been shown to induce a physiologic and/or tumor lymphangiogenesis and tumor spreading.4 Transforming growth factor-β-induced protein (TGFBIp, also known as βig-H3 and keratoepithelin) is a secreted extracellular matrix protein and is expressed in a wide range of cells, including fibroblasts, chondrocytes, smooth muscle cells, corneal epithelial cells and various types of cancer cells.6
Secreted TGFBIp interacts with other matrix proteins, such as fibronectin, collagen and laminin, and mediates cell adhesion, migration, spread and proliferation through interaction with integrins.7,8 TGFBIp is also known to be involved in cell growth, differentiation, wound healing, apoptosis, tumorigenesis, angiogenesis, tumor progression and metastasis.9–14 In tumorigenesis, however, TGFBIp has dual functions and can act as both a tumor suppressor and promoter, depending on the tumor microenvironment.15 However, its precise function remains obscure. Although TGFBIp can be a doubled edged sword with respect to tumorigenesis, its precise role in tumor lymphangiogenesis and metastasis is still unclear. In this study, we show TGFBIp-induced adhesion, migration, tube formation and sprouting of lymphatic vessels via the activation of SRC, FAK, AKT, JNK and ERK signaling pathways. Moreover, we demonstrate that TGFBIp increases not only the expression of CCL21, which recruits tumor cells to lymphatic vessels, but also lymphatic endothelial cell (LEC) permeability by inducing the dissociation of VE-cadherin junctions between LECs via the activation of SRC signaling. Finally, we show that mice bearing TGFBIp-expressing tumors develop more metastases via an increased lymphatic density. Taken together, these data demonstrate that TGFBIp functions as a pro-lymphangiogenic factor. RESULTS TGFBIp promotes lymphangiogenesis in vitro TGFBIp was identified as a regulator of angiogenesis both in vitro and in vivo.16 As blood and lymphatic systems share common
1 Corneal Dystrophy Research Institute, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea; 2Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA and 3Severance Biomedical Science Institute, Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea. Correspondence: Professor EK Kim, Department of Ophthalmology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea. E-mail: [email protected] Received 28 August 2014; revised 17 December 2014; accepted 5 February 2015
Inhibition of TGFBIp and reduction in tumor metastasis Y-S Maeng et al
2 structural and functional features, we asked whether the lymphatic system could also be regulated by TGFBIp. We initially focused on the effects of TGFBIp on cell proliferation, migration, adhesion, tube formation and survival of LECs. As shown in Figures 1a–c, TGFBIp increased cell migration, tube formation and adhesion in a dose-dependent manner. Several lines of evidence have identified integrins as the cellular receptors for TGFBIp;16,17 however, the integrin subtype responsible for mediating LEC binding to TGBFIp remains unknown. As such, we set out to elucidate the integrin subtype mediating this important interaction between LECs and TGFBIp. To accomplish this, we pretreated the cells with integrin αvβ3 or β3 blocking antibody, and performed in vitro lymphangiogenesis assay. As shown in Figures 1d–f, TGFBIp-induced LEC migration, tube formation and adhesion were inhibited by both αvβ3 and β3 blocking antibodies, with β3 integrin having a greater inhibitory effect on cellular function as compared with integrin αvβ3 suggesting an important role for β3 in facilitating TGFBIp-mediated LEC activation. These results suggest that TGFBIp promotes LEC migration, tube formation and adhesion through specific binding of β3 integrin. In our assessment of TGFBIp expression pattern in LECs, we noticed that LECs only produced a low basal level of TGFBIp.
To assess the effects of TGFBIp, which were produced by LECs themselves, on lymphangiogenic activity, TGFBIp-specific shRNA lentivirus was used to infect LECs, successfully reducing both TGFBIp mRNA (Figure 2a) and protein levels (Figure 2b). In LECs infected with TGFBIp shRNA, migration, tube formation, wound healing and adhesion activity were all significantly reduced (Figures 2c–j). Moreover, TGFBIp shRNA, but not control shRNA, significantly inhibited VEGF-C-induced LEC migration and tube formation (Figures 2c–f). In addition, to study whether TGFBIp was involved in lymphatic vessel sprouting, TGFBIp shRNA-infected LEC-coated beads were embedded in 3D fibrin gels and covered by WI-38 human fibroblasts. As shown in Figures 3a and b, control shRNA-infected LECs showed robust sprouting of lymphatic vessels, whereas TGFBIp shRNA-infected LEC-coated beads did not form any sprouts. A lymphatic ring assay was used to assess the sprouting capacity of LECs from a preexisting vessel, as well as the ability of these cells to proliferate, migrate and differentiate into capillaries.18 To evaluate the effects of TGFBIp on sprouting of lymphatic vessels from mouse thoracic ducts, isolated thoracic ducts were infected with mouse TGFBIp shRNA, and the lymphatic ring assay was performed. After 7 days, control shRNA-infected lymphatic ring cultures exhibited an outgrowth of cells that
Figure 1. TGFBIp promotes LEC migration, tube formation, adhesion through binding to integrin β3. (a–c) LECs were treated with TGFBIp (1, 5 and 10 μg/ml). (d–f) LECs were preincubated for 30 min with or without anti-integrin αvβ3 or β3 (1 μg/ml) antibody and stimulated with TGFBIp (10 μg/ml). Migration (a, d), tube formation (b, e) and adhesion (c, f) were quantified. All data are presented as the mean ± s.e. from three different experiments in duplicate. **P o0.01 vs TGFBIp alone. Oncogene (2015) 1 – 10
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Figure 2. Knockdown of TGFBIp expression inhibits LEC migration, tube formation, wound healing and adhesion. (a and b) TGFBIp mRNA and protein levels in LECs were measured by real-time qRT–PCR (a) and western blotting (b). (c–f) TGFBIp shRNA-infected LECs were treated with VEGF-C (20 ng/ml). (c and d) After 5 h, migration was quantified by counting the cells that migrated to the lower side of the filter with optical microscopy at × 200 magnification. (e and f) Tubular network formation on Matrigel was photographed and quantified at 24 h. (g and h) TGFBIp shRNA-infected LECs were seeded on 35-mm dishes. After confluence, cells were scratched with micropipette tips and washed to remove any debris. Images were captured at 0 and 16 h after wounding. For quantitative analysis, five fields per plate were photographed and distances between front lines were measured by using ImageJ software. (i and j) The 96-well plates were coated overnight (4 °C) with 0.1 mg/ml human fibronectin. TGFBIp shRNA-infected LECs in adhesion buffer were seeded at 105 cells/well in 100-μl volume and incubated for 30 min at 37 °C. Cell adhesion was quantified by counting the cells that attached to the fibronectin-coated matrix with optical microscopy at × 200 magnification. All data are presented as the mean ± s.e. from three different experiments in duplicate. **P o0.01 vs shControl.
organized into capillary-like structures. However, the cell outgrowth of the TGFBIp shRNA-infected lymphatic rings was significantly attenuated (Figure 3c). Moreover, we quantified the lymphatic vessel sprouts and their interconnected network by image binarization using a computer-assisted image analysis. As shown in Figure 3d, TGFBIp shRNA-infected lymphatic rings showed a marked reduction in the number of intersecting microvessels with a grid of concentric outlines of the thoracic duct boundary as a function of the distance to the ring compared with controls. In addition, lymphatic vessels sprouting from the thoracic duct were positively stained with CD31 and LYVE1, specific markers for LECs (Figure 3e). Real-time qRT–PCR and western blot analysis confirmed that the expression of TGFBIp mRNA and protein was significantly decreased in TGFBIp shRNAinfected lymphatic vessels as compared with control shRNA (Figures 3f and g). Collectively, these results indicate an important role for TGFBIp in the regulation of in vitro lymphangiogenic activity and lymphatic vessel sprouting. Stimulation of LECs in vitro by TGFBIp suggests that this factor can activate intracellular signaling pathways, as has been reported for VEGF-C.4 Therefore, we investigated the phosphorylation of intracellular lymphangiogenic signaling molecules in response to TGFBIp. Western blot analysis showed that TGFBIp increased the phosphorylation of ERK, AKT, FAK, JNK, FOXO1 and SRC in a timedependent manner (Figures 4a and b), but did not induced p38 © 2015 Macmillan Publishers Limited
activation (data not shown). Furthermore, to determine whether these intracellular signaling pathways had a functional relevance to TGFBIp’s biological activity, we infected LECs with TGFBIp shRNA and treated TGFBIp in the absence or presence of anti-integrin β3, CAS4506-66-5, wortmannin, SP600125, PD98059 and SB202190, which are integrin β3, FAK, PI3K, JNK, ERK and p38 inhibitors, respectively. An LEC 3D spheroid sprouting assay showed that TGFBIp shRNA significantly inhibited sprouting of lymphatic vessels, whereas the addition of recombinant TGFBIp to TGFBIp shRNA LECs rescued the lymphatic vessel sprouting reduced by shTGFBIp infection (Figures 4c and d). Moreover, pretreatment of LECs with integrin β3, FAK, PI3K, JNK and ERK inhibitor markedly inhibited lymphatic vessel sprouting stimulated by treatment with TGFBIp, but a p38 inhibitor that was not activated by TGFBIp had no effect (Figures 4c and d). These data show that TGFBIp induces phosphorylation of intracellular signaling molecules in pathways that are essential for TGFBIpmediated lymphangiogenesis. TGFBIp increases CCL21 expression through an ERK-dependent pathway In the cancer environment, chemokines (CXCL12, CCL19 and CCL21) secreted by LECs are key players in the active metastatic dissemination of tumor cells via the lymphatic system.19,20 Oncogene (2015) 1 – 10
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Figure 3. Knockdown of TGFBIp expression inhibits the lymphatic vessel sprouting and lymphangiogenesis in vitro. (a) 3D in vitro lymphangiogenesis with fibrin gel-embedded microbeads of TGFBIp shRNA-infected LECs. (b) Cumulative length of all sprouts originating from an individual spheroid was quantified after 5 days. **P o 0.01 vs shControl-infected group. (c) Thoracic duct explants embedded in type I collagen gel were infected with TGFBIp shRNA. After 7 days, a representative micrograph of an outgrowing explant is shown (left). The grid obtained by dilatation of the thoracic duct boundary is used for quantification (right). (d) The number of intersections between capillaries and the grid is plotted as a function of distance to the ring. (e) Immunostaining of lymphatic rings with anti-LYVE-1 and anti-CD31 antibodies. (f and g) TGFBIp mRNA and protein levels in sprouting lymphatic vessels were measured by real-time qRT–PCR (f) and western blotting (g). All data are presented as the mean ± s.e. from three different experiments with n = 6 per group per experiment. **P o 0.01 vs shControl.
They serve as chemoattractants for tumor cells, thus promoting metastasis. We thus examined whether TGFBIp induces the expression of chemokines that promote tumor metastasis. As shown in Figure 5a, TGFBIp increased the expression of CCL21 mRNA, and maximal expression was observed after 6 h, whereas mRNA levels of CCL19 and CXCL12 were unchanged in response to TGFBIp. Western blot analysis also confirmed the increased expression of CCL21 protein in the presence of TGFBIp (Figure 5b). Mitogen-activated protein kinase, signal transducers and activators of transcription 3, and nuclear factor-kappa B signaling pathways are known to be involved in modulating the expression of CCL21 through the interleukin family of cytokines in LECs.21 To determine whether these pathways are also involved in Oncogene (2015) 1 – 10
modulating CCL21 expression in the TGFBIp-stimulated LECs, we first examined the phosphorylation of IκBα, an upstream signaling molecule of nuclear factor-kappa B. As shown in Figure 5c, TGFBIp had no effect on IκBα phosphorylation over time. Furthermore, signal transducers and activators of transcription 3 activation was also not detected (data not shown). However, we already found strong ERK phosphorylation in stimulated cells in the presence of TGFBIp (Figure 4). To investigate the significance of the ERK signaling pathway in regulating CCL21 expression in TGFBIp-stimulated LECs, LECs were pretreated with PD98059, an ERK inhibitor and stimulated with TGFBIp. TGFBIp-stimulated LECs showed an increased expression of CCL21 and the effect was effectively blocked by PD98059 in a dose-dependent manner © 2015 Macmillan Publishers Limited
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Figure 4. TGFBIp-induced sprouting of LECs is mediated through integrin β3, FAK, PI3K, JNK and ERK signaling pathways. Representative western blotting images (a) and densitometric analyses (b) of phosphorylated ERK, AKT, FAK, JNK, FOXO1 and SRC. After serum-starved LECs were treated with TGFBIp (10 μg/ml) for the indicated periods of times (5, 10, 15, 30 or 60 min), cell lysates were subjected to western blotting by using IgGs against pERK, pAKT, pFAK, pJNK, pFOXO1 and pSRC. The membranes were then stripped and reprobed with IgGs against ERK, AKT, FAK, JNK, FOXO1 and SRC to estimate the total protein loaded. The relative ratios were normalized by arbitrarily setting the phosphorylation ratio at time 0 as 1. Experiments were performed in triplicate. Data are presented as the mean ± s.e.. *P o0.05 vs time 0. (c) TGFBIp shRNA-infected LEC spheroids were treated with TGFBIp (10 μg/ml), anti-integrin β3 antibody (1 μg/ml), CAS4506-66-5 (1 μM), wortmannin (50 nM), SP600125 (5 μM), PD98059 (10 μM), SB202190 (10 μM) or their combination as indicated and stained for PECAM-1. (d) Quantification of sprout number in the experiment. All data are presented as the mean ± s.e. from three different experiments with n = 7 per group per experiment. **P o0.01 vs shTGFBIp+rhTGFBIp.
(Figure 5d). These results suggest that the ERK signaling pathway is important for the regulation of CCL21 expression in TGFBIptreated LECs. TGFBIp stimulates lymphatic permeability Specialized cell–cell junctions expressing VE-cadherin and tight junction-associated proteins such as zona occludens 1 (ZO-1) have been reported to localize between LECs.22 Disruption of functional organization of such structures may lead to vascular fragility and increase permeability. We therefore examined whether TGFBIp could alter lymphatic permeability. Phosphorylation of SRC (on tyrosine 418) and VE-cadherin is required for stimulation of endothelial cell permeability.23 Similarly, we found that TGFBIp increased the phosphorylation of SRC and VE-cadherin in a timedependent manner (Figures 6a and b). To further investigate the regulation of VE-cadherin turnover in response to TGFBIp, the localization of VE-cadherin was examined. Without stimulation, VE-cadherin localized to the plasma membrane and displayed a linear pattern in the cell borders (Figure 6c, top left). In contrast, in LECs treated with TGFBIp for 30 min, VE-cadherin levels diminished at the plasma membrane and the linear pattern disrupted, and disorganized at cell borders, presumably because of increased VE-cadherin endocytosis or internalization (Figure 6c, © 2015 Macmillan Publishers Limited
top right). Additionally, to determine whether cell-surface-derived VE-cadherin was internalized, cell-surface VE-cadherin was labeled in live LECs and then cells were acid-washed (low pH). This approach demonstrated that a cell-surface pool of VE-cadherin was internalized by TGFBIp (Figure 6c, bottom right). However, in non-TGFBIp-treated cells, internalized VE-cadherin was not detected (Figure 6c, bottom left). Similar results were also obtained from trypsin digestion experiments. As shown in Figure 6d, treatment of LECs with TGFBIp increased the amount of trypsin-resistant VE-cadherin in a time-dependent manner, suggesting that TGFBIp stimulates internalization of cell-surface VE-cadherin, because internalized cell-surface proteins are resistant to trypsin digestion. This finding raises the possibility that TGFBIp is capable of stimulating the permeability of LEC monolayers by inducing the opening of intercellular junctions. Using a Transwell system, we determined the permeability of LEC monolayers stimulated with TGFBIp. Stimulation of LECs with TGFBIp significantly increased the permeability of FITC-dextran across the cell monolayer in a dose- and time-dependent manner compared with that of the positive control VEGF-C or vehicle alone (Figures 6e and f). These results indicate that TGFBIp induces lymphatic permeability by disorganizing lymphatic VE-cadherin junctions. Oncogene (2015) 1 – 10
Inhibition of TGFBIp and reduction in tumor metastasis Y-S Maeng et al
6 blood vessel-specific markers, LYVE-1 and CD31, respectively. shTGFBIp tumors showed a very low density of lymphatic vessels stained with LYVE-1/CD31 in the peri-tumoral (margin of tumor) (Figure 7e) and intratumoral (central region of tumor) areas (Figure 7f). Quantification of LYVE-1/CD31-positive lymphatic structures confirmed that vessel density was significantly reduced in shTGFBIp-expressing tumors as compared with controls (Figure 7g). However, only CD31-positive blood vessel density was slightly decreased in shTGFBIp tumors (Figure 7g). To further validate the effects of TGFBIp on tumor metastasis via the lymphatic system, shTGFBIp tumor bearing mice were sacrificed and examined for metastases in the lung and other organs. Microscopic analysis of the lungs revealed a significant decrease in the number of metastatic nodules per lung from shTGFBIp-expressing tumors versus control tumors (Figure 8a), which was confirmed by quantification of the number of metastatic nodules per lung (Figure 8b). Moreover, tumor cell-specific CCR7 staining and immunohistochemical analysis revealed significantly reduced lung (Figures 8c and d) and lateral axillary LN metastases (Figures 8e and f) in the shTGFBIp tumor bearing mice as compared with control mice. Overall, our data suggest that inhibition of TGFBIp expression is useful to suppress the metastatic potential of colon cancer cells via the lymphatic system without affecting the growth rate of tumors.
Figure 5. TGFBIp increases CCL21 expression through an ERKdependent pathway. (a) Total mRNA was isolated from LECs treated with TGFBIp (10 μg/ml) or untreated for the indicated times, and CCL21, CXCL12 and CCL19 genes expression was assessed by realtime qRT–PCR analysis. (b and c) LECs were treated with TGFBIp (10 μg/ml). (d) LECs were preincubated for 30 min with or without PD98059 (1, 5 and 10 μM) and stimulated with TGFBIp (10 μg/ml). Cell lysates were subjected to western blotting by using IgGs against CCL21 (b and d) and phospho-IκBα (c). The membranes were then stripped and reprobed with IgGs against actin and IκBα to estimate the total protein loaded. Experiments were performed in triplicate. Data are presented as the mean ± s.e. **P o0.01 vs control.
Inhibition of TGFBIp expression reduces tumor lymphangiogenesis and tumor metastasis Metastasis is the principal cause of cancer mortality. In the past decade, converging data has highlighted the importance of the tumor lymphatic vasculature in colon cancer dissemination. We investigated the capacity of TGFBIp to promote tumor lymphangiogenesis and subsequent dissemination in TGFBIpexpressing colon cancer models: a subcutaneous xenograft of the human colon cancer SW620 cell line that displays an aggressive metastatic pattern.24 Stable expression of TGFBIp shRNA constructs considerably reduced the protein levels of TGFBIp in SW620 cells (Figure 7a). Equal numbers of TGFBIp shRNA- or control shRNA-expressing SW620 cells were implanted subcutaneously into NOD-SCID mice, and the growth of the resultant primary tumors was monitored. Interestingly, there was no significant difference in the average size and weight of the tumors derived from the shTGFBIp-SW620 and shcontrol-SW620 cells (Figures 7b and d). Additionally, in vitro growth rates were not modified by inhibition of TGFBIp expression (unpublished data). To determine whether TGFBIp could induce tumor lymphangiogenesis, tumors were sectioned and stained for lymphatic- or Oncogene (2015) 1 – 10
DISCUSSION Tumor-induced lymphangiogenesis is known to promote lymphatic metastasis, and lymphangiogenic growth factors that are produced and secreted by the tumors themselves are critical activators of tumor lymphangiogenesis during the process of metastasis.25–27 In the present study, we provide an evidence that TGFBIp acts both in vitro and in vivo as a lymphangiogenic factor. First, we show that TGFBIp promotes in vitro lymphangiogenesis. TGFBIp induces migration, tube formation and adhesion of human LECs in both a time- and dose-dependent manner; these effects are mediated by the binding of TGFBIp with integrin β3 specifically. This finding is consistent with a report showing that inhibition of TGFBIp expression significantly inhibited in vitro 3D lymphatic bead sprouting, 3D spheroid sprouting and thoracic duct lymphatic ring sprouting. In addition, the lymphatic vessel sprouting recovered by the addition of recombinant TGFBIp was markedly inhibited by anti-integrin β3 antibody and specific inhibitors of FAK, AKT, JNK and ERK, but the p38 inhibitor had no effect. These results suggest that TGFBIp produced by LECs is secreted into the extracellular matrix, where it binds to integrin β3 and activates the intracellular signaling pathways essential for TGFBIp-mediated lymphangiogenesis, thus generating a positive feedback mechanism. Chemotactic gradients of chemokine ligands established by tumor-associated LECs and LNs actively guide chemokine receptor-positive metastatic cells initially into lymphatic vessels and thereby further enhance metastasis.20 Our data show that the expression of CCL21 was significantly increased by TGFBIp in LECs via an ERK-dependent pathway, whereas that of other chemokines (that is, CCL19 and CXCL12) did not change in response to TGFBIp. These data indicate that the expression of CCL21 is regulated by TGFBIp via an ERK-dependent signaling pathway and may subsequently promote the active migration of chemokine receptor-positive cancer cells into the lymphatic vessels. Control of lymphatic permeability might also be a crucial element contributing to metastasis.22 Here we showed that TGFBIp activates the phosphorylation of SRC and VE-cadherin, which has been shown to correlate with increased permeability of LECs similar to lymphatic factors.28,29 VE-cadherin that was diminished at the cell surface by TGFBIp internalized into the cytosol, which led to open gaps between LECs and increased the permeability of FITC-dextran across the cell monolayer in both © 2015 Macmillan Publishers Limited
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Figure 6. TGFBIp increases LEC permeability by disorganizing lymphatic VE-cadherin junctions. (a and b) Confluent LECs were treated with TGFBIp (10 μg/ml) for the indicated times. (a) Western blot analysis was performed by using an antibody specific for phospho-SRC (tyrosine 418) and anti-SRC antibody. (b) Cell lysates were subject to affinity precipitation by using anti-VE-cadherin antibody. Western blot analysis was performed by using an antibody specific for phospho-tyrosine and VE-cadherin. (c) Confluent LECs were stimulated with TGFBIp (10 μg/ml) for 30 min. The cells were then fixed and stained with anti-VE-cadherin antibody. Arrows indicate the disruption of VE-cadherin junction at the plasma membrane (top). VE-cadherin internalization was monitored by internal acid-resistant vesicles, as described in the Materials and methods. Arrows point to VE-cadherin in internal vesicle-like compartments (bottom). (d) Confluent LECs were treated with TGFBIp (10 μg/ml). At the indicated times, cells were treated with trypsin, pelleted and lysed for further analysis of VE-cadherin levels present in these trypsin-resistant fractions. Total cell lysates without trypsin treatment were used as controls. Cell lysates were then analyzed by western blot analysis by using anti-VE-cadherin antibody. (e and f) LECs were plated onto a Transwell filter. After reaching confluence, LECs monolayers were treated with TGFBIp (2, 5 and 10 μg/ml) or VEGF-C (20 ng/ml) for 30 min (e). LECs monolayers were treated with TGFBIp (10 μg/ml) for the indicated times (f). FITC-dextran permeability was performed. Experiments were performed in triplicate. Data are presented as the mean ± s.e. **P o0.01 vs control.
a dose- and time-dependent manner. Our results indicate that TGFBIp induces lymphatic permeability by disorganizing lymphatic VE-cadherin junctions and thereby may facilitate intravasation of tumor cells into lymphatic vessels. Finally, inhibition of TGFBIp expression in colon cancer cells decreased tumor lymphangiogenesis and metastasis without a significant effect on tumor growth rate. Lymphatic vessels with wider lumens were observed in high TGFBIp-expressing tumors and were correlated with increased tumor metastasis to the lung and LNs. Analysis of LYVE-1/CD31-positive lymphatic structures confirmed that vessel density was significantly reduced in shTGFBIp-expressing tumors as compared to controls expressing high TGFBIp levels. However, CD31-positive blood vessel density © 2015 Macmillan Publishers Limited
was slightly decreased in shTGFBIp tumors, suggesting that TGFBIp derived from tumor cells has a stronger effect on lymphatic vessels than on blood vessels. Furthermore, analysis of tumor cell metastasis in mouse organs revealed a significant decrease in the number of metastatic nodules per lung and reduced lateral axillary LN metastasis in shTGFBIp-expressing tumors. Our data suggest that inhibition of TGFBIp expression is useful to suppress the metastatic potential of colon cancer cells via the lymphatic system. In summary, our findings clearly show that TGFBIp is a lymphangiogenic factor that induces metastasis via three probable mechanisms: (1) by increasing lymphatic density and consequently augmenting surface contact with cancer cells; (2) by Oncogene (2015) 1 – 10
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Figure 7. Inhibition of TGFBIp expression reduces tumor lymphangiogenesis in colon cancer. (a) TGFBIp protein levels in TGFBIp shRNAinfected SW620 cells were measured by western blotting. SW620 cells were injected subcutaneously into the midline of the backs of NOS-SCID mice (n = 5 per group). Tumors were removed after 33 days, excised and serially sectioned. (b) Representative tumors. (c and d) Analysis of tumor growth rates and weights. (e) Margins of tumor sections were stained for infiltrating LECs using anti-LYVE1 and anti-CD31 antibodies. Nuclei were stained with DAPI (blue fluorescence). Arrows indicate lymphatic vessels. Yellow dotted lines point to margins of the tumor. Images were viewed using an Olympus IX81-ZDC microscope with a LUCPL FLN 10 × /0.40 NA lens. (f) Central regions of tumor sections were stained for infiltrating LECs using anti-LYVE1 and anti-CD31 antibodies. Nuclei were stained with DAPI (blue fluorescence). Arrows indicate lymphatic vessels. Images were viewed by using an Olympus IX81-ZDC microscope with a LUCPL FLN 10 × /0.40 NA lens. (g) Quantitative assessment of LYVE1 and CD31-positive lymphatic vessels per field for each tumor section. Data are presented as the mean ± s.e. **P o 0.01 vs shControl group.
increasing CCL21 expression in LECs and promoting migration of cancer cells; and (3) by increasing lymphatic permeability and facilitating intravasation and/or extravasation of tumor cells (Figure 8g). Our data suggest, for the first time, that TGFBIp may contribute to lymphatic metastasis, supporting the strategy of targeting not only tumor-derived functions, but also targeting functions of the host that support the tumor. Overall, our findings suggest that TGFBIp can be a potential therapeutic target for treating metastatic colon cancer. MATERIALS AND METHODS Cell culture Primary human LECs were purchased from PromoCell (Heidelberg, Germany) and cultured in endothelial basal medium (EBM; Lonza, Walkersville, MD, USA) supplemented with 10% FBS and other factors as previously described.30 LECs less than eight passages were used for all experiments. SW620 cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and antibiotics.
In vitro tube formation assay Tube formation was assayed as previously described.31 In brief, 250 μl Matrigel (BD Biosciences, Bedford, MA, USA) was added to a 16-mm diameter tissue culture well and allowed to polymerize for 30 min at 37 °C. After trypsinization, the harvested LECs were resuspended in EBM containing rhTGFBIp (5 ~ 10 μg/ml, Sino Biological Inc., Beijing, China) or recombinant human VEGF-C (Upstate Biotechnology, Lake Placid, NY, USA), and plated onto the layer of Matrigel (1.2 × 105 cells/well). Matrigel cultures were incubated at 37 °C and photographed at various time points Oncogene (2015) 1 – 10
(×200 magnification). The area covered by the tube network was determined with an optical imaging technique: Pictures of the tubes were scanned into Adobe Photoshop and quantified by ImageJ software (National Institutes of Health).
Cell-matrix adhesion Cell-matrix adhesion assays were performed as described previously.32 The 96-well plates were coated overnight (4 °C) with 1–10 μg/ml rhTGFBIp or 0.1 mg/ml human fibronectin (Sigma-Aldrich, St Louis, MO, USA). LECs in adhesion buffer (serum free media) were seeded at 105 cells/well in 100-μl volume and incubated for 30 min at 37 °C. After the removal of nonadherent cells after two washes, adherent cells were measured by hematoxylin and eosin staining, and quantified in triplicate by counting adherent cells in five randomly selected fields per well (Axiovert 100; Carl Zeiss Micro-Imaging, Thornwood, NY, USA). Results are representative of three different experiments in duplicate.
Thoracic duct collection and 3D lymphatic ring assay Identification and harvesting of thoracic ducts were conducted as described previously.33 Briefly, the dissected thoracic duct was cut transversely to generate lymphatic duct fragments, which then were embedded into type I collagen gels. An shRNA lentivirus against the mouse TGFBIp gene (Santa Cruz Biotechnology Inc.) was added to the culture medium at the beginning of the experiment as appropriate. Seven days later, these lymphatic ring cultures exhibited an outgrowth of cells that organized into capillary-like structures. Quantification using computerized image analysis was based on the elaboration of a grid obtained by the successive dilatation of the explant boundary, after image binarization. Rat Anti-mouse CD31 monoclonal antibody (Clone: MEC 13.3, 1:200 dilution, BD PharMingen, San Diego, CA, USA) or rabbit anti-mouse LYVE1 © 2015 Macmillan Publishers Limited
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Figure 8. Inhibition of TGFBIp expression suppresses the tumor metastasis to LN and lung. (a) Images of lung metastases of mice injected with SW620 cells. (b) The number of metastatic nodules per lung was quantified by a microscopic inspection. **P o 0.01 vs shControl group. (c and e) CCR7 and hematoxylin and eosin staining with frozen sections of lung and lateral axillary LN tissues isolated from mice subcutaneously injected with SW620 cells. (d and f) The incidence of metastasis per lung or lateral axillary LN was quantified by microscopic analysis. Red dotted lines indicate tumors in the mouse lateral axillary LN. Data are presented as the mean ± s.e. **P o0.01 vs shControl group. (g) A schematic diagram of a proposed mechanism of TGFBIp on tumor lymphangiogenesis and metastasis. polyclonal antibody (1:200 dilution, Angiobio Co., Del Mar, CA, USA) was used to identify the sprouting of lymphatic vessels from the thoracic duct.
Immunofluorescence staining and immunohistochemistry Confluent LECs were fixed in 3.7% formaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS); LECs were then preincubated with a blocking solution of PBS containing 5% normal donkey serum and 0.05% Tween-20. Cells were incubated with goat anti-VE-cadherin polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology Inc.) for 2 h at room temperature and labeled with a fluorescein-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA). Samples were observed with a fluorescence microscope (Olympus, Tokyo, Japan). Eight micrometer-thick frozen tumor sections were washed in PBS and blocked in 5% normal donkey serum in an antibody dilution buffer consisting of PBS containing 0.1% Triton X-100. Sections were then incubated overnight in primary antibody (Rat anti-mouse CD31 monoclonal antibody (Clone: MEC 13.3, 1:200 dilution) or rabbit anti-mouse LYVE1 polyclonal antibody (1:200 dilution, Angiobio Co.)) at 4 °C and labeled with a fluorescein-conjugated secondary antibody (Molecular Probes). Nuclei were counterstained with DAPI and are seen in blue. Samples were observed with a fluorescence microscope (Olympus). The number of lymphatic vessels within the tumor was counted in six fields per section: the center regions of the tumor (intratumor vessel density) and within an area 1 mm from the tumor border (peri-tumor vessel density). Seven slide sections per mouse were analyzed. To determine the metastasis of SW620 tumor cells to mouse lateral axillary LNs and lungs, mouse LNs and lungs sections (seven slide sections per mouse) were © 2015 Macmillan Publishers Limited
stained with hematoxylin and eosin stain and rabbit anti-CCR7 monoclonal antibody (Clone:Y59, 1:200 dilution, Abcam Inc. Cambridge, MA, USA).
VE-cadherin internalization assay (immunofluorescence label) VE-cadherin internalization assays were performed as described previously.34 A mouse monoclonal antibody directed against the VE-cadherin extracellular domain (Clone: BV6, 1:200, Merck Millipore, Darmstadt, Germany) was dialyzed into MCDB 131 medium containing 20 mM HEPES and 3% BSA. The dialyzed antibody was incubated with LEC cultures at 4 °C for 1 h. Unbound antibody was removed by rinsing cells in ice-cold MCDB 131. Cells were incubated at 37 °C for 30 min in the absence or presence of 10 μg/ml TGFBIp. To remove cell surface-bound antibody while retaining internalized antibody, cells were washed for 15 min in PBS (pH 2.7) containing 25 mM glycine and 3% BSA. The cells were rinsed, fixed, and processed for immunofluorescence labeling.
Internalization of cell-surface VE-cadherin (trypsin digestion) LECs grown in six-well plates were treated with vehicle only or TGFBIp (10 μg/ml) at 37 °C for 30 min. Following treatment, cells were untreated or treated with trypsin as previously described35 to remove cell surface proteins. Trypsin-undigested and trypsin-digested cells were subsequently lysed, subjected to reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and electro-transferred onto polyvinylidene difluoride membranes. Internalized or intracellular VE-cadherin, which was resistant to trypsin digestion, on polyvinylidene difluoride membranes was visualized using streptavidin-HRP and the ECL system. Oncogene (2015) 1 – 10
Inhibition of TGFBIp and reduction in tumor metastasis Y-S Maeng et al
10 Mouse tumor models and in vivo procedures SW620 cancer cells were harvested from subconfluent cell culture plates, washed with PBS, and resuspended at a concentration of 2.5 × 107 cells per ml DMEM containing 10% FBS. Of the suspended cells, an aliquot of 0.2 ml was injected subcutaneously into the right posterior flank of 5-week-old NOD-SCID mice (Orient Company, Seongnam, Korea) with five mice in each group. Tumors were measured with calipers to estimate volumes on days 1–33 after injection. Four weeks after injection, mice were sacrificed and examined for the growth of subcutaneous tumors. All organs were removed for examination, and lateral axillary LN around tumor region and lung metastases were detected by hematoxylin and eosin staining and quantified by counting metastatic lesions in each section. All studies were repeated twice to ensure reproducibility (with a minimum of five mice per group).
Statistical analysis All experiments were repeated at least three times. Data are presented as the means ± s.e., and statistical comparisons between groups were performed by one-way ANOVA followed by Tukey’s test. Supplementary Information shows the additional materials and methods.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0028699).
AUTHOR CONTRIBUTIONS Conceived and designed the experiments: YSM, BA, SIC. Performed the experiments: YSM, SIC. Analyzed the data: YSM, SIC, EKK. Contributed reagents/ materials/analysis tools: YSM, SIC, EKK. Wrote the manuscript: YSM, BA, EKK.
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12 Kim JE, Kim SJ, Jeong HW, Lee BH, Choi JY, Park RW et al. RGD peptides released from beta ig-h3, a TGF-beta-induced cell-adhesive molecule, mediate apoptosis. Oncogene 2003; 22: 2045–2053. 13 Zhang Y, Wen G, Shao G, Wang C, Lin C, Fang H et al. TGFBI deficiency predisposes mice to spontaneous tumor development. Cancer Res 2009; 69: 37–44. 14 Becker J, Erdlenbruch B, Noskova I, Schramm A, Aumailley M, Schorderet DF et al. Keratoepithelin suppresses the progression of experimental human neuroblastomas. Cancer Res 2006; 66: 5314–5321. 15 Ween MP, Oehler MK, Ricciardelli C. Transforming growth factor-beta-induced protein (TGFBI)/(betaig-H3): a matrix protein with dual functions in ovarian cancer. Int J Mol Sci 2012; 13: 10461–10477. 16 Nam JO, Kim JE, Jeong HW, Lee SJ, Lee BH, Choi JY et al. Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect. J Biol Chem 2003; 278: 25902–25909. 17 Irigoyen M, Anso E, Salvo E, Dotor de las Herrerias J, Martinez-Irujo JJ, Rouzaut A. TGFbeta-induced protein mediates lymphatic endothelial cell adhesion to the extracellular matrix under low oxygen conditions. Cell Mol Life Sci 2008; 65: 2244–2255. 18 Lee AS, Kim DH, Lee JE, Jung YJ, Kang KP, Lee S et al. Erythropoietin induces lymph node lymphangiogenesis and lymph node tumor metastasis. Cancer Res 2011; 71: 4506–4517. 19 Christiansen A, Detmar M. Lymphangiogenesis and cancer. Genes Cancer 2011; 2: 1146–1158. 20 Albrecht I, Christofori G. Molecular mechanisms of lymphangiogenesis in development and cancer. Int J Dev Biol 2011; 55: 483–494. 21 Miyagaki T, Sugaya M, Okochi H, Asano Y, Tada Y, Kadono T et al. Blocking MAPK signaling downregulates CCL21 in lymphatic endothelial cells and impairs contact hypersensitivity responses. J Invest Dermatol 2011; 131: 1927–1935. 22 Baluk P, Fuxe J, Hashizume H, Romano T, Lashnits E, Butz S et al. Functionally specialized junctions between endothelial cells of lymphatic vessels. J Exp Med 2007; 204: 2349–2362. 23 Dejana E, Orsenigo F, Lampugnani MG. The role of adherens junctions and VEcadherin in the control of vascular permeability. J Cell Sci 2008; 121: 2115–2122. 24 Hewitt RE, McMarlin A, Kleiner D, Wersto R, Martin P, Tsokos M et al. Validation of a model of colon cancer progression. J Pathol 2000; 192: 446–454. 25 Mohammed RA, Green A, El-Shikh S, Paish EC, Ellis IO, Martin SG. Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio- and lymphangiogenesis. Br J Cancer 2007; 96: 1092–1100. 26 Schoppmann SF, Fenzl A, Nagy K, Unger S, Bayer G, Geleff S et al. VEGF-C expressing tumor-associated macrophages in lymph node positive breast cancer: impact on lymphangiogenesis and survival. Surgery 2006; 139: 839–846. 27 Wartiovaara U, Salven P, Mikkola H, Lassila R, Kaukonen J, Joukov V et al. Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation. Thromb Haemost 1998; 80: 171–175. 28 Cao R, Eriksson A, Kubo H, Alitalo K, Cao Y, Thyberg J. Comparative evaluation of FGF-2-, VEGF-A-, and VEGF-C-induced angiogenesis, lymphangiogenesis, vascular fenestrations, and permeability. Circ Res 2004; 94: 664–670. 29 Weis S, Cui J, Barnes L, Cheresh D. Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis. J Cell Biol 2004; 167: 223–229. 30 Aguilar B, Choi I, Choi D, Chung HK, Lee S, Yoo J et al. Lymphatic reprogramming by Kaposi sarcoma herpes virus promotes the oncogenic activity of the virus-encoded G-protein-coupled receptor. Cancer Res 2012; 72: 5833–5842. 31 Min JK, Cho YL, Choi JH, Kim Y, Kim JH, Yu YS et al. Receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) increases vascular permeability: impaired permeability and angiogenesis in eNOS-deficient mice. Blood 2007; 109: 1495–1502. 32 Maeng YS, Choi HJ, Kwon JY, Park YW, Choi KS, Min JK et al. Endothelial progenitor cell homing: prominent role of the IGF2-IGF2R-PLCbeta2 axis. Blood 2009; 113: 233–243. 33 Bruyere F, Melen-Lamalle L, Blacher S, Roland G, Thiry M, Moons L et al. Modeling lymphangiogenesis in a three-dimensional culture system. Nat Methods 2008; 5: 431–437. 34 Paterson AD, Parton RG, Ferguson C, Stow JL, Yap AS. Characterization of E-cadherin endocytosis in isolated MCF-7 and chinese hamster ovary cells: the initial fate of unbound E-cadherin. J Biol Chem 2003; 278: 21050–21057. 35 Boensch C, Huang SS, Connolly DT, Huang JS. Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells. J Biol Chem 1999; 274: 10582–10589.
Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)
Oncogene (2015) 1 – 10
© 2015 Macmillan Publishers Limited
ORIGINAL ARTICLE
Inhibition of TGFBIp expression reduces lymphangiogenesis and tumor metastasis Y-S Maeng1, B Aguilar2, S-I Choi1 and EK Kim1,3 Transforming growth factor-β-induced protein (TGFBIp) is an extracellular matrix protein that has a role in a wide range of pathological conditions. However, the role of TGFBIp signaling in lymphangiogenesis is poorly understood. The purpose of this study was therefore to analyze the effects of TGFBIp on lymphangiogenesis and determine whether TGFBIp-related lymphangiogenesis is important for the metastasis of tumor cells. TGFBIp increased adhesion, migration, and morphologic differentiation of human lymphatic endothelial cells (LECs), consistent with an increase in lymphatic vessel sprouting in a three-dimensional lymphatic ring assay. TGFBIp also induced phosphorylation of intracellular signaling molecules SRC, FAK, AKT, JNK and ERK. TGFBIp-induced lymphatic vessel sprouting was inhibited by addition of anti-integrin β3 antibody and pharmacologic inhibitors of FAK, AKT, JNK or ERK. TGFBIp increased both CCL21 expression in LECs, a chemokine that actively recruits tumor cells expressing the cognate chemokine receptors to lymphatic vessels and LEC permeability by inducing the dissociation of VE-cadherin junctions between LECs via the activation of SRC signaling. In vivo, inhibition of TGFBIp expression in SW620 cancer cells dramatically reduced tumor lymphangiogenesis and metastasis. Collectively, our findings demonstrate that TGFBIp is a lymphangiogenic factor contributing to tumor dissemination and represents a potential target to inhibit metastasis. Oncogene advance online publication, 16 March 2015; doi:10.1038/onc.2015.73
INTRODUCTION Metastatic spread of tumor cells to distant organs is the leading cause of mortality from cancer.1,2 Although metastatic tumor spread can occur via a variety of mechanisms, including direct local invasion of tissue or the seeding of body cavities, most metastases arise following the invasion of and dissemination via the circulatory systems. Although both blood and lymphatic vascular systems have been implicated, preclinical experimental systems suggest the most common pathway of initial metastasis is via the lymphatic system.3,4 Indeed, in many human cancers, the detection of tumor metastases in the tumor-draining lymph node (LN) is the first step in tumor dissemination and is one of the most important markers of both patient prognosis and therapeutic strategy decisions. Historically, lymphatic vessels were considered as passive participants in tumor metastasis by simply providing channels through which tumor cells could transit. However, the discovery of several key lymphatic-specific molecular markers and the increased availability of in vitro and in vivo experimental systems to study lymphatic biology have highlighted a much more complex, active role for the lymphatic vasculature in metastatic tumor spread. A vast number of lymphangiogenic factors, some previously identified as regulators of blood vascular endothelium,5 have been shown to induce a physiologic and/or tumor lymphangiogenesis and tumor spreading.4 Transforming growth factor-β-induced protein (TGFBIp, also known as βig-H3 and keratoepithelin) is a secreted extracellular matrix protein and is expressed in a wide range of cells, including fibroblasts, chondrocytes, smooth muscle cells, corneal epithelial cells and various types of cancer cells.6
Secreted TGFBIp interacts with other matrix proteins, such as fibronectin, collagen and laminin, and mediates cell adhesion, migration, spread and proliferation through interaction with integrins.7,8 TGFBIp is also known to be involved in cell growth, differentiation, wound healing, apoptosis, tumorigenesis, angiogenesis, tumor progression and metastasis.9–14 In tumorigenesis, however, TGFBIp has dual functions and can act as both a tumor suppressor and promoter, depending on the tumor microenvironment.15 However, its precise function remains obscure. Although TGFBIp can be a doubled edged sword with respect to tumorigenesis, its precise role in tumor lymphangiogenesis and metastasis is still unclear. In this study, we show TGFBIp-induced adhesion, migration, tube formation and sprouting of lymphatic vessels via the activation of SRC, FAK, AKT, JNK and ERK signaling pathways. Moreover, we demonstrate that TGFBIp increases not only the expression of CCL21, which recruits tumor cells to lymphatic vessels, but also lymphatic endothelial cell (LEC) permeability by inducing the dissociation of VE-cadherin junctions between LECs via the activation of SRC signaling. Finally, we show that mice bearing TGFBIp-expressing tumors develop more metastases via an increased lymphatic density. Taken together, these data demonstrate that TGFBIp functions as a pro-lymphangiogenic factor. RESULTS TGFBIp promotes lymphangiogenesis in vitro TGFBIp was identified as a regulator of angiogenesis both in vitro and in vivo.16 As blood and lymphatic systems share common
1 Corneal Dystrophy Research Institute, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea; 2Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA and 3Severance Biomedical Science Institute, Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea. Correspondence: Professor EK Kim, Department of Ophthalmology, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea. E-mail: [email protected] Received 28 August 2014; revised 17 December 2014; accepted 5 February 2015
Inhibition of TGFBIp and reduction in tumor metastasis Y-S Maeng et al
2 structural and functional features, we asked whether the lymphatic system could also be regulated by TGFBIp. We initially focused on the effects of TGFBIp on cell proliferation, migration, adhesion, tube formation and survival of LECs. As shown in Figures 1a–c, TGFBIp increased cell migration, tube formation and adhesion in a dose-dependent manner. Several lines of evidence have identified integrins as the cellular receptors for TGFBIp;16,17 however, the integrin subtype responsible for mediating LEC binding to TGBFIp remains unknown. As such, we set out to elucidate the integrin subtype mediating this important interaction between LECs and TGFBIp. To accomplish this, we pretreated the cells with integrin αvβ3 or β3 blocking antibody, and performed in vitro lymphangiogenesis assay. As shown in Figures 1d–f, TGFBIp-induced LEC migration, tube formation and adhesion were inhibited by both αvβ3 and β3 blocking antibodies, with β3 integrin having a greater inhibitory effect on cellular function as compared with integrin αvβ3 suggesting an important role for β3 in facilitating TGFBIp-mediated LEC activation. These results suggest that TGFBIp promotes LEC migration, tube formation and adhesion through specific binding of β3 integrin. In our assessment of TGFBIp expression pattern in LECs, we noticed that LECs only produced a low basal level of TGFBIp.
To assess the effects of TGFBIp, which were produced by LECs themselves, on lymphangiogenic activity, TGFBIp-specific shRNA lentivirus was used to infect LECs, successfully reducing both TGFBIp mRNA (Figure 2a) and protein levels (Figure 2b). In LECs infected with TGFBIp shRNA, migration, tube formation, wound healing and adhesion activity were all significantly reduced (Figures 2c–j). Moreover, TGFBIp shRNA, but not control shRNA, significantly inhibited VEGF-C-induced LEC migration and tube formation (Figures 2c–f). In addition, to study whether TGFBIp was involved in lymphatic vessel sprouting, TGFBIp shRNA-infected LEC-coated beads were embedded in 3D fibrin gels and covered by WI-38 human fibroblasts. As shown in Figures 3a and b, control shRNA-infected LECs showed robust sprouting of lymphatic vessels, whereas TGFBIp shRNA-infected LEC-coated beads did not form any sprouts. A lymphatic ring assay was used to assess the sprouting capacity of LECs from a preexisting vessel, as well as the ability of these cells to proliferate, migrate and differentiate into capillaries.18 To evaluate the effects of TGFBIp on sprouting of lymphatic vessels from mouse thoracic ducts, isolated thoracic ducts were infected with mouse TGFBIp shRNA, and the lymphatic ring assay was performed. After 7 days, control shRNA-infected lymphatic ring cultures exhibited an outgrowth of cells that
Figure 1. TGFBIp promotes LEC migration, tube formation, adhesion through binding to integrin β3. (a–c) LECs were treated with TGFBIp (1, 5 and 10 μg/ml). (d–f) LECs were preincubated for 30 min with or without anti-integrin αvβ3 or β3 (1 μg/ml) antibody and stimulated with TGFBIp (10 μg/ml). Migration (a, d), tube formation (b, e) and adhesion (c, f) were quantified. All data are presented as the mean ± s.e. from three different experiments in duplicate. **P o0.01 vs TGFBIp alone. Oncogene (2015) 1 – 10
© 2015 Macmillan Publishers Limited
Inhibition of TGFBIp and reduction in tumor metastasis Y-S Maeng et al
3
Figure 2. Knockdown of TGFBIp expression inhibits LEC migration, tube formation, wound healing and adhesion. (a and b) TGFBIp mRNA and protein levels in LECs were measured by real-time qRT–PCR (a) and western blotting (b). (c–f) TGFBIp shRNA-infected LECs were treated with VEGF-C (20 ng/ml). (c and d) After 5 h, migration was quantified by counting the cells that migrated to the lower side of the filter with optical microscopy at × 200 magnification. (e and f) Tubular network formation on Matrigel was photographed and quantified at 24 h. (g and h) TGFBIp shRNA-infected LECs were seeded on 35-mm dishes. After confluence, cells were scratched with micropipette tips and washed to remove any debris. Images were captured at 0 and 16 h after wounding. For quantitative analysis, five fields per plate were photographed and distances between front lines were measured by using ImageJ software. (i and j) The 96-well plates were coated overnight (4 °C) with 0.1 mg/ml human fibronectin. TGFBIp shRNA-infected LECs in adhesion buffer were seeded at 105 cells/well in 100-μl volume and incubated for 30 min at 37 °C. Cell adhesion was quantified by counting the cells that attached to the fibronectin-coated matrix with optical microscopy at × 200 magnification. All data are presented as the mean ± s.e. from three different experiments in duplicate. **P o0.01 vs shControl.
organized into capillary-like structures. However, the cell outgrowth of the TGFBIp shRNA-infected lymphatic rings was significantly attenuated (Figure 3c). Moreover, we quantified the lymphatic vessel sprouts and their interconnected network by image binarization using a computer-assisted image analysis. As shown in Figure 3d, TGFBIp shRNA-infected lymphatic rings showed a marked reduction in the number of intersecting microvessels with a grid of concentric outlines of the thoracic duct boundary as a function of the distance to the ring compared with controls. In addition, lymphatic vessels sprouting from the thoracic duct were positively stained with CD31 and LYVE1, specific markers for LECs (Figure 3e). Real-time qRT–PCR and western blot analysis confirmed that the expression of TGFBIp mRNA and protein was significantly decreased in TGFBIp shRNAinfected lymphatic vessels as compared with control shRNA (Figures 3f and g). Collectively, these results indicate an important role for TGFBIp in the regulation of in vitro lymphangiogenic activity and lymphatic vessel sprouting. Stimulation of LECs in vitro by TGFBIp suggests that this factor can activate intracellular signaling pathways, as has been reported for VEGF-C.4 Therefore, we investigated the phosphorylation of intracellular lymphangiogenic signaling molecules in response to TGFBIp. Western blot analysis showed that TGFBIp increased the phosphorylation of ERK, AKT, FAK, JNK, FOXO1 and SRC in a timedependent manner (Figures 4a and b), but did not induced p38 © 2015 Macmillan Publishers Limited
activation (data not shown). Furthermore, to determine whether these intracellular signaling pathways had a functional relevance to TGFBIp’s biological activity, we infected LECs with TGFBIp shRNA and treated TGFBIp in the absence or presence of anti-integrin β3, CAS4506-66-5, wortmannin, SP600125, PD98059 and SB202190, which are integrin β3, FAK, PI3K, JNK, ERK and p38 inhibitors, respectively. An LEC 3D spheroid sprouting assay showed that TGFBIp shRNA significantly inhibited sprouting of lymphatic vessels, whereas the addition of recombinant TGFBIp to TGFBIp shRNA LECs rescued the lymphatic vessel sprouting reduced by shTGFBIp infection (Figures 4c and d). Moreover, pretreatment of LECs with integrin β3, FAK, PI3K, JNK and ERK inhibitor markedly inhibited lymphatic vessel sprouting stimulated by treatment with TGFBIp, but a p38 inhibitor that was not activated by TGFBIp had no effect (Figures 4c and d). These data show that TGFBIp induces phosphorylation of intracellular signaling molecules in pathways that are essential for TGFBIpmediated lymphangiogenesis. TGFBIp increases CCL21 expression through an ERK-dependent pathway In the cancer environment, chemokines (CXCL12, CCL19 and CCL21) secreted by LECs are key players in the active metastatic dissemination of tumor cells via the lymphatic system.19,20 Oncogene (2015) 1 – 10
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Figure 3. Knockdown of TGFBIp expression inhibits the lymphatic vessel sprouting and lymphangiogenesis in vitro. (a) 3D in vitro lymphangiogenesis with fibrin gel-embedded microbeads of TGFBIp shRNA-infected LECs. (b) Cumulative length of all sprouts originating from an individual spheroid was quantified after 5 days. **P o 0.01 vs shControl-infected group. (c) Thoracic duct explants embedded in type I collagen gel were infected with TGFBIp shRNA. After 7 days, a representative micrograph of an outgrowing explant is shown (left). The grid obtained by dilatation of the thoracic duct boundary is used for quantification (right). (d) The number of intersections between capillaries and the grid is plotted as a function of distance to the ring. (e) Immunostaining of lymphatic rings with anti-LYVE-1 and anti-CD31 antibodies. (f and g) TGFBIp mRNA and protein levels in sprouting lymphatic vessels were measured by real-time qRT–PCR (f) and western blotting (g). All data are presented as the mean ± s.e. from three different experiments with n = 6 per group per experiment. **P o 0.01 vs shControl.
They serve as chemoattractants for tumor cells, thus promoting metastasis. We thus examined whether TGFBIp induces the expression of chemokines that promote tumor metastasis. As shown in Figure 5a, TGFBIp increased the expression of CCL21 mRNA, and maximal expression was observed after 6 h, whereas mRNA levels of CCL19 and CXCL12 were unchanged in response to TGFBIp. Western blot analysis also confirmed the increased expression of CCL21 protein in the presence of TGFBIp (Figure 5b). Mitogen-activated protein kinase, signal transducers and activators of transcription 3, and nuclear factor-kappa B signaling pathways are known to be involved in modulating the expression of CCL21 through the interleukin family of cytokines in LECs.21 To determine whether these pathways are also involved in Oncogene (2015) 1 – 10
modulating CCL21 expression in the TGFBIp-stimulated LECs, we first examined the phosphorylation of IκBα, an upstream signaling molecule of nuclear factor-kappa B. As shown in Figure 5c, TGFBIp had no effect on IκBα phosphorylation over time. Furthermore, signal transducers and activators of transcription 3 activation was also not detected (data not shown). However, we already found strong ERK phosphorylation in stimulated cells in the presence of TGFBIp (Figure 4). To investigate the significance of the ERK signaling pathway in regulating CCL21 expression in TGFBIp-stimulated LECs, LECs were pretreated with PD98059, an ERK inhibitor and stimulated with TGFBIp. TGFBIp-stimulated LECs showed an increased expression of CCL21 and the effect was effectively blocked by PD98059 in a dose-dependent manner © 2015 Macmillan Publishers Limited
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Figure 4. TGFBIp-induced sprouting of LECs is mediated through integrin β3, FAK, PI3K, JNK and ERK signaling pathways. Representative western blotting images (a) and densitometric analyses (b) of phosphorylated ERK, AKT, FAK, JNK, FOXO1 and SRC. After serum-starved LECs were treated with TGFBIp (10 μg/ml) for the indicated periods of times (5, 10, 15, 30 or 60 min), cell lysates were subjected to western blotting by using IgGs against pERK, pAKT, pFAK, pJNK, pFOXO1 and pSRC. The membranes were then stripped and reprobed with IgGs against ERK, AKT, FAK, JNK, FOXO1 and SRC to estimate the total protein loaded. The relative ratios were normalized by arbitrarily setting the phosphorylation ratio at time 0 as 1. Experiments were performed in triplicate. Data are presented as the mean ± s.e.. *P o0.05 vs time 0. (c) TGFBIp shRNA-infected LEC spheroids were treated with TGFBIp (10 μg/ml), anti-integrin β3 antibody (1 μg/ml), CAS4506-66-5 (1 μM), wortmannin (50 nM), SP600125 (5 μM), PD98059 (10 μM), SB202190 (10 μM) or their combination as indicated and stained for PECAM-1. (d) Quantification of sprout number in the experiment. All data are presented as the mean ± s.e. from three different experiments with n = 7 per group per experiment. **P o0.01 vs shTGFBIp+rhTGFBIp.
(Figure 5d). These results suggest that the ERK signaling pathway is important for the regulation of CCL21 expression in TGFBIptreated LECs. TGFBIp stimulates lymphatic permeability Specialized cell–cell junctions expressing VE-cadherin and tight junction-associated proteins such as zona occludens 1 (ZO-1) have been reported to localize between LECs.22 Disruption of functional organization of such structures may lead to vascular fragility and increase permeability. We therefore examined whether TGFBIp could alter lymphatic permeability. Phosphorylation of SRC (on tyrosine 418) and VE-cadherin is required for stimulation of endothelial cell permeability.23 Similarly, we found that TGFBIp increased the phosphorylation of SRC and VE-cadherin in a timedependent manner (Figures 6a and b). To further investigate the regulation of VE-cadherin turnover in response to TGFBIp, the localization of VE-cadherin was examined. Without stimulation, VE-cadherin localized to the plasma membrane and displayed a linear pattern in the cell borders (Figure 6c, top left). In contrast, in LECs treated with TGFBIp for 30 min, VE-cadherin levels diminished at the plasma membrane and the linear pattern disrupted, and disorganized at cell borders, presumably because of increased VE-cadherin endocytosis or internalization (Figure 6c, © 2015 Macmillan Publishers Limited
top right). Additionally, to determine whether cell-surface-derived VE-cadherin was internalized, cell-surface VE-cadherin was labeled in live LECs and then cells were acid-washed (low pH). This approach demonstrated that a cell-surface pool of VE-cadherin was internalized by TGFBIp (Figure 6c, bottom right). However, in non-TGFBIp-treated cells, internalized VE-cadherin was not detected (Figure 6c, bottom left). Similar results were also obtained from trypsin digestion experiments. As shown in Figure 6d, treatment of LECs with TGFBIp increased the amount of trypsin-resistant VE-cadherin in a time-dependent manner, suggesting that TGFBIp stimulates internalization of cell-surface VE-cadherin, because internalized cell-surface proteins are resistant to trypsin digestion. This finding raises the possibility that TGFBIp is capable of stimulating the permeability of LEC monolayers by inducing the opening of intercellular junctions. Using a Transwell system, we determined the permeability of LEC monolayers stimulated with TGFBIp. Stimulation of LECs with TGFBIp significantly increased the permeability of FITC-dextran across the cell monolayer in a dose- and time-dependent manner compared with that of the positive control VEGF-C or vehicle alone (Figures 6e and f). These results indicate that TGFBIp induces lymphatic permeability by disorganizing lymphatic VE-cadherin junctions. Oncogene (2015) 1 – 10
Inhibition of TGFBIp and reduction in tumor metastasis Y-S Maeng et al
6 blood vessel-specific markers, LYVE-1 and CD31, respectively. shTGFBIp tumors showed a very low density of lymphatic vessels stained with LYVE-1/CD31 in the peri-tumoral (margin of tumor) (Figure 7e) and intratumoral (central region of tumor) areas (Figure 7f). Quantification of LYVE-1/CD31-positive lymphatic structures confirmed that vessel density was significantly reduced in shTGFBIp-expressing tumors as compared with controls (Figure 7g). However, only CD31-positive blood vessel density was slightly decreased in shTGFBIp tumors (Figure 7g). To further validate the effects of TGFBIp on tumor metastasis via the lymphatic system, shTGFBIp tumor bearing mice were sacrificed and examined for metastases in the lung and other organs. Microscopic analysis of the lungs revealed a significant decrease in the number of metastatic nodules per lung from shTGFBIp-expressing tumors versus control tumors (Figure 8a), which was confirmed by quantification of the number of metastatic nodules per lung (Figure 8b). Moreover, tumor cell-specific CCR7 staining and immunohistochemical analysis revealed significantly reduced lung (Figures 8c and d) and lateral axillary LN metastases (Figures 8e and f) in the shTGFBIp tumor bearing mice as compared with control mice. Overall, our data suggest that inhibition of TGFBIp expression is useful to suppress the metastatic potential of colon cancer cells via the lymphatic system without affecting the growth rate of tumors.
Figure 5. TGFBIp increases CCL21 expression through an ERKdependent pathway. (a) Total mRNA was isolated from LECs treated with TGFBIp (10 μg/ml) or untreated for the indicated times, and CCL21, CXCL12 and CCL19 genes expression was assessed by realtime qRT–PCR analysis. (b and c) LECs were treated with TGFBIp (10 μg/ml). (d) LECs were preincubated for 30 min with or without PD98059 (1, 5 and 10 μM) and stimulated with TGFBIp (10 μg/ml). Cell lysates were subjected to western blotting by using IgGs against CCL21 (b and d) and phospho-IκBα (c). The membranes were then stripped and reprobed with IgGs against actin and IκBα to estimate the total protein loaded. Experiments were performed in triplicate. Data are presented as the mean ± s.e. **P o0.01 vs control.
Inhibition of TGFBIp expression reduces tumor lymphangiogenesis and tumor metastasis Metastasis is the principal cause of cancer mortality. In the past decade, converging data has highlighted the importance of the tumor lymphatic vasculature in colon cancer dissemination. We investigated the capacity of TGFBIp to promote tumor lymphangiogenesis and subsequent dissemination in TGFBIpexpressing colon cancer models: a subcutaneous xenograft of the human colon cancer SW620 cell line that displays an aggressive metastatic pattern.24 Stable expression of TGFBIp shRNA constructs considerably reduced the protein levels of TGFBIp in SW620 cells (Figure 7a). Equal numbers of TGFBIp shRNA- or control shRNA-expressing SW620 cells were implanted subcutaneously into NOD-SCID mice, and the growth of the resultant primary tumors was monitored. Interestingly, there was no significant difference in the average size and weight of the tumors derived from the shTGFBIp-SW620 and shcontrol-SW620 cells (Figures 7b and d). Additionally, in vitro growth rates were not modified by inhibition of TGFBIp expression (unpublished data). To determine whether TGFBIp could induce tumor lymphangiogenesis, tumors were sectioned and stained for lymphatic- or Oncogene (2015) 1 – 10
DISCUSSION Tumor-induced lymphangiogenesis is known to promote lymphatic metastasis, and lymphangiogenic growth factors that are produced and secreted by the tumors themselves are critical activators of tumor lymphangiogenesis during the process of metastasis.25–27 In the present study, we provide an evidence that TGFBIp acts both in vitro and in vivo as a lymphangiogenic factor. First, we show that TGFBIp promotes in vitro lymphangiogenesis. TGFBIp induces migration, tube formation and adhesion of human LECs in both a time- and dose-dependent manner; these effects are mediated by the binding of TGFBIp with integrin β3 specifically. This finding is consistent with a report showing that inhibition of TGFBIp expression significantly inhibited in vitro 3D lymphatic bead sprouting, 3D spheroid sprouting and thoracic duct lymphatic ring sprouting. In addition, the lymphatic vessel sprouting recovered by the addition of recombinant TGFBIp was markedly inhibited by anti-integrin β3 antibody and specific inhibitors of FAK, AKT, JNK and ERK, but the p38 inhibitor had no effect. These results suggest that TGFBIp produced by LECs is secreted into the extracellular matrix, where it binds to integrin β3 and activates the intracellular signaling pathways essential for TGFBIp-mediated lymphangiogenesis, thus generating a positive feedback mechanism. Chemotactic gradients of chemokine ligands established by tumor-associated LECs and LNs actively guide chemokine receptor-positive metastatic cells initially into lymphatic vessels and thereby further enhance metastasis.20 Our data show that the expression of CCL21 was significantly increased by TGFBIp in LECs via an ERK-dependent pathway, whereas that of other chemokines (that is, CCL19 and CXCL12) did not change in response to TGFBIp. These data indicate that the expression of CCL21 is regulated by TGFBIp via an ERK-dependent signaling pathway and may subsequently promote the active migration of chemokine receptor-positive cancer cells into the lymphatic vessels. Control of lymphatic permeability might also be a crucial element contributing to metastasis.22 Here we showed that TGFBIp activates the phosphorylation of SRC and VE-cadherin, which has been shown to correlate with increased permeability of LECs similar to lymphatic factors.28,29 VE-cadherin that was diminished at the cell surface by TGFBIp internalized into the cytosol, which led to open gaps between LECs and increased the permeability of FITC-dextran across the cell monolayer in both © 2015 Macmillan Publishers Limited
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Figure 6. TGFBIp increases LEC permeability by disorganizing lymphatic VE-cadherin junctions. (a and b) Confluent LECs were treated with TGFBIp (10 μg/ml) for the indicated times. (a) Western blot analysis was performed by using an antibody specific for phospho-SRC (tyrosine 418) and anti-SRC antibody. (b) Cell lysates were subject to affinity precipitation by using anti-VE-cadherin antibody. Western blot analysis was performed by using an antibody specific for phospho-tyrosine and VE-cadherin. (c) Confluent LECs were stimulated with TGFBIp (10 μg/ml) for 30 min. The cells were then fixed and stained with anti-VE-cadherin antibody. Arrows indicate the disruption of VE-cadherin junction at the plasma membrane (top). VE-cadherin internalization was monitored by internal acid-resistant vesicles, as described in the Materials and methods. Arrows point to VE-cadherin in internal vesicle-like compartments (bottom). (d) Confluent LECs were treated with TGFBIp (10 μg/ml). At the indicated times, cells were treated with trypsin, pelleted and lysed for further analysis of VE-cadherin levels present in these trypsin-resistant fractions. Total cell lysates without trypsin treatment were used as controls. Cell lysates were then analyzed by western blot analysis by using anti-VE-cadherin antibody. (e and f) LECs were plated onto a Transwell filter. After reaching confluence, LECs monolayers were treated with TGFBIp (2, 5 and 10 μg/ml) or VEGF-C (20 ng/ml) for 30 min (e). LECs monolayers were treated with TGFBIp (10 μg/ml) for the indicated times (f). FITC-dextran permeability was performed. Experiments were performed in triplicate. Data are presented as the mean ± s.e. **P o0.01 vs control.
a dose- and time-dependent manner. Our results indicate that TGFBIp induces lymphatic permeability by disorganizing lymphatic VE-cadherin junctions and thereby may facilitate intravasation of tumor cells into lymphatic vessels. Finally, inhibition of TGFBIp expression in colon cancer cells decreased tumor lymphangiogenesis and metastasis without a significant effect on tumor growth rate. Lymphatic vessels with wider lumens were observed in high TGFBIp-expressing tumors and were correlated with increased tumor metastasis to the lung and LNs. Analysis of LYVE-1/CD31-positive lymphatic structures confirmed that vessel density was significantly reduced in shTGFBIp-expressing tumors as compared to controls expressing high TGFBIp levels. However, CD31-positive blood vessel density © 2015 Macmillan Publishers Limited
was slightly decreased in shTGFBIp tumors, suggesting that TGFBIp derived from tumor cells has a stronger effect on lymphatic vessels than on blood vessels. Furthermore, analysis of tumor cell metastasis in mouse organs revealed a significant decrease in the number of metastatic nodules per lung and reduced lateral axillary LN metastasis in shTGFBIp-expressing tumors. Our data suggest that inhibition of TGFBIp expression is useful to suppress the metastatic potential of colon cancer cells via the lymphatic system. In summary, our findings clearly show that TGFBIp is a lymphangiogenic factor that induces metastasis via three probable mechanisms: (1) by increasing lymphatic density and consequently augmenting surface contact with cancer cells; (2) by Oncogene (2015) 1 – 10
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Figure 7. Inhibition of TGFBIp expression reduces tumor lymphangiogenesis in colon cancer. (a) TGFBIp protein levels in TGFBIp shRNAinfected SW620 cells were measured by western blotting. SW620 cells were injected subcutaneously into the midline of the backs of NOS-SCID mice (n = 5 per group). Tumors were removed after 33 days, excised and serially sectioned. (b) Representative tumors. (c and d) Analysis of tumor growth rates and weights. (e) Margins of tumor sections were stained for infiltrating LECs using anti-LYVE1 and anti-CD31 antibodies. Nuclei were stained with DAPI (blue fluorescence). Arrows indicate lymphatic vessels. Yellow dotted lines point to margins of the tumor. Images were viewed using an Olympus IX81-ZDC microscope with a LUCPL FLN 10 × /0.40 NA lens. (f) Central regions of tumor sections were stained for infiltrating LECs using anti-LYVE1 and anti-CD31 antibodies. Nuclei were stained with DAPI (blue fluorescence). Arrows indicate lymphatic vessels. Images were viewed by using an Olympus IX81-ZDC microscope with a LUCPL FLN 10 × /0.40 NA lens. (g) Quantitative assessment of LYVE1 and CD31-positive lymphatic vessels per field for each tumor section. Data are presented as the mean ± s.e. **P o 0.01 vs shControl group.
increasing CCL21 expression in LECs and promoting migration of cancer cells; and (3) by increasing lymphatic permeability and facilitating intravasation and/or extravasation of tumor cells (Figure 8g). Our data suggest, for the first time, that TGFBIp may contribute to lymphatic metastasis, supporting the strategy of targeting not only tumor-derived functions, but also targeting functions of the host that support the tumor. Overall, our findings suggest that TGFBIp can be a potential therapeutic target for treating metastatic colon cancer. MATERIALS AND METHODS Cell culture Primary human LECs were purchased from PromoCell (Heidelberg, Germany) and cultured in endothelial basal medium (EBM; Lonza, Walkersville, MD, USA) supplemented with 10% FBS and other factors as previously described.30 LECs less than eight passages were used for all experiments. SW620 cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and antibiotics.
In vitro tube formation assay Tube formation was assayed as previously described.31 In brief, 250 μl Matrigel (BD Biosciences, Bedford, MA, USA) was added to a 16-mm diameter tissue culture well and allowed to polymerize for 30 min at 37 °C. After trypsinization, the harvested LECs were resuspended in EBM containing rhTGFBIp (5 ~ 10 μg/ml, Sino Biological Inc., Beijing, China) or recombinant human VEGF-C (Upstate Biotechnology, Lake Placid, NY, USA), and plated onto the layer of Matrigel (1.2 × 105 cells/well). Matrigel cultures were incubated at 37 °C and photographed at various time points Oncogene (2015) 1 – 10
(×200 magnification). The area covered by the tube network was determined with an optical imaging technique: Pictures of the tubes were scanned into Adobe Photoshop and quantified by ImageJ software (National Institutes of Health).
Cell-matrix adhesion Cell-matrix adhesion assays were performed as described previously.32 The 96-well plates were coated overnight (4 °C) with 1–10 μg/ml rhTGFBIp or 0.1 mg/ml human fibronectin (Sigma-Aldrich, St Louis, MO, USA). LECs in adhesion buffer (serum free media) were seeded at 105 cells/well in 100-μl volume and incubated for 30 min at 37 °C. After the removal of nonadherent cells after two washes, adherent cells were measured by hematoxylin and eosin staining, and quantified in triplicate by counting adherent cells in five randomly selected fields per well (Axiovert 100; Carl Zeiss Micro-Imaging, Thornwood, NY, USA). Results are representative of three different experiments in duplicate.
Thoracic duct collection and 3D lymphatic ring assay Identification and harvesting of thoracic ducts were conducted as described previously.33 Briefly, the dissected thoracic duct was cut transversely to generate lymphatic duct fragments, which then were embedded into type I collagen gels. An shRNA lentivirus against the mouse TGFBIp gene (Santa Cruz Biotechnology Inc.) was added to the culture medium at the beginning of the experiment as appropriate. Seven days later, these lymphatic ring cultures exhibited an outgrowth of cells that organized into capillary-like structures. Quantification using computerized image analysis was based on the elaboration of a grid obtained by the successive dilatation of the explant boundary, after image binarization. Rat Anti-mouse CD31 monoclonal antibody (Clone: MEC 13.3, 1:200 dilution, BD PharMingen, San Diego, CA, USA) or rabbit anti-mouse LYVE1 © 2015 Macmillan Publishers Limited
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Figure 8. Inhibition of TGFBIp expression suppresses the tumor metastasis to LN and lung. (a) Images of lung metastases of mice injected with SW620 cells. (b) The number of metastatic nodules per lung was quantified by a microscopic inspection. **P o 0.01 vs shControl group. (c and e) CCR7 and hematoxylin and eosin staining with frozen sections of lung and lateral axillary LN tissues isolated from mice subcutaneously injected with SW620 cells. (d and f) The incidence of metastasis per lung or lateral axillary LN was quantified by microscopic analysis. Red dotted lines indicate tumors in the mouse lateral axillary LN. Data are presented as the mean ± s.e. **P o0.01 vs shControl group. (g) A schematic diagram of a proposed mechanism of TGFBIp on tumor lymphangiogenesis and metastasis. polyclonal antibody (1:200 dilution, Angiobio Co., Del Mar, CA, USA) was used to identify the sprouting of lymphatic vessels from the thoracic duct.
Immunofluorescence staining and immunohistochemistry Confluent LECs were fixed in 3.7% formaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS); LECs were then preincubated with a blocking solution of PBS containing 5% normal donkey serum and 0.05% Tween-20. Cells were incubated with goat anti-VE-cadherin polyclonal antibody (1:100 dilution, Santa Cruz Biotechnology Inc.) for 2 h at room temperature and labeled with a fluorescein-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA). Samples were observed with a fluorescence microscope (Olympus, Tokyo, Japan). Eight micrometer-thick frozen tumor sections were washed in PBS and blocked in 5% normal donkey serum in an antibody dilution buffer consisting of PBS containing 0.1% Triton X-100. Sections were then incubated overnight in primary antibody (Rat anti-mouse CD31 monoclonal antibody (Clone: MEC 13.3, 1:200 dilution) or rabbit anti-mouse LYVE1 polyclonal antibody (1:200 dilution, Angiobio Co.)) at 4 °C and labeled with a fluorescein-conjugated secondary antibody (Molecular Probes). Nuclei were counterstained with DAPI and are seen in blue. Samples were observed with a fluorescence microscope (Olympus). The number of lymphatic vessels within the tumor was counted in six fields per section: the center regions of the tumor (intratumor vessel density) and within an area 1 mm from the tumor border (peri-tumor vessel density). Seven slide sections per mouse were analyzed. To determine the metastasis of SW620 tumor cells to mouse lateral axillary LNs and lungs, mouse LNs and lungs sections (seven slide sections per mouse) were © 2015 Macmillan Publishers Limited
stained with hematoxylin and eosin stain and rabbit anti-CCR7 monoclonal antibody (Clone:Y59, 1:200 dilution, Abcam Inc. Cambridge, MA, USA).
VE-cadherin internalization assay (immunofluorescence label) VE-cadherin internalization assays were performed as described previously.34 A mouse monoclonal antibody directed against the VE-cadherin extracellular domain (Clone: BV6, 1:200, Merck Millipore, Darmstadt, Germany) was dialyzed into MCDB 131 medium containing 20 mM HEPES and 3% BSA. The dialyzed antibody was incubated with LEC cultures at 4 °C for 1 h. Unbound antibody was removed by rinsing cells in ice-cold MCDB 131. Cells were incubated at 37 °C for 30 min in the absence or presence of 10 μg/ml TGFBIp. To remove cell surface-bound antibody while retaining internalized antibody, cells were washed for 15 min in PBS (pH 2.7) containing 25 mM glycine and 3% BSA. The cells were rinsed, fixed, and processed for immunofluorescence labeling.
Internalization of cell-surface VE-cadherin (trypsin digestion) LECs grown in six-well plates were treated with vehicle only or TGFBIp (10 μg/ml) at 37 °C for 30 min. Following treatment, cells were untreated or treated with trypsin as previously described35 to remove cell surface proteins. Trypsin-undigested and trypsin-digested cells were subsequently lysed, subjected to reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and electro-transferred onto polyvinylidene difluoride membranes. Internalized or intracellular VE-cadherin, which was resistant to trypsin digestion, on polyvinylidene difluoride membranes was visualized using streptavidin-HRP and the ECL system. Oncogene (2015) 1 – 10
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10 Mouse tumor models and in vivo procedures SW620 cancer cells were harvested from subconfluent cell culture plates, washed with PBS, and resuspended at a concentration of 2.5 × 107 cells per ml DMEM containing 10% FBS. Of the suspended cells, an aliquot of 0.2 ml was injected subcutaneously into the right posterior flank of 5-week-old NOD-SCID mice (Orient Company, Seongnam, Korea) with five mice in each group. Tumors were measured with calipers to estimate volumes on days 1–33 after injection. Four weeks after injection, mice were sacrificed and examined for the growth of subcutaneous tumors. All organs were removed for examination, and lateral axillary LN around tumor region and lung metastases were detected by hematoxylin and eosin staining and quantified by counting metastatic lesions in each section. All studies were repeated twice to ensure reproducibility (with a minimum of five mice per group).
Statistical analysis All experiments were repeated at least three times. Data are presented as the means ± s.e., and statistical comparisons between groups were performed by one-way ANOVA followed by Tukey’s test. Supplementary Information shows the additional materials and methods.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011-0028699).
AUTHOR CONTRIBUTIONS Conceived and designed the experiments: YSM, BA, SIC. Performed the experiments: YSM, SIC. Analyzed the data: YSM, SIC, EKK. Contributed reagents/ materials/analysis tools: YSM, SIC, EKK. Wrote the manuscript: YSM, BA, EKK.
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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)
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