Brain metastasis (BM) is a major cause of mortality in small-cell lung cancer (SCLC) patients; however, the molecular pathway of SCLC BM remains largely unknown because of a lack of investigation. Here we screen the levels of some candidate-soluble factors in the serum of SCLC patients and find that SCLC patients with high levels of placental growth factor (PLGF) are prone to BM. Using in vitro blood–brain barrier model, we show that PLGF derived from SCLC cells triggers vascular endothelial growth factor receptor-1-Rho-extracellular regulated protein kinase 1/2 signaling axis activation, results in disassembly of tight junction in brain endothelial cells and promotes SCLC cell transendothelial migration. Furthermore, the downregulation of PLGF suppresses SCLC cell metastasis to the brain in an experimental BM model. These data suggest that PLGF is a potential signature of SCLC BM and a prospective therapeutic target for SCLC BM.
Small-cell lung cancer (SCLC) is the most aggressive histological subtype of lung cancer, with a strong predilection for brain metastasis (BM). The prognosis of SCLC patients with BM is consistently poor, with median survival of 4–6 months.1, 2 A better understanding of the mechanisms of SCLC BM is important to improve current therapies and design new treatment modalities.
Owing to the existence of the blood–brain barrier (BBB), the mechanisms of BM are believed to be different from that of metastasis to other sites. The BBB mainly consists of a network of closely opposed endothelial cells in brain microvessels, characterized by the presence of continuous tight junctions (TJs).3 Magnetic resonance imaging showed that BBB is breached at the metastatic sites and >70% of BM has leakage of contrast agent from blood vessels.4 We previously found that SCLC cells induce disassembly of brain endothelial TJ, leading to SCLC cell transendothelial migration.5 These suggested that the ‘opening’ of TJ at BBB is a key event in the SCLC extravasation into the brain parenchyma. However, the detailed mechanisms remain largely unclear.
Placental growth factor (PLGF) belongs to the vascular endothelial growth factor (VEGF) subfamily and has a crucial role in inducing angiogenesis that associated with pathological events in the adult.6 It has been reported that PLGF was involved in cellular cytoskeleton rearrangements, leading to cell migration in an acute myeloid leukemia cell line, human breast cancer cell line and human non-SCLC cell line.7, 8, 9 Clinical studies showed that the highly expressed PLGF in tumor tissue was associated with disease progression and poor prognosis in patients with colorectal cancer, breast cancer and gastric cancer,10, 11, 12 yet, the involvement of PLGF in the pathogenesis of SCLC remains unknown.
In the present study, we found that PLGF was significantly elevated in the serum of SCLC patients with BM. Our data demonstrated that PLGF secreted by SCLC cells promoted disassembly of TJ in brain endothelial cells for SCLC cell transendothelial migration and entry into the brain.
Elevated levels of PLGF in the serum of SCLC patients are associated with BM
Recent studies implicated that soluble factors, including cytokines, might participate in metastatic colonization in the brain.13 In this study, the serum levels of some candidate-soluble factors in SCLC patients with BM were screened using enzyme-linked immunosorbent assay. The results showed that the level of PLGF was significantly increased in SCLC patients with BM compared with SCLC patients without BM and normal specimens (Figure 1a). This suggested that PLGF may be associated with SCLC metastasis to brain. To further determine potential values of serum PLGF for prediction of BM, we performed the receiver operating curve analysis of serum PLGF. For screening method, serum PLGF value of >136 ng/ml seemed to be the appropriate cutoff level for the prediction of BM with 77.78% of sensitivity, 66.67% of specificity and 59.3% of accuracy. The associated value of area under the curve of PLGF was 0.7778 (P=0.0018, 95% confidence interval 0.6247–0.9309), which indicates the potential promising predictive value of BM (Figure 1b). In addition, the expression of PLGF in the tumor tissues from SCLC patients was determined by immunohistochemical analysis and the relationships between the level of PLGF expression and the clinicopathological characteristics of SCLC were summarized in Table S1. The data suggested that PLGF was correlated with progression and poor prognosis of SCLC. Also, our results showed strong positive staining of PLGF in the tumor tissues obtained from lung and brain (Figure 1c). As shown in Figure 1d, the expression levels of PLGF were significantly higher in the tumor from SCLC BM than it was in the lung tumor from the SCLC without BM. Thus, the high levels of PLGF in the serum of SCLC patients with BM, together with the highly expressed PLGF in the metastatic brain tumor tissues from SCLC patients, suggested that PLGF may be involved in the BM of SCLC.
PLGF promotes SCLC cell migration across the brain endothelial monolayer
It has been suggested that penetration of cancer cells through the BBB is a key step in BM. To investigate whether PLGF was involved in SCLC cells crossing the BBB, we employed in vitro BBB model to assess the transendothelial migration ability of SCLC cells as described in Materials and methods. Six lung cancer cell lines were used in this study, including three SCLC cell lines (NCI-H250, NCI-H209 and NCI-H446) and three non-SCLC cell lines (A549, NCI-H460 and NCI-H292). The results showed that PLGF was highly expressed in the SCLC cell lines (Figure 2a). PLGF expression in NCI-H250 cells derived from SCLC brain metastatic site14 was significantly higher than NCI-H209 and NCI-H446 cells, which were from bone marrow metastatic site and pleural effusion metastatic site of SCLC,15 respectively. Further results showed that the transendothelial migration ability of NCI-H250 cells was higher than that of NCI-H209 and NCI-H446 cells (Figure 2b, Supplementary Figure S1). There was a significant correlation between the mRNA levels of PLGF and the transendothelial migration ability in these lung cancer cell lines (P<0.01) (Figure 2c). Furthermore, transendothelial migration of NCI-H446 cells was significantly increased in the presence of recombinant human PLGF proteins (Figure 2d, Supplementary Figure S2A), whereas the transendothelial migration of NCI-H250 cells was significantly decreased in the presence of neutralized antibody against PLGF (Figure 2e, Supplementary Figure S2B).
Next, PLGF knockdown in SCLC cells was carried out using adenovirus-based RNA interference (Supplementary Figures S3A–D) followed by transendothelial migration assay. We found that downregulation of PLGF expression led to a significantly decreased transendothelial migration of SCLC cells (Figure 2f, Supplementary Figures S2C and S4). Additional results showed that knockdown of PLGF had no effect on the proliferation of SCLC cells (Supplementary Figures S3E–G). Thus, these results indicated that PLGF is required for the migration of SCLC cells across the brain endothelial cells.
PLGF triggers disassembly of TJ in brain endothelial cells for SCLC cell transendothelial migration
We previously reported that the disassembly of TJ in human brain microvascular endothelial cell (HBMEC) contributed to SCLC cell transendothelial migration.5 Here we evaluated the effect of PLGF on the integrity of TJ in HBMEC. The results showed that PLGF induced horse radish peroxidase (HRP) leakage through the HBMEC monolayers in a time- and dose-dependent manner (Figure 2g, Supplementary Figure S5A). Meanwhile, the distribution of TJ structural proteins, occludin and ZO-1, was visualized by immunofluorescence. Normal HBMEC showed a characteristic polygonal shape and linear pattern of immunostaining for occludin and ZO-1 at the cell–cell borders. In HBMEC treated with PLGF, the distribution of occludin and ZO-1 became discontinuous, segmented and dotted (Figure 2h, Supplementary Figure S5B). Also, there was an obvious shift of occludin distribution from insoluble to soluble fractions in HBMEC treated with PLGF (Figure 2i, Supplementary Figure S5C). These results indicated that PLGF could disrupt TJ complexes, leading to disassembly of TJ in brain endothelial cells. Accumulating evidences suggested that the phosphorylation of TJ proteins attenuated endothelial barrier integrity.16, 17, 18 Therefore, we further examined the phosphorylation of occludin by immunoprecipitation, utilizing antibodies against phosphorylated serine (Ser) and threonine (Thr) residues, respectively. As shown in Figure 2j, PLGF induced an obviously increased phosphorylation of occludin on Ser/Thr residues. Our results pointed out that PLGF could induce the Ser/Thr phosphorylation of occludin, resulting in the disassembly of TJ in brain endothelial cells.
PLGF-promoted TJ disassembly is mediated by VEGF receptor-1 (VEGFR-1) in brain endothelial cells
VEGFR-1 (also known as Flt-1) is a specific receptor for PLGF. The results showed that PLGF triggered the tyrosine phosphorylation of VEGFR-1 in HBMEC with a time-dependent manner (Figure 3a). To evaluate whether VEGFR-1 is activated in brain endothelial cells in the process of transendothelial migration of SCLC cells, HBMEC was cocultured with NCI-H250 cells and the activation of VEGFR-1 in HBMEC was detected. The results showed that NCI-H250 cells, expressing high levels of PLGF, induced an evident increase of tyrosine phosphorylation of VEGFR-1 in HBMEC after 2 h of incubation (Figure 3b). The PLGF-neutralizing antibody effectively blocked the NCI-H250 cell-induced tyrosine phosphorylation of VEGFR-1 in HBMEC (Figure 3c). We also observed the activation of VEGFR-1 in HBMEC incubated with NCI-H209 and NCI-H446 cells, respectively, which was blocked by PLGF antibody (Supplementary Figures S6A and B, and S7A and B). These results suggested that VEGFR-1 is activated in brain endothelial cells in the process of PLGF-promoted transendothelial migration of SCLC cells.
To further investigate whether VEGFR-1 is required for the PLGF-induced TJ disassembly in brain endothelial cells, we constructed stable HBMEC cell line with VEGFR-1 knockdown (Supplementary Figure S8). Knockdown of VEGFR-1 in HBMEC abolished the PLGF-induced HRP leakage through endothelial monolayers (Figure 3d), redistribution of ZO-1 and occludin (Figure 3e), recruitment of detergent-soluble occludin (Figure 3f) and Ser/Thr phosphorylation of occludin (Figure 3g) in HBMEC. Furthermore, VEGFR-1 knockdown in HBMEC resulted in a noticeable decrease of NCI-H250 cells transendothelial migration compared with the control (Figure 3h, Supplementary Figure S9A). Additionally, a significant reduction in transendothelial migration of NCI-H250 cells was detected in the presence of VEGFR-1-neutralizing antibody (Figure 3i, Supplementary Figure S9B). The similar results were also observed in the transendothelial migration of NCI-H209 and NCI-H446 cells (Supplementary Figures S6C and D, and S7C and D). These findings demonstrated that VEGFR-1 activation in brain endothelial cells is necessary for PLGF-mediated disassembly of TJ in the process of transendothelial migration of SCLC cells.
PLGF-induced TJ disassembly in brain endothelial cells requires activation of Rho kinase (ROCK) and ERK signaling
A significant body of evidence indicated that the assembly and the maintenance of TJ are regulated by several components of the intracellular signaling pathways, including ROCK,19 phosphoinositide 3-kinase,20 protein kinase C,21 extracellular regulated protein kinase (ERK)22 and c-Src kinase.23 To define the intracellular effectors that were responsible for the PLGF-induced TJ disassembly in brain endothelial cells, HBMEC monolayers were pretreated with the specific inhibitors for the signaling molecules mentioned above. The results showed that ROCK (Y27632) and ERK1/2 inhibitor (PD98059) significantly blocked PLGF-induced HRP leakage through endothelial monolayers (Figure 4a), redistribution of ZO-1 and occludin (Figure 4b), recruitment of detergent-soluble occludin (Figure 4c) and Ser/Thr phosphorylation of occludin (Figure 4d) in HBMEC. In contrast, phosphoinositide 3-kinase inhibitor (LY294002), protein kinase C inhibitor (Gö 6976) and Src inhibitor (PP1) had no such effect. Because the activation of ROCK is mostly regulated by small GTPase Rho, the levels of active Rho-guanosine-5'-triphosphate were measured and the Rho activation was observed in HBMEC after 5 min of PLGF treatment (Figure 4e). In addition, we also found an increase of ERK1/2 phosphorylation in HBMEC treated with PLGF in a time-dependent manner (Figure 4f). These results suggested that the activation of ROCK and ERK1/2 was involved in PLGF-induced disassembly of TJ in brain endothelial cells.
To confirm the role of Rho/ROCK signaling in PLGF-induced disassembly of TJ in brain endothelial cells, stable HBMEC cell line transfected with pCAG vector containing wild-type ROCK and Rho-binding-defective mutant of ROCK (dominant-negative ROCK)24 were used. The results showed that overexpression of dominant-negative ROCK in HBMEC significantly blocked PLGF-induced HRP leakage through endothelial monolayers (Figure 5a), redistribution of ZO-1 and occludin (Figure 5b), recruitment of detergent-soluble occludin (Figure 5c) and Ser/Thr phosphorylation of occludin (Figure 5d) in HBMEC. To provide further evidence for the contribution of ERK1/2 to PLGF-induced TJ disassembly in brain endothelial cells, downregulation of ERK1/2 in HBMEC was carried out using specific small interfering RNAs.25 The depletion of endogenous ERK1/2 was identified by western blot (Supplementary Figure S10). Our results showed that depletion of ERK1/2 expression in HBMEC significantly blocked PLGF-induced HRP leakage through endothelial monolayers (Figure 5e), redistribution of ZO-1 and occludin (Figure 5f), recruitment of detergent-soluble occludin (Figure 5g) and Ser/Thr phosphorylation of occludin (Figure 5h) in HBMEC. These results indicated that Rho/ROCK and ERK1/2 signaling were exactly required for PLGF-induced TJ disassembly in brain endothelial cells.
Additional results showed that the PLGF-induced activation of Rho and ERK1/2 in HBMEC was attenuated by pretreatment with SU5416, a selective inhibitor of the VEGFR receptor tyrosine kinase (Figure 6a) and knockdown of VEGFR-1 in HBMEC (Figure 6b), respectively. These results supported that PLGF derived from SCLC cells bound with VEGFR-1 in brain endothelial cells, leading to the activation of Rho/ROCK and ERK1/2 signaling.
ERK is a downstream effector of ROCK in PLGF-mediated disassembly of TJ in brain endothelial cells
We noticed that Rho activation was prior to ERK1/2 activation in HBMEC treated with PLGF (Figures 4e and f). Therefore, the phosphorylation of ERK1/2 was detected in PLGF-treated HBMEC in which ROCK activity was inhibited by Y27632 and transfection of dominant-negative ROCK mutant, respectively. The results showed that inhibition of ROCK significantly diminished ERK1/2 activation in HBMEC stimulated with PLGF (Figures 6c and d). Then, the active Rho-guanosine-5'-triphosphate was assessed in PLGF-treated HBMEC in which ERK1/2 activity was inhibited by PD98059 and transfection of ERK1/2 small interfering RNA, respectively. The results showed that inhibition of ERK1/2 had no effect on the activation of Rho/ROCK in HBMEC stimulated with PLGF (Figures 6e and f). In addition, inhibition of ERK1/2 in HBMEC with ERK1/2 small interfering RNA and PD98059 resulted in significant reductions in transendothelial migration of SCLC cells (Figures 6g and h, Supplementary Figures S11 and S12). These data demonstrated that PLGF derived from SCLC cells bound with VEGFR-1 in brain endothelial cells, and then induced Rho/ROCK-dependent ERK1/2 activation, leading to the disassembly of TJ, which is necessary for the transendothelial migration of SCLC cells.
Downregulation of PLGF suppresses SCLC cell BM in mice model
To further confirm the role of PLGF in BM in vivo, first, NCI-H250 cells, which were derived from a male, were transfected with the Ad-PLGF short hairpin RNA. Enzyme-linked immunosorbent assay showed that the level of PLGF secreted by NCI-H250 cells transfected with Ad-PLGF short hairpin RNA was significantly decreased compared with the control (Figure 7a). Then, the transfected NCI-H250 cells were injected into the internal carotid artery of female nude mice and the pattern of BM was characterized by the formation of circumscribed lesions in the brain parenchyma (Figure 7b). Hematoxylin and Eosin staining showed that the metastatic tumors formed in the brain had morphological characteristics of SCLC in the mice injected with transfected NCI-H250 cells (Figure 7c). Fluorescence in situ hybridization assay using Y-chromosome-specific probe was performed and the results showed that the positive signal was solely observed in the area of brain metastatic lesions (Supplementary Figure S13). These data demonstrated that the metastatic nodule within mice brain was the result of exogenously injected NCI-H250 cells. Our results showed that BM was present in 5 out of 18 mice injected with NCI-H250/Ad-scramble cells within 90 days (Table 1). However, no BM was observed in 12 mice injected with NCI-H250/Ad-PLGF short hairpin RNA cells within 90 days after the injection, despite the fact that tumor nodules in lung were observed. There was a statistical significance in the incidence of BM in mice injected with NCI-H250/Ad-PLGF short hairpin RNA cells compared with the control group (P<0.05). These results demonstrated that downregulation of PLGF suppressed BM of SCLC cells in mice.
To explore the relationship between PLGF expression in the primary lung tumor and the prognosis in SCLC patients, a retrospective study was performed. Interestingly, we found that the probability of survival was significantly lower in the high PLGF expression group (mean survival 15 months) than that of the low PLGF expression group (mean survival 25 months) (P<0.05) (Figure 7d).
BM is a major cause of mortality in patients with SCLC; however, the underlying mechanisms remain unknown. Our previous study showed that the disassembly of TJ between brain endothelial cells was an important biological event during transendothelial migration of SCLC cells.5 In this study, we provide first evidence that PLGF, secreted by SCLC cells, promoted SCLC BM through TJ of BBB.
It is known that some soluble factors, including VEGF,26 stromal cell-derived factor 1α,27 cyclooxygenase 228 and transforming growth factor-β2,29 might participate in breast cancer and melanoma metastasis to the brain. Interestingly, our results revealed that PLGF may be a potential candidate factor correlated with SCLC metastasis to brain instead of the factors mentioned above. Subsequently, the receiver operating curve analysis clearly indicated the potential predictive value of serum PLGF for SCLC metastasis to brain. Our further data revealed that PLGF expression were increased in tumor tissue of SCLC patients, especially in the tissues of BM. We therefore consider that elevated PLGF levels in the serum, which is contributed by the primary tumor, might be an important signature for the brain metastastic state at the initial diagnosis of SCLC patients. These clinical data indicated that the involvement of PLGF in SCLC metastasis to brain should be a novel cellular and molecular mechanism that facilitates cancer cells metastasis to brain. Further clinical investigations should be done to validate the value of PLGF as a serum biomarker associated with BM in SCLC.
Using in vitro BBB model, we demonstrated that PLGF, derived from SCLC cells, acted on brain endothelial cells to promote transendothelial migration of SCLC cells. Our previous study indicated that TJ disassembly in brain endothelial cells is necessary for the transendothelial migration of SCLC cells.5 In this study, for the first time, we indicated that PLGF induced the Ser/Thr phosphorylation of occludin in brain endothelial cells, leading to disassembly of TJ, which contributed to transendothelial migration of SCLC cells. It was noticed that there is a controversy over whether phosphorylation or dephosphorylation of occludin at Thr residues is linked to the integrity of TJ.17, 18, 30, 31 In our study, phosphorylation of occludin at Thr residues is associated with attenuated TJ integrity. This could be due to the type of cell (endothelial or epithelial) and the type of stimulus (for example, growth factors, inflammatory cytokines or oxidative stress).16, 30, 32, 33, 34
Most of the studies focused on the function of PLGF in the angiogenesis of tumor progression. In our context, a novel role of PLGF in the BM of SCLC was identified. That is, PLGF, derived from SCLC cells, breached brain endothelial TJ to allow the SCLC cells’ penetration through the BBB and entry into the brain. Our results showed that the transendothelial migration of SCLC cells was prevented by neutralization of PLGF in the medium and knockdown of PLGF in SCLC cells. Interestingly, the addition of exogenous PLGF to the medium still enhanced the transendothelial migration of SCLC cells, even if the SCLC cells themselves expressed the high levels of PLGF, which excluded the chemotaxis of PLGF to SCLC cells. Thus, these data further demonstrated that PLGF might directly affect the function of BBB to promote SCLC cells transendothelial migration. Using BM mouse model in vivo, we found that downregulation of PLGF in SCLC cells effectively blocked the formation of tumor nodules in the brain. Recent studies suggested that neo-angiogenesis is not required for brain metastatic tumor formation in breast cancer and melanoma.35, 36, 37 These lead us to propose that the decreased BM in the mouse model caused by PLGF knockdown is mainly due to the attenuated penetration of SCLC cells through the BBB, without the involvement of neo-angiogenesis. In addition, we found a statistical correlation between the levels of PLGF expression in the tumor tissues and the survival of SCLC patients. These data suggested that PLGF is a novel target to block the penetration of SCLC cells through BBB and the metastasis to brain.
Our findings demonstrated that PLGF-promoted transendothelial migration of SCLC cells was driven by VEGFR-1 on brain endothelial cells. VEGFR-1 was the sole high-affinity receptor for PLGF.38 To our knowledge, there was no report about the involvement of individual VEGFR-1 in TJ integrity. Here we convincingly demonstrated that VEGFR-1 was required for the PLGF-induced disruption of TJ integrity in brain endothelial cells. This is somewhat consistent with a recent study, showing that recombinant PLGF induced an increase of filtration in isolated cerebral vein of Galen, which was abolished by neutralizing VEGFR-1.39
It has been shown that intracellular signaling molecules, including phospholipase C, phosphoinositide 3-kinase/Akt and Ras/Raf/MAPK, were involved in VEGFR-1-mediated biological responses.40, 41, 42 In this study, we found that Rho/ROCK signaling in brain endothelial cells is necessary for the TJ disassembly induced by PLGF-triggered VEGFR-1 activation. It is known that ERK activation is associated with loss of endothelial TJ integrity.16, 43 Here, we found that ERK1/2 activation was the downstream signal of Rho/ROCK in PLGF-induced TJ disassembly in brain endothelial cells, which contributed to transendothelial migration of SCLC cells.
In summary, this study provides a clear scenario for the PLGF-mediated SCLC cell migration across the BBB. First, PLGF derived from SCLC cells bind to its receptor VEGFR-1 on brain endothelial cells, and then, the intracellular Rho/ROCK signaling is activated followed by ERK1/2 activation. Activated ERK1/2 causes Ser/Thr phosphorylation of TJ protein (occludin), which results in TJ disassembly for the transendothelial migration of SCLC cells (Figure 7e). This study identifies a novel target, PLGF, to prevent penetration of SCLC through the BBB and metastasis to the brain.
Materials and methods
Patients and specimens
Primary lung cancer specimens were from 79 patients with SCLC who attended Liaoning Cancer Hospital from 2006 to 2009. The specimens were obtained at the time of surgery. None of the patients had received preoperative neo-adjuvant chemotherapy or radiation therapy. Clinicopathological characteristics were defined according to the tumor-node-metastasis (TNM) criteria of the Universal Integrated Circuit Card. The clinical data of patients were described in Supplementary Table S1.
Specimens of BM and serum samples were collected from 21 SCLC patients with BM who attended the First Hospital of China Medical University and Shengjing Hospital of China Medical University from 2007 to 2010.
Additional collection of serum samples were from 30 SCLC patients without BM who attended the Shengjing Hospital of China Medical University from 2007 to 2010. A total of 30 serum samples of normal human were from Physical Examination Center in Shengjing Hospital of China Medical University. The patients of SCLC with BM and without BM were homogenous with respect to clinical features. Institutional Review Board approval was obtained to procure and analyze all the samples used in this study.
Cell lines and reagents
Human SCLC cell lines, NCI-H250, NCI-H209 and NCI-H446, as well as human non-SCLC cell lines, NCI-H460, NCI-H292 and A549, were obtained from the American Type Culture Collection (Rockville, MD, USA). HBMEC was a generous gift from Dr KS Kim44 (Johns Hopkins University School of Medicine, Baltimore, MD, USA). The reagents are described in Supplementary data.
Cell Culture and transfection
HBMECs were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA, USA), 10% Nu-serum (BD Biosciences, Franklin Lakes, NJ, USA). A549 cells were maintained in DMEM medium containing 10% fetal bovine serum, and the other cancer cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum. Stable HBMEC cell lines transfected with pCAG-myc-wild-type ROCK and pCAG-myc-dominant-negative ROCK were constructed as described previously.5
The PLGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for immunohistochemistry. The results were reviewed by two independent researchers and analyzed using the MetaMorph/DP10/BX41 image analyzing system (UIC/Olympus, NY, USA/Japan).
The HBMEC monolayers grown on glass coverslips were fixed with 4% paraformaldehyde. The cells were incubated with mouse anti-ZO-1, rabbit anti-occludin (Invitrogen Corporation) to visualize the distribution of ZO-1 and occludin. Nuclear staining using DAPI was used to visualize all the cells. The glass slides were analyzed using immunofluorescence microscopy (Olympus, Tokyo, Japan).
Enzyme-linked immunosorbent assay
Serum levels of human PLGF, VEGF, SDF-1α, TGF-β2 and COX-2 were quantified using enzyme-linked immunosorbent assay assay kit (Uscn Life Science Inc., Wuhan, China) according to the manufacturer’s instruction.
Adenovirus-based RNA interference of PLGF in SCLC cells and pRNA-U6.1/Neo (GeneScript)-mediated RNA interference of VEGFR-1 in HBMEC were performed as described in Supplementary data. The small interfering RNA sequences for ERK1/2 were used as described elsewhere,25 and the detail procedure were listed in Supplementary data.
Real-time PCR was performed as described previously.45 The sequences of primers and probes were listed in Supplementary Table 2. The relative expression level of PLGF was normalized to the expression level of GAPDH.
Cell fractionation and western blot
The cells were lysed and subjected to western blot analysis as described previously.5
Transendothelial migration assay
A total of 1 × 105 HBMECs were seeded on the fibronectin-coated 24-well Transwell inserts with pore sizes of 8 μm (Corning Costar Corp., Cambridge, MA, USA) and grew for 4 days. Experiments were conducted when transendothelial electrical resistance was >200 ohm cm2. SCLC cells were labeled with Vybrant carboxyfluorescein diacetate succinimidyl ester Cell Tracer Kit (Molecular Probes, Inc., Eugene, OR, USA). Prior to the assays, 2 × 105 CFDA SE dye-labeled cancer cells were added to the upper chamber. After incubation for 8 h, the upper chambers were fixed with 3.7% formaldehyde. To remove non-migrating cells, the apical side of the upper chamber was scraped gently with cotton wool and the transmigrated cells were counted under a fluorescence microscope. Experiments in the presence of inhibitors were performed as described.5, 45
HRP flux measurement
The HBMEC monolayers cultured in Transwell inserts (0.4-μm pore size, Costar, Corning, NY, USA) were incubated with PLGF in the presence of HRP (0.4 mg/ml; Sigma Aldrich, St Louis, MO, USA). Then, the media from the lower chamber were collected and the HRP leakage through the HBMEC monolayers were assessed colorimetrically as described previously.5 The HRP flux was expressed as nanogram passed per cm2 surface area per hour.
After overnight serum starvation, the SCLC cells were stimulated with PLGF (100 ng/ml) at 37 °C. Cell lysates were prepared with NP-40 lysis buffer (20 mM Tris–HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA, 10 mM NaF and 0.1 mM Na3VO4) with proteinase inhibitors. The cell lysates were incubated with appropriate antibodies and the immunocomplexes were captured by incubating with protein A/G-PLUS-Agarose (Santa Cruz Biotechnology). The immunoprecipitated proteins were subjected to western blot analysis.
The activation of Rho in HBMEC incubated with PLGF (100 ng/ml) was detected by Rho Assay Kit (Upstate Biotechnology, Lake Placid, NY, USA) according to the manufacturer’s instructions. Activated guanosine-5'-triphosphate-Rho was detected by western blot.
Experimental BM in mice
Pathogen-free female athymic BALB/c nude mice were purchased from Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China). In vivo model of BM was established as described.46, 47 Briefly, the nude mice were anesthetized by methoxyflurane. Then the tissue is separated by means of blunt dissection to visualize the internal carotid artery under a dissecting microscope. A total of 5 × 105 SCLC cells in 0.1ml Hank’s Balanced Salt Solution were slowly injected into the internal carotid artery. After inoculation, the mice were observed daily to 90 days and were killed when they became moribund or at the end of the observation period. The brain and lung tissue in mice were obtained and the presence of BM was confirmed by histology.
Fluorescence in situ hybridization
Sections from paraffin-embedded tissues were prepared and in situ hybridization was performed using labeled Y-chromosome probe, CEP Y (DYZ1) SpectrumGreen Probe and Paraffin Pretreatment Reagent Kit (Vysis, Inc., Downers Grove, IL, USA) according to the manufacturer’s protocol.
Two-way ANOVA was used to compare multiple groups. Pairwise comparisons were performed using the student’s t-test method. All the experiments were repeated at least three times. Data were presented as means of determinants (mean±s.e.m.) and P<0.05 values were considered statistically significant.
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We are grateful to Drs Monique Stins and Kwang Sik Kim (Department of Pediatrics, John Hopkins University School of Medicine) for providing HBMEC and Dr Shuh Narumiya (Kyoto University Faculty of Medicine, Kyoto, Japan) for providing pCAG-myc, pCAG-myc-ROCK-WT and pCAG-myc-ROCK-KDIA. This work was supported by the Trans-Century Training Program Foundation for Talents, Ministry of Education of China (JJH2002–48), the Innovation Team Program Foundation of Liaoning Province (2006T131, LT2011011), the National Natural Science Foundation of China (30600270) and the Science and Technology Foundation of Liaoning Province (20102291).
The authors declare no conflict of interest
Supplementary Information accompanies the paper on the Oncogene website
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Li, B., Wang, C., Zhang, Y. et al. Elevated PLGF contributes to small-cell lung cancer brain metastasis. Oncogene 32, 2952–2962 (2013) doi:10.1038/onc.2012.313
- blood–brain barrier
- brain metastasis
- Rho kinase
- small-cell lung cancer
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