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CORRESPONDENCE Patrick Mehlen: mehlen{at}lyon.fnclcc.fr
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Abbreviations used: CAM, chorioallantoic membrane; DAPK, DAP kinase; DCC, deleted in colorectal cancer; MNA, MYCN amplification; mRNA, messenger RNA; Myoc, myocardium; NB, neuroblastoma; PTX, primary tumor xenograft; Q-RT-PCR, quantitative RT-PCR; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling.
© 2009 Delloye-Bourgeois et al.
Dependence receptors now number more than a dozen, including deleted in colorectal cancer (DCC) (1), UNC5H (2), Patched (3), some integrins (4), neogenin (5), p75NTR (6), RET (7), ALK (8), and TrkC (9). Although they have no structural homology (other than possibly in a domain referred to as the DART [dependence-associated receptor transmembrane] domain) (10), they all share the functional property of inducing cell death when disengaged from their trophic ligands, whereas the presence of their trophic ligands blocks this proapoptotic activity. Such receptors thus create cellular states of dependence on their respective ligands (11, 12).
The prototype dependence receptors are the netrin-1 receptors. Netrin-1, a diffusible laminin-related protein, has been shown to play a major role in the control of neuronal navigation during the development of the nervous system by interacting with its main receptors, DCC (13, 14, 15) and UNC5H (16, 17). However, DCC and UNC5H (i.e., UNC5H1, UNC5H2, UNC5H3, and UNC5H4) have been shown to belong to the dependence receptor family (1, 2). This dependence effect upon netrin-1 has been suggested to act as a mechanism for eliminating tumor cells that would develop in settings of ligand unavailability (for reviews see references 18, 19). Along this line, disruption of the proapoptotic signaling of these netrin-1 receptors in the gastrointestinal tracts of mice, by netrin-1 overexpression or by inactivation of UNC5H3, is associated with intestinal tumor progression (20, 21).
Thus, loss of the dependence receptors' proapoptotic activity represents a selective advantage for tumor cells. In this respect, DCC was proposed in the early 1990s to function as a tumor suppressor gene, whose expression is lost in the vast majority of human cancers (22, 23). This hypothesis also fits with the observation that UNC5H genes are down-regulated in most colorectal tumors, hence suggesting that loss of UNC5H genes represents a selective advantage for tumor development (21, 24, 25). We have analyzed expression of netrin-1 and its receptors in neuroblastoma (NB), the most frequent extracranial solid tumor of early childhood. The aggressive and metastatic stage 4 NB displays three distinct clinical patterns at presentation and dissemination sites based on patients' ages. Indeed, neonates and infants (<1 yr of age) with stage 4S and stage 4 without 4S features have an overall good prognosis, whereas stage 4 in children (>1 yr of age) shows a poor prognosis. We describe in this paper that, rather than the loss of netrin-1 receptor expression, a large fraction of aggressive NBs has evolved to select a gain of ligand expression that apparently represents a similar selective growth advantage. We therefore propose to use disruption of this selective advantage as an anticancer strategy in NB.
We first analyzed the expression of netrin-1 and its dependence receptors, DCC, UNC5H1, UNC5H2, UNC5H3, and UNC5H4, by quantitative RT-PCR (Q-RT-PCR) in a panel of 102 stage 4 NB tumors including 24 stage 4S and 12 [1yr–] stage 4. As shown in Fig. 1 A, netrin-1 is up-regulated in [1yr+] stage 4 as compared with both stage 4S (P < 0.05) and [1yr–] stage 4 (P < 0.01). Similar results were obtained when comparing netrin-1 protein level by immunohistochemistry (Fig. 1 B and quantification in Fig. S1 A). Interestingly, netrin-1 is detected mainly in tumor cells and is barely detected in stroma cells (Fig. 1 B and Fig. S1 B). Conversely, netrin-1 dependence receptor expression analysis showed that DCC was only weakly expressed in the different stage 4 NB (Fig. S1 C) as reported (29), whereas UNC5H1, UNC5H2, UNC5H3, and UNC5H4 expression showed no significant differences when comparing [1yr–] versus [1yr+] stage 4 (Fig. 1 C). However, we observed that the different UNC5H receptors are up-regulated specifically in stage 4S (mean increase in UNC5H expression in stage 4S vs. other stage 4 NBs: 2.98-fold, P < 0.007), suggesting UNC5H receptors as hallmarks of stage 4S NB. The UNC5H1 and UNC5H4, which show the highest messenger RNA (mRNA) expression, could also be seen at the protein level by immunohistochemistry (Fig. 1 D).
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Netrin-1 is up-regulated in a large fraction of aggressive NB
We focused on stage 4 NB with a specific interest in comparing netrin-1 and its receptors' expression levels between the three distinct clinical patterns of stage 4, based on disease distribution and age of the patients (26). On the one hand, there are the neonates and infants (<1 yr of age) with stage 4S (2–5% of all NB) and the similarly young stage 4 without 4S features, hereafter termed [1yr–] stage 4, who make up 10% of the NB population. On the other hand, there are the stage 4 children (>1 yr of age), comprising 45% of all NBs, who will hereafter be termed [1yr +] stage 4. These three clinical aspects of stage 4 NB differ in their respective malignant behaviors and associated prognoses: good for stage 4S and [1yr –] stage 4 (5-yr event-free survival >80%), and dismal for [1yr +] stage 4 (5-yr event-free survival of 
30%) despite intensive treatment including high-dose chemotherapy and hematopoietic stem cell transplantation (27, 28).
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Despite a largely favorable prognosis among infants with stage 4 NB (i.e, stage 4S and [1yr–] stage 4) with no MNA, many succumb to the disease. Thus, we assessed whether netrin-1 expression may serve as a prognostic marker for the infants with stage 4 NB. As shown in Fig. 1 E, the overall survival of infants with stage 4S differed markedly based on whether the tumor displayed high levels of netrin-1 expression (netrin-1 high) or low-level expression (netrin-1 low), with the netrin-1 expression threshold being its median expression value in the 102 cases. Indeed, although 100% of the infants survived after 10 yr (including 1 MNA out of 17), when the NB 4S was netrin-1 low, the 5-yr overall survival was only 46% when the NB 4S was netrin-1 high (P = 0.0109). Furthermore, 43% of the non-MNA patients with high-level netrin-1 expression died. More generally, when a similar overall survival analysis was performed on all infants with stage 4 NB (i.e, stage 4S and [1yr–] stage 4), a similar dichotomy was observed. Indeed, 5-yr overall survival was found to be 90% for the netrin-1–low infants yet only 48% for netrin-1–high infants (P = 0.032; Fig. 1 F). These data suggest that netrin-1 is a potential prognostic marker for aggressiveness in stage 4 NB diagnosed in infants. Whether or not it constitutes an independent prognostic marker of stage 4 NB in neonates and infants deserves to be tested in a larger patient cohort. Nevertheless, these data indicate that a netrin-1 threshold may turn as an alternative determinant for the biological behavior of stage 4 NB in infants, potentially suggesting its involvement in a cell death process of very early childhood neuroblasts, reminiscent of that operating during nervous system development (31).
Netrin-1 high expression is not only detected in 38% of [1yr+] stage 4 and in poor outcome [1yr–] stage 4 primary NB tumors but also in a fraction of NB cell lines mainly derived from stage 4 tumor material (Fig. 2 A and Fig. S2 A). Two human NB cell lines, IMR32 (netrin-1 high) and CLB-Ge2 (netrin-1 low), were evaluated further. In spite of a marked difference in netrin-1 and DCC expression, the UNC5H levels are similar in IMR32 and CLB-Ge2 cells; UNC5H1, UNC5H3, and UNC5H4 show the highest expression (Fig. 2 B). Specifically, UNC5H1, UNC5H3, and UNC5H4 proteins could be detected at the plasma membrane by confocal analysis (Fig. 2 C). To test the hypothesis that the high netrin-1 mRNA levels detected in IMR32 cells are associated with an autocrine netrin-1 production, we next performed netrin-1 immunohistochemistry on IMR32 and CLB-Ge2 cells. As shown in Fig. 2 D, a netrin-1–specific membrane staining was detected in a homogeneous pattern in IMR32 cells, whereas no specific staining was detected for CLB-Ge2 cells. Confocal analysis further confirmed the presence of netrin-1 at the cell membrane (Fig. 2 E and Fig. S2 B). To further analyze whether netrin-1 is secreted from IMR32 cells, netrin-1 ELISA assay was used to detect netrin-1 in the conditioned medium. As shown in Fig. 2 F, 11.7 ng/ml netrin-1 was recovered from the conditioned medium of IMR32 cells, whereas no netrin-1 was detected from the conditioned medium of CLB-Ge2 cells. Thus, together these data suggest that the high netrin-1 content observed in aggressive NB could result from an autocrine expression of netrin-1 in NB cells.
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Netrin-1 up-regulation is a selective advantage for NB cell survival
To investigate whether the netrin-1 autocrine expression observed in IMR32 cells provides a selective advantage for survival, as would be expected from the dependence receptor theory, cell death was analyzed in response to the disruption of this autocrine loop. As a first approach, netrin-1 was down-regulated by RNA interference. As shown in Fig. 3 A, the addition of netrin-1 small interfering RNA (siRNA) to IMR32 cells was associated with a significant reduction in netrin-1 mRNA. This mRNA reduction was associated with a decrease of netrin-1 protein as observed by immunohistochemistry (Fig. 3 B). Although scramble siRNA failed to affect IMR32 cell survival, as measured by trypan blue exclusion, netrin-1 siRNA treatment was associated with IMR32 cell death (Fig. 3 C). In contrast, CLB-Ge2 cell survival was unaffected after netrin-1 siRNA treatment (Fig. 3 C). To determine whether this increase in cell death was in part caused by an increase in apoptotic cell death, caspase-3 activity was measured in response to netrin-1 siRNA treatment. As shown in Fig. 3 D, although significant apoptotic cell death was detected upon netrin-1 siRNA treatment in IMR32 cells, no such effect was observed in CLB-Ge2 cells. A similar proapoptotic effect of the netrin-1 siRNA was observed in CLB-VolMo cells, another netrin-1 high cell line (unpublished data).
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Furthermore, in IMR32 cells, the proapoptotic serine threonine kinase DAP kinase (DAPK), which is shown to be required for UNC5H-induced cell death, exhibited a loss of its inhibitory autophosphorylation (38) upon DCC-5Fbn treatment (Fig. 5 F) or netrin-1 siRNA transfection (Fig. 5 G). Accordingly, autophosphorylation was restored by a treatment with excess netrin-1 or by a combination of UNC5H1, UNC5H3, and UNC5H4 siRNAs. Moreover, the transfection of the antiapoptotic protein BCL-2 was sufficient to inhibit netrin-1 siRNA-induced cell death but did not inhibit DAPK dephosphorylation, hence suggesting that DAPK activation is not a result of cell death but is specifically engaged by UNC5H after netrin-1 inhibition (unpublished data).
Interference with netrin-1 inhibits NB progression and dissemination
We next assessed whether in vivo modulation of netrin-1 could be used to limit/inhibit NB progression and dissemination. An elegant chicken model has been developed in which graft of NB cells in the chorioallantoic membrane (CAM) of 10-d-old chick embryos recapitulates both tumor growth at a primary site, i.e., within the CAM, and tumor invasion and dissemination at a secondary site, metastasis to the lung (Fig. 6 A). In a first approach, IMR32 or CLB-Ge2 cells were loaded in 10-d-old CAM and embryos were treated on days 11 and 14 with PBS or DCC-5Fbn. 17-d-old chicks were then analyzed for primary tumor growth and metastasis to the lung. As shown in Fig. 6 (B and C), specifically in CAMs grafted with IMR32 but not with CLB-Ge2 cells, DCC-5Fbn significantly reduced primary tumor size. This size reduction was associated with increased tumor apoptosis, as shown by an increased caspase-3 activity in the tumor lysate (Fig. 6 D). More importantly, DCC-5Fbn also reduced lung metastasis formation, as shown in Fig. 6 E. Similar results were obtained when CAM-grafted embryos were treated with netrin-1 siRNA (unpublished data). To next assess whether DCC-5Fbn could also induce the regression of metastatic lesions, IMR32 cells were CAM grafted and DCC-5Fbn (or PBS) treatment started after metastasis to the lung is known to occur, i.e., treatments were performed on days 14 and 15 because pulmonary metastases are routinely detectable at day 13. As shown in Fig. 6 F, pulmonary metastases were markedly reduced, suggesting that DCC-5Fbn not only inhibits tumor dissemination but also induces regression of metastatic lesions at the secondary site.
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3β1 (42). It is then interesting to wonder why such a large fraction of aggressive NBs have selected a gain of netrin-1 rather than a loss of the netrin-1 dependence receptor death pathways. A possible explanation is that netrin-1 expression not only confers a gain in survival, but may also lead to enhancement of nonapoptotic signaling mediated by netrin-1 receptors. Netrin-1 was indeed shown to bind a complex that includes some integrins (43). These integrins regulate cell migration and invasiveness and, thus, may play a role in cancer progression. Netrin-1 was also proposed to play a role in angiogenesis, although whether netrin-1 is proangiogenic or antiangiogenic is controversial (44–47) and this effect on angiogenesis may increase NB metastases development. It is also of interest to note that NB is a complex disease that originates from migrating neural crest cells. Netrin-1 up-regulation may then be implicated in the main function played by netrin-1 during nervous system development that is neuronal navigation. Along this line, netrin-1 and DCC have been shown to play an important role during neural crest cell migration (48), and it is then tempting to suggest that the gain in netrin-1 expression also promotes NB cell migration.
As different types of stage 4 NBs are distinguished, not only by the age of the children but also by the tissues in which metastases are found, one may wonder about the implication of netrin-1 produced in the normal tissues in which the tumor cells spread, and this would be interesting to explore. In particular, it would be interesting to evaluate whether, according to the classical "seed and soil" theory for metastasis (for review see reference 18), netrin-1 expression in specific tissues may favor metastasis development more specifically in these tissues. It is also intriguing to note that others have shown that in some particular cell lines, netrin-1 is able to promote apoptosis rather than inhibit apoptosis (49), so that the view of netrin-1 up-regulation being only a survival-selective advantage to block apoptosis via dependence receptors is probably only part of the overall regulatory mechanisms that links NB, netrin-1, and its receptors.
The observation that low levels of netrin-1 in NB correlate with good outcome is of clinical interest, in particular in NB diagnosed in neonates and infants. Indeed, low netrin-1 expression predicts long-term survival in infants (100% in 4S stage and 90% in infants in general) in a type of pathology in which therapeutic management is highly dependent on presentation and MNA (50, 51). This is particularly true with stage 4S infants who receive no (or minimal) treatment based on the lack of MNA, even though current statistics show that 1 in 10 of these infants will eventually die of NB. In this paper, we propose that determination of low netrin-1 level confirms a good prognosis for these infants without therapy, whereas the infants with high netrin-1 expression should be considered for more intensive treatment. Regarding infants or children with high netrin-1–expressing NB tumors, an alternative or supplementary targeted treatment based on disruption of the netrin-1 autocrine survival loop may improve standard high-dose chemotherapy regimen efficiency. We propose that a treatment based on inhibition of the interaction between netrin-1 and its dependence receptors, or inhibition of the ability of netrin-1 to multimerize its receptors, could potentially improve the survival of a large fraction of the patients suffering from aggressive NB. Moreover, it is interesting to note that no correlation between netrin-1 up-regulation and molecular signature of apoptosis and invasion was observed in NB tumors (Fig. S1 D), strengthening the case for netrin-1 as an original target for NB. Future preclinical and clinical studies should assess whether such therapeutic strategies, which could include small molecules (drugs), monoclonal antibodies, or the DCC-5Fbn recombinant protein presented in this paper, used alone or in combination with standard chemotherapy, could be of therapeutic benefit for infants and children with NB.
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Human NB tumor samples and biological annotations.
According to parental consent, surgical human NB tumor material was immediately frozen. Material and annotations were obtained from the Biological Resources Centers of both national referent Institutions for NB treatment (Centre Léon Bérard and at Institut Gustave Roussy). Protocols using human material were approved by the local ethics Committees of Lyon University and Paris XI University. MYCN genomic content was assessed on histologically qualified tumors as previously described (52). For immunohistochemistries, 5-µm sections were prepared and frozen at –80°C.
Plasmid constructs, siRNA, and DCC-5Fbn production.
The dominant-negative mutant for UNC5H and DCC (pCR-UNC5H2-IC-D412N and pCR-DCC-IC-D1290N, respectively) and the plasmids encoding Neogenin (pCDNA3-Neogenin) and UNC5H1 (pCDNA3.1-UNC5H1-HA) have been previously described (1, 2, 5). The plasmids encoding UNC5H2 (pCDNA3.1-UNC5B-HA), UNC5H3 (pCDNA3-UNC5C-HA), and UNC5H4 (pCAG3-hU5H4-His) were gifts from H. Arakawa (National Cancer Institute, Tokyo, Japan), M. Tessier-Lavigne (Genentech, San Francisco, CA), K.L. Guan (University of Michigan, Ann Arbor, MI), and N. Yamamoto (Osaka University, Osaka, Japan). Human netrin-1–encoding plasmid (peak8-hNTN1-His) was obtained from D.E. Bredesen (The Buck Institute for Age Research, Novato, CA). Ps974-DCC-5Fbn allowing bacterial expression of the fifth fibronectin type III domain of DCC was obtained by inserting a Pst1–BamH1 DNA fragment generated by PCR using pDCC-CMV-S as a template. DCC-5Fbn production was performed using a standard procedure. In brief, BL21 cells were forced to express DCC-5Fbn in response to imidazole, and the BL21 lysate was subjected to affinity chromatography using FLAG-Sepharose (Sigma-Aldrich). A peptide corresponding to the ectodomain of IL3R was produced in the same conditions and used as a control. For cell culture use, netrin-1, DCC, and neogenin siRNAs (Santa Cruz Biotechnology, Inc.) were designed as a pool of three target-specific 20–25-nt siRNAs. UNC5H1, UNC5H2, UNCH3, and UNC5H4 siRNAs were designed by Sigma-Aldrich. MYCN siRNA was designed by Thermo Fisher Scientific.
Cell death assays.
2 x 105 cells were grown in serum-poor medium and were treated (or not) with 1 µg/ml DCC-5Fbn or transfected with siRNA using Lipofectamine 2000. Cell death was analyzed using trypan blue staining procedures as previously described (1). The extent of cell death is presented as the percentage of trypan blue–positive cells in the different cell populations. Apoptosis was monitored by measuring caspase-3 activity as described previously (1) using the Caspase 3/CPP32 Fluorimetric Assay kit (Gentaur). For detection of DNA fragmentation, treated cells were cytospun, and TUNEL was performed with 300 U/ml TUNEL enzyme and 6 µM biotinylated dUTP (Roche) as previously described (53).
Q-RT-PCR.
To assay netrin-1, DCC, and UNC5H receptor expression in NB samples, total RNA was extracted from histologically qualified tumor biopsies (>60% immature neuroblasts) using the NucleoSpin RNAII kit (Macherey-Nagel), and 200 ng were reverse transcribed using 1U Superscript II reverse transcription (Invitrogen), 1U RNase inhibitor (Roche), and 250 ng of random hexamer (Roche). Total RNA was extracted from mouse and human cell lines using the NucleoSpin RNAII kit and 1 µg was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad Laboratories). Real-time Q-RT-PCR was performed on a LightCycler 2.0 apparatus (Roche) using the LightCycler FastStart DNA Master SYBER Green I kit (Roche). Reaction conditions for all optimal amplifications, as well as primer selection for murine and human netrin-1, DCC, and UNC5H1-4, were determined as already described. The ubiquitously expressed human HPRT genes showing the least variability in expression in NB was used as an internal control (54). The sequences of the primers are the following: NTN1, 5'-TGCAAGAAGGACTATGCCGTC-3' and 5'-GCTCGTGCCCTGCTTATACAC-3'; UNC5H1, 5'-CATCACCAAGGACACAAGGTTTGC-3' and 5'-GGCTGGAAATTATCTTCTGCCGAA-3'; UNC5H2, 5'-GGGCTGGAGGATTACTGGTG-3' and 5'-TGCAGGAGAACCTCATGGTC-3'; UNC5H3, 5'-GCAAATTGCTGGCTAAATATCAGGAA-3' and 5'-GCTCCACTGTGTTCAGGCTAAATCTT-3'; UNC5H4, 5'-GGTGAACCCAGCCTCCAGTCAG-3' and 5'-CTTCCACTGACATCACTTCCTCCC-3'; DCC, 5'-AGCCAATGGGAAAATTACTGCTTAC-3' and 5'-AGGTTGAGATCCATGATTTGATGAG-3'; and HPRT, 5'-TGACACTGGCAAAACAATGCA-3' and 5'-GGTCCTTTTCACCAGCAAGCT-3'.
Genomic DNA quantification.
Genomic DNA from IMR32 and CLB-Ge2 cells was extracted with the NucleoSpin Tissue kit (Macherey-Nagel). 50 ng of genomic DNA was used to perform quantitative PCR using primers specific to NTN1 and MYCN genomic sequences. Real-time quantitative PCR was performed on a LightCycler 2.0 apparatus using the Light Cycler FastStart DNA Master SYBER Green I kit. NAGK (the N-acetylglucosamine kinase gene), which is located on chromosome 2 similarly to the MYCN gene but separated from the MYCN amplicon, was used as an internal control gene to determine the gene dosage (55). For each pair of primers, genomic DNA amplification was assessed by polymerase activation at 95°C for 10 min, followed by 35 cycles at 95°C for 10 s, 65°C for 30 s, and 72°C for 10 s. The sequences of the primers are the following: NTN1, 5'-CTGTGTCCCCCACTTGTTCT-3' and 5'-CCATGAACCCCACTGACTCT-3'; MYCN, 5'-GTGCTCTCCAATTCTCGCCT-3' and 5'-GATGGCCTAGAGGAGGGCT-3'; and NAGK, 5'-TGGGCAGACACATCGTAGCA-3' and 5'-CACCTTCACTCCCACCTCAAC-3'.
Immunohistochemistry and immunoblotting analysis.
105 cells were centrifugated on coverslips with a cytospinner (Shandon Cytospin 3; Thermo Fisher Scientific). Tumor slides and cells were fixed in 4% paraformaldehyde. The slides were then incubated at room temperature for 1 h with an antibody recognizing the human netrin-1 (1:150; R&D Systems), UNC5H1 (1:100; Abcam), UNC5H3 (1:100; R&D system), or UNC5H4 (1:100; Santa Cruz Biotechnology, Inc.). After rinsing in PBS, the slides were incubated with an Alexa 488 donkey anti–rat antibody (Invitrogen), an Alexa 488 donkey anti–rabbit antibody (Invitrogen), a Cy3 donkey anti–mouse antibody (Jackson ImmunoResearch Laboratories), or an Alexa 488 donkey anti–goat antibody (Invitrogen), respectively. For tumor slides, netrin-1 and UNC5H4 signals were amplified using biotinyl-tyramide (TSA; Thermo Fisher Scientific) and Alexa 488–streptavidin (Invitrogen). Nuclei were visualized with Hoechst staining. Densitometric value corresponding to netrin-1 signal was quantified with AxioVision Release 4.6 software. Immunoblots were performed as already described using anti–phospho-DAPK and anti-DAPK (Sigma-Aldrich) (38), anti-DCC (1:500; Santa Cruz Biotechnology, Inc.), anti-neogenin (1:500; Santa Cruz Biotechnology, Inc.), anti-HA (1:7,500; Sigma-Aldrich), anti-HIS (1:1,000; QIAGEN), anti-MYCN (1:1000; BD), or anti-β-actin (1:1,000; Millipore) antibodies.
Netrin-1 ELISA assay.
Detection of netrin-1 protein in IMR32 and CLB-Ge2 cell culture medium was performed using a modified ELISA assay. In brief, 96-well plates (Nunc-Immuno plate MaxiSorp; Thermo Fisher Scientific) were coated with 200 ng/well of purified recombinant extracellular domain of DCC (DCC-Ec-Fc). To minimize aspecific binding, each well was incubated with 100 µl of blocking solution, containing 5% (wt/vol) BSA (Sigma-Aldrich) in 0.05% PBS-Tween. 3 ml FBS free cell culture medium was added sequentially (300 µl/well) to coated 96-well plates and incubated for 1 h at 37°C. After three washes with 0.5% BSA/PBS, 100 µl of rat anti–netrin-1 antibody (diluted 1:500 in blocking solution) was added to each well and incubated for 30 min at 37°C. After extensive washing, each well was incubated with 100 µl HRP-conjugated goat anti–rat antibody (1:1,000; Jackson ImmunoResearch Laboratories) for 30 min at 37°C. After removal of unbound antibody by three washes in 0.5% BSA/PBS, the plates were incubated for 5 min at room temperature with ECL Western Blotting Substrate (Thermo Fisher Scientific). Luminescent signal was measured using a Luminoskan Ascent apparatus (Thermo Fisher Scientific).
Reporter assay.
105 cells were plated in 12-well plates and transfected with the firefly luciferase reporter under the control of the netrin-1 promoter (pGL3-NetP-Luc) or the pGL3 empty vector. All transfections were done in triplicate and the Dual-Luciferase Reporter Assay system (Promega) was performed 48 h after transfection according to the manufacturer's protocol, using the Luminoskan Ascent apparatus. As an internal control of transfection efficiency, the renilla luciferase-encoding plasmid (pRL-CMV; Promega) was cotransfected, and for each sample firefly luciferase activity was normalized to the renilla luciferase activity.
Chicken model for NB progression and dissemination.
107 NB cells suspended in 40 µl of complete medium were seeded on 10-d-old chick CAM. 10 µg DCC-5Fbn or the same PBS volume was injected in the tumor on days 11 and 14. For siRNA treatment, 4 µg of scramble or netrin-1 siRNA was injected under the same conditions as for DCC-5Fbn. On day 17, tumors were resected and the area was measured with AxioVision Release 4.6 software (Carl Zeiss, Inc.). To test the effect of DCC-5Fbn on metastasis regression, 3 µg DCC-5Fbn or PBS was injected on days 14 and 15 in a chorioallantoic vessel. To assess metastasis, lungs were harvested from the tumor-bearing embryos and genomic DNA was extracted with a NucleoSpin Tissue kit (Macherey-Nagel). Metastasis was quantified by PCR-based detection of the human Alu sequence using the primers 5'-ACGCCTGTAATCCCAGCACTT-3' (sense) and 5'-TCGCCCAGGCTGGAGTGCA-3' (antisense) with chick GAPDH-specific primers (sense, 5'-GAGGAAAGGTCGCCTGGTGGATCG-3'; antisense, 5'-GGTGAGGACAAGCAGTGAGGAACG-3') as controls. For both couples of primers, metastasis was assessed by polymerase activation at 95°C for 2 min followed by 30 cycles at 95°C for 30 s, 63°C for 30 s, and 72°C for 30 s. Genomic DNA extracted from lungs of healthy chick embryos was used to determine the threshold between NB cell–invaded and –noninvaded lungs. To monitor apoptosis in primary tumors, primary tumors and surrounding CAM were resected and broken up in lysis buffer and caspase-3 activity was measured using the Caspase 3/CPP32 Fluorimetric Assay kit.
NB metastasis in nude mice.
7-wk-old (20–22 g body weight) female athymic nu/nu mice were obtained from Charles River Laboratories. The mice were housed in sterilized filter-topped cages and maintained in a pathogen-free animal facility. IGR-N-91–derived PTX and Myoc cell lines were implanted by i.v. injection of 106 cells in 130 µl of PBS into a tail vein (day 0). 20 µg DCC-5Fbn or PBS with equal volume was i.p. injected daily during 22 d. Lungs were harvested on day 23. Lung genomic DNA was extracted with the NucleoSpin Tissue kit, and quantification of human tumor cells in lungs was done by PCR-based detection of the human Alu sequence using the primers 5'-CACCTGTAATCCCAGCACTTT-3' (sense) and 5'-CCCAGGCTGGAGTGCAGT-3' (antisense), using 25 ng of genomic DNA as previously described (56). PCR was performed under the following conditions: 95°C for 2 min, 30 cycles at 95°C for 30 s, 65°C for 20 s, and 72°C for 20 s. Quantification of human DNA in mice lungs was based on a standard curve using human genomic DNA isolated from PTX and Myoc cell lines.
Online supplemental material.
Fig. S1 associates netrin-1 and DCC expression in NB tumors with their apoptosis and invasion molecular signatures obtained with microarrays. Fig. S2 presents netrin-1 mRNA and protein expression in NB cell lines and shows CLB-VolMo netrin-1–high cell line sensitivity to DCC-5Fbn decoy fragment. In Fig. S3, MYCN and neogenin implication in netrin-1 siRNA-induced cell death is studied, and specificity and efficiency of UNC5H siRNAs are presented at the mRNA, protein, and cellular levels. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20082299/DC1.
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Acknowledgments |
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This work was supported by the Ligue Contre le Cancer (P. Mehlen), the Agence Nationale de la Recherche (P. Mehlen), the Institut National du Cancer (J. Bénard, A. Puisieux, and P. Mehlen), the Société Francaise des Cancers de l'Enfant (J. Bénard), The French Health Minister (J. Bénard and D. Valteau-Couanet), The EU fundings Hermione (P. Mehlen) and APOSYS (P. Mehlen), the Centre National de la Recherche Scientifique, and the Centre Léon Bérard.
The authors have no conflicting financial interests.
Submitted: 14 October 2008
Accepted: 3 March 2009
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