WO2009141440A1

WO2009141440A1 - Netrin-1 overexpression as a biological marker and a survival factor for aggressive neuroblastoma

Info

Publication number: WO2009141440A1
Application number: PCT/EP2009/056252
Authority: WO
Inventors: Patrick Mehlen; Agnès BERNET; Céline DELLOYE
Original assignee: Centre National De La Recherche Scientifique (Cnrs); Centre Leon Berard; Ens - Ecole Normale Superieure De Lyon; Universite Claude Bernard Lyon 1
Priority date: 2008-05-21
Filing date: 2009-05-22
Publication date: 2009-11-26

Abstract

The present invention comprises an in vitro method for predicting whether neuroblastoma (NB) identified in a patient is a metastatic and/or an aggressive NB, and/or a NB having a poor prognosis, or for predicting a NB having a good prognosis. The present invention further comprises a method for preventing and/or treating such NB cancer and a method for selecting a therapeutic compound for the prevention or the treatment of such metastatic and/or aggressive NB. Finally, the present invention is directed to a method for determining in vitro the efficiency of an anti-NB cancer treatment or for in vitro selecting patients who respond to a specific anti-NB treatment and wherein said NB cancer is a metastatic and/or aggressive NB related to the presence of an overexpression of netrin-1 in the tumoral tissue.

Description

Netrin-1 overexpression as a biological marker and a survival factor for aggressive neuroblastoma

The present invention comprises an in vitro method for predicting whether neuroblastoma (NB) identified in a patient is a metastatic and/or an aggressive NB, and/or a NB having a poor prognosis. The invention also encompasses a method for predicting whether neuroblastoma (NB) identified in a patient has a good prognosis. The present invention further comprises a method for preventing and/or treating such NB cancer and a method for selecting a therapeutic compound for the prevention or the treatment of such metastatic and/or aggressive NB. Finally, the present invention is directed to a method for determining in vitro the efficiency of an anti-NB cancer treatment or for in vitro selecting patients who respond to a specific anti-NB treatment and wherein said NB cancer is a metastatic and/or aggressive NB related to the presence of an overexpression of netrin-1 in the tumoral tissue.

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 (Deleted in Colorectal Cancer) 1, 2, 3 and UNC5H. However, more recently, netrin-1 has emerged as a completely different molecule that regulates cell survival. Indeed, the netrin-1 receptors DCC and UNC5H, - i.e., UNC5H1, UNC5H2, and UNC5H3- belong to the so-called dependence receptor family. Dependence receptors form a group of receptors that share the ability to induce cell death when expressed in settings in which their ligand is not available. As pro- apoptotic entities, they have been shown to regulate tumorigenesis as described in different solid tumors like colorectal or breast cancer (see PCT patent application Mehlen et al, published on September 7, 2007 under the number WO 2007/099133).

Dependence receptors now number more than a dozen that include DCC (Deleted in Colorectal Cancer) \ UNC5H ², Patched ³, some integrins ⁴, neogenin ⁵, p75NTR ⁶, RET \ ALK ⁸ and TrkC ⁹. While they have no structural homology, they all share the functional property of inducing cell death when disengaged from their ligands, while the presence of their ligand blocks this pro-apoptotic activity. Such receptors thus create cellular states of dependence on their respective ligands ¹⁰' ⁿ. 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 ¹²' ¹³' ¹⁴ and UNC5H ¹⁵' ¹⁶. However, DCC and UNC5H, - i.e., UNC5H1, UNC5H2, UNC5H3 and UNC5H4- have been shown to belong to the dependence receptor family ^{h 2}; this dependence effect upon netrin-1 has been suggested to act as a mechanism for eliminating tumour cells that would develop in settings of ligand unavailability (for reviews ¹⁷'¹⁸). Along this line, disruption of the pro-apoptotic signaling of these netrin-1 receptors in mice gastrointestinal tract - by netrin-1 overexpression or by inactivation of UNC5H3- is associated with intestinal tumour progression ¹⁹' ²⁰.

A selective advantage for a tumour cell would then be to lose the pro-apoptotic activity of its dependence receptors. In this respect, DCC was proposed in the early 1990s to function as a tumour suppressor gene, whose expression is lost in the vast majority of human cancers ²¹' ²². This hypothesis also fits with the observation that UNC5H genes are down-regulated in most colorectal tumours, hence suggesting that the loss of UNC5H genes represents a selective advantage for tumour development ²⁰' ²³' ²⁴.

It has been found that a large fraction of metastatic breast cancers overexpress netrin-1. Moreover, it has been found that netrin-1 -expressing mammary metastatic tumor cell lines undergo apoptosis when netrin-1 expression is experimentally decreased or when decoy soluble receptor ectodomains are added. Such treatments prevent metastasis formation both in a syngenic mouse model of lung colonization of a mammary cancer cell line and in a model of spontaneous lung metastasis of xenografted human breast tumor (Fitamant et al, Proc Natl Acad Sci U S A. 2008, 25; 105 (12):4850-4855:Netrin-l expression confers a selective advantage for tumor cell survival in metastatic breast cancer).

Neuroblastoma (NB), the most frequent solid tumour of early childhood is diagnosed as a disseminated disease in more than 60% of cases (stage 4). NB displays 3 distinct clinical patterns based on patients' age at presentation and dissemination sites. Indeed neonates and infants (< 1 year of age) with stage 4S and stage 4 without 4S features have an overall good prognosis and often show a spontaneous regression of the disease, whereas stage 4 in children (> 1 year of age) shows a poor prognosis (30% survival at 5 years, despite intensive treatment).

The inventors have investigated whether netrin-1 dependence receptors, DCC (Deleted in Colorectal Cancer) and UNC5H, may also control tumorigenesis of NB, the most common extracranial solid tumour of early childhood that is often diagnosed as a disseminated disease (stage 4).

Indeed, it is particular desirable to provide a marker associated to the aggressiveness of NB with poor prognosis especially for patients diagnosed before one year of age, or to provide a marker associated to the good prognosis of NB. Surprisingly, the inventors have first demonstrated that that a large fraction of aggressive NB have selected a gain of netrin-1 expression, and that high netrin-1 expression is associated with poor outcome especially for patients diagnosed before one year of age. They have also observed that Stage 4S NB that are very often associated with good prognosis is associated with a gain of UNC5H. They have also demonstrated that autocrine production of netrin-1 is a tumour growth and dissemination selective advantage in aggressive NB as it blocks the pro-apoptotic activity of the netrin-1 dependence receptors UNC5H. The inventors have shown that rather than losing netrin- 1 receptors expression, a large fraction of aggressive NB has selected a gain of ligand expression that represents a similar selective growth advantage. They have shown that interference with the netrin-1 autocrine loop in malignant neuroblasts represent an alternative therapeutic strategy, as disruption of this loop triggers in vitro NB cell death and inhibits NB metastasis.

The present invention is thus directed to the use of the netrin-1 up-regulation or overexpression as a potent marker for poor prognosis in NB, particularly for stage 4, more particularly stage 4S, NB diagnosed infants. Alternatively, the present invention is directed to the use of UNC5H (H1-H2-H3-H4) up-regulation as a potent marker for good prognosis in stage 4 NB and more particularly in stage 4S.

The present invention is also directed to the use of compound capable of interfering on the netrin-1 autocrine loop in NB cells as therapeutic compound for the prevention and/or the treatment of metastatic and/or aggressiveness NB. In a first aspect, the present invention is directed to an in vitro method for predicting whether a neuroblastoma (NB) identified in a patient is a metastatic or an aggressive neuroblastoma, or a neuroblastoma having a poor prognosis from a biological sample of said patient containing tumors cells, said method comprising the following step of:

(a) obtaining an isolated biological sample from said patient wherein said isolated biological sample is selected from body fluid, from a tissue sample or a combination thereof;

(b)measuring of the netrin-1 expression level in said biological sample. The method according to the invention wherein said biological sample is a body fluid selected from the group consisting of blood, serum, plasma, saliva, urine, milk, cerebrospinal fluid, tears, nasal secrete, semen, bile, lymph, sweat and/or faeces. Blood, serum, plasma, urine, milk or cerebrospinal fluid are the most preferred body fluids.

When the biological sample is a biological fluid it is also preferred that the step (b) of measuring the netrin-1 expression level in said biological sample is carried out by determining the RNA coding the netrin-1 or by determining the netrin-1 protein present in said biological fluid, particularly when the body fluid is selected from the group consisting of blood, serum, plasma, urine, milk or cerebrospinal fluid.

In a more preferred embodiment, the presence of netrin-1 protein is determined by ELISA, immunoblot or by a proteomic method such as by mass spectroscopy.

The netrin-1 RNA can be determined by method comprising a reverse transcriptase polymerase chain reaction assay.

A method according to the invention, wherein the biological sample is a tissue sample, is preferred. In a preferred embodiment, said tissue sample is a biopsy of tissue selected from brain, muscle, cutis, subcutis, kidney, brain, and liver or a sample of hair or nails. The most preferred are tissue sample or biopsy from the neuroblastoma tumor.

The amino acid sequence of human netrin-1 or human netrin receptor such as

DCC (deleted in Colorectal Cancer) or UNC5H1, UNC5H2, UNC5H3 and UNC5H4 (Unc-5 homo log 1, 2 and 3, 4 equivalent to Unc-5 homo log A, B, C and D) are well known by the skilled man. Example of these amino acid sequences with the localization of their particular domain can be found in Genbank under the accession number AAD09221 or NP 004813 for human netrin-1, NP 588610 for human netrin receptor Unc-5 homo log 1, Q8IZJ1 for netrin receptor Unc-5 homo log 2 and 095185 for Unc-5 homo log 3.

By aggressive cancer, it is intended to designate a tumor quickly growing, tending to spread rapidly, as, for example, an aggressive tumor. Based on the microscopic appearance of cancer cells, pathologists commonly describe tumor grade by four degrees of severity: Grades 1, 2, 3, and 4. The cells of Grade 1 tumors resemble normal cells, and tend to grow and multiply slowly. Grade 1 tumors are generally considered the least aggressive in behavior. Conversely, the cells of Grade 3 or Grade 4 tumors do not look like normal cells of the same type. Grade 3 and 4 tumors tend to grow rapidly and spread faster (aggressive tumors or cancers) than tumors with a lower grade.

The American Joint Commission on Cancer recommends the following guidelines for grading tumors: Grade

GX Grade cannot be assessed (Undetermined grade)

Gl Well-differentiated (Low grade)

G2 Moderately differentiated (Intermediate grade)

G3 Poorly differentiated (High grade) G4 Undifferentiated (High grade)

In a preferred embodiment of the method according to invention, an increase of the netrin-1 expression level or a high level of the netrin-1 expression in said biopsy, compared with expression of netrin-1 in a non-metastatic primary NB tumor biopsies or in a non-aggressive NB biopsies is significant of the presence of a metastatic NB or an aggressive NB, or a NB having a poor prognosis.

In a preferred embodiment of the method according to invention, a low level of the netrin-1 expression in said biopsy, less or equivalent to the level of expression of netrin-1 in a non-metastatic primary NB tumor biopsies or in a non-aggressive NB biopsies is significant of the presence of a non aggressive or non metastatic NB, or a NB having a good prognosis. In a more preferred embodiment, the method for predicting according to the present invention is characterized in that a ratio superior to 1.5, 2, preferably to 2.5, to 3, to 3.5, to 4, to 4.5 and to 5, between netrin-1 expression in the biopsy to be tested and in the non-metastatic or non-aggressive reference biopsy is significant of the presence of a metastatic NB or an aggressive NB, or a NB having a poor prognosis.

In a preferred embodiment of the method according to invention, the measured netrin-1 expression product is the RNA encoding netrin-1.

In a preferred embodiment of the method according to invention, the RNA encoding netrin-1 is measured by a quantitative real time reverse PCR method. In a preferred embodiment of the method according to invention, the measured netrin-1 expression product is the netrin-1 protein level.

In a preferred embodiment of the method according to invention, the netrin- 1 protein level is measured by a method using specific antibodies able to specifically recognize said netrin-1 protein. In a preferred embodiment of the method according to invention, the netrin-1 protein level is measured by an Elisa method, by immunohistochemistry or immunoblot.

In a preferred embodiment of the method according to invention, said patients are selected from patients having a stage 4 neuroblastoma.

In a preferred embodiment of the method according to invention, said patients are patients having a stage 4S neuroblastoma.

In a preferred embodiment of the method according to invention, said patients are infants.

In a preferred embodiment, the method for prediction is characterized in that the measured netrin-1 expression product is the RNA encoding netrin-1, particularly measured by a quantitative real time reverse PCR method, or in that the expression level of netrin-1 which is measured is the measure of the netrin-1 protein level, particularly by a method using specific antibodies able to specifically recognize said netrin-1 protein.

Alternatively, the present invention is directed to the use of the up-regulation of a netrin-1 receptor selected from the group consisting of DCC (deleted in Colorectal Cancer) or UNC5H1, UNC5H2, UNC5H3 and UNC5H4 UNC5H as a potent marker for good prognosis in stage 4 NB and more particularly in stage 4S.

Thus, in an alternative preferred embodiment of the method according to invention, an increase of the netrin-1 receptor expression level or a high level of the netrin-1 receptor expression in said biological sample, particularly in a biological fluid, more particularly at the membrane surface of cells of tissue sample, preferably at the membrane surface of the tumor cells.

The netrin-1 receptor expression level to be evaluated in the biological sample, is compared in the same conditions with the expression of netrin-1 receptor in a biological sample from reference healthy patient or patient having non-metastatic primary NB or non-aggressive NB, an increase of the netrin-1 receptor expression level or a high level in said biological sample, compared to the reference sample, is significant of a good prognosing of the NB. Preferably, the ratio of netrin-1 receptor expression level evaluated for the two sample (patient to be tested/reference) is equal or superior to 1.5, preferably 2.0, 2.5, 3.0 and 3.5 to conclude for a good prognosis.

In a more preferred embodiment, the method for predicting according to the present invention is characterized in that a ratio superior to 1.5, 2, preferably to 2.5, to 3, to 3.5, to 4, to 4.5 and to 5, between netrin-1 receptor expression in the biological sample to be tested and in the reference biological sample (preferably a non-metastatic or non-aggressive NB sample reference) is significant of a good prognosis.

In a preferred embodiment of the method according to invention, the measured netrin-1 receptor expression product is a mRNA encoding a netrin-1 receptor. In a preferred embodiment of the method according to invention, the mRNA encoding netrin-1 receptor is measured by a quantitative real time reverse PCR method. In a preferred embodiment of the method according to invention, the measured netrin-1 receptor expression product is the netrin-1 netrin-1 receptor protein level.

In a preferred embodiment of the method according to invention, the netrin- 1 protein level is measured by a method using specific antibodies able to specifically recognize said netrin-1 protein or netrin-1, preferably labeled. In a preferred embodiment of the method according to invention, the netrin- 1 netrin-1 receptor protein level is measured by an ELISA method, by immunohistochemistry or immunoblot.

In a preferred embodiment of the method according to invention, said patients are infants. In a preferred embodiment, the method for prediction is characterized in that the measured netrin-1 receptor expression product is the mRNA encoding netrin-1 receptor, particularly measured by a quantitative real time reverse PCR method, or in that the expression level of netrin-1 receptor which is measured is the measure of the netrin-1 receptor protein level, particularly by a method using specific antibodies able to specifically recognize said netrin-1 receptor protein.

In a preferred embodiment, said netrin-1 receptor is selected from the group consisting of DCC (deleted in Colorectal Cancer) or UNC5H1, UNC5H2, UNC5H3 and UNC5H4, the UNC5H (Hl, H2, H3 and H4) being the more preferred.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) the netrin- 1 protein or its receptor.

The term "antibody" comprises monoclonal or polyclonal antibodies but also chimeric or humanized antibodies. An isolated netrin-1 protein or netrin-1 receptor protein, or a specific fragment thereof can be used as an immunogen to generate antibodies that bind such protein using standard techniques for polyclonal and monoclonal antibody preparation. It may be also possible to use any fragment of these protein which contains at least one antigenic determinant may be used to generate these specific antibodies. A protein immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain said protein, or fragment thereof, and further can include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.

Thus, antibody for use in accordance with the invention include either polyclonal, monoclonal chimeric or humanized antibodies, antibodies able to selectively bind, or which selectively bind to an epitope-containing a polypeptide comprising a contiguous span of at least 8 to 10 amino acids of an amino acid sequence of the netrin-

1 protein or its receptor.

A preferred agent for detecting and quantifying mRNA or cDNA encoding netrin-1 protein, is a labeled nucleic acid probe or primers able to hybridize this mRNA or cDNA. The nucleic acid probe can be an oligonucleotide of at least 10, 15, 30, 50 or 100 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA or cDNA. The nucleic acid primer can be an oligonucleotide of at least 10, 15 or 20 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA or cDNA, or complementary sequence thereof. A preferred agent for detecting and quantifying the netrin-1 protein, is an antibody able to bind specifically to this protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescent Iy labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

For example, in vitro techniques for detection of candidate mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of the candidate protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of candidate cDNA include Southern hybridizations. When the invention encompasses method for quantifying the level of netrin-1 protein, the method can comprise a labeled compound or agent capable of quantifying these proteins.

In certain embodiments of the method of the present invention, the determination of the netrin-1 transcripts involves the use of a probe/primer in a polymerase chain reaction (PCR), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., 1988, Science 241 :23- 1080; and Nakazawa et al., 1994, Proc. Natl. Acad. Sci. USA, 91 :360-364), or alternatively quantitative real time RT-PCR This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g. mRNA) from the cells of the sample, optionally transforming mRNA into corresponding cDNA, contacting the nucleic acid sample with one or more primers which specifically hybridize to the netrin-1 or mRNA or their corresponding cDNA under conditions such that hybridization and amplification of the netrin-1 mRNA or cDNA occurs, and quantifying the presence of the amplification products. It is anticipated that PCR and/or LCR may be desirable to use as an amplification step in conjunction with any of the techniques used for quantifying nucleic acid detecting.

The methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or set of primer or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to follow-up or diagnose patients.

In a second aspect, the present invention is directed to a method for preventing and/or treating metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the NB tumoral tissue, comprising administering an amount of a compound effective to inhibit the binding of netrin-1 to its dependence receptor.

The present invention also comprises the use of a compound effective to inhibit the binding of netrin-1 to its dependence receptor, for the manufacture of a medicament for the prevention or the treatment of metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the tumoral tissue. In a preferred embodiment, said dependence receptor is selected from the group consisting of DCC, UNC5H1, UNC5H2, UNC5H3 or UNC5H4.

The present invention also comprises a method for preventing and/or treating metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the NB tumoral tissue, comprising administering an amount of a compound effective to inhibit the expression or the activity of the netrin-1 protein.

The present invention also comprises the use of a compound effective to inhibit the expression or the activity of the netrin-1 protein, for the manufacture of a medicament for the prevention or the treatment of metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin- 1 in the tumoral tissue.

In a preferred embodiment, said compound to be administered is selected from one or more of an anti-netrin-1 antibody, an UNC5H receptor, an UNC5H receptor ectodomain, a DCC receptor, a DCC ectodomain, or an siRNA capable of inhibiting the expression of said netrin-1 by the tumors cells.

Antibodies can be used as inhibitors of the activity of a particular protein (e.g., blocking antibodies). Antibodies can have extraordinary affinity and specificity for particular epitopes. Antibodies that bind to a particular protein in such a way that the binding of the antibody to the epitope on the protein can interfere with the function of that protein. For example, an antibody may inhibit the function of the protein by sterically hindering the proper protein-protein interactions or occupying active sites. Alternatively the binding of the antibody to an epitope on the particular protein may alter the conformation of that protein such that it is no longer able to properly function. In the context of the present application, a preferred antibody may bind to and inhibit the function of a receptor required for netrin-1 signaling in a cell. Alternatively, the antibody may bind to a different site on the enzyme to sterically hinder the protein- protein interactions required for function. In still another example, the antibody may bind to a different site on the protein and alter the conformation of the protein such that the protein is no longer able to function.. Monoclonal or polyclonal antibodies can be made using standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster, a rat, a goat, or a rabbit can be immunized with an immunogenic form of the peptide. Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. Following immunization of an animal with an antigenic preparation of a polypeptide, antisera can be obtained and, if desired, polyclonal antibodies isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a particular polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells. In the context of the present invention, antibodies can be screened and tested to identify those antibodies that can inhibit the function of a particular protein. One of skill in the art will recognize that not every antibody that is specifically imrmmoreactive with a particular protein will interfere with the function of that protein. However, one of skill in the art can readily test antibodies to identify those that are capable of blocking the function of the netrin-1 protein.

In a preferred embodiment, said compound said is selected in the group consisting of the compound comprising the extracellular domain of a netrin-1 receptor or fragment thereof, said-1 receptors being selected from the group of DCC, UNC5H (particularly UNC5H1 , UNC5H2, UNC5H3 and UNC5H4).

In a preferred embodiment, said compound comprises an extracellular domain of netrin-1 receptor is from DCC, preferably said compound is DCC-EC-Fc or DCC-5Fbn. In another embodiment, the compound that inhibits the expression and/or activity of a netrin polypeptide is selected from an antisense oligonucleotide that binds to and inhibits the expression and/or activity of netrin, or a ribozym that inhibits the expression and/or activity of netrin, a small molecule that binds to and inhibits the expression and/or activity of netrin, or a small molecule that inhibits the expression and/or activity of netrin by interfering with the binding of netrin to a netrin receptor.

Said compound to be administered can be an antisense or iRNA (interfering RNA) oligonucleotides specific of the nucleic acid encoding netrin- 1 protein, particularly a siRNA (small interfering RNA).

Interfering RNA (iRNA) is a phenomenon in which a double stranded RNA (dsRNA) specifically suppresses the expression of a gene bearing its complementary sequence. iRNA has since become a useful research tool for many organisms. Although the mechanism by which dsRNA suppresses gene expression is not entirely understood, experimental data provide important insights. This technology has great potential as a tool to study gene function in mammalian cells and may lead to the development of pharmacological agents based upon siRNA The RNAi constructs contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript for the gene to be inhibited (i.e., the "target" gene). The double- stranded RNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi. Thus, the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 31 end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art.

In a third aspect, the present invention relates to a method for selecting a compound for the prevention or the treatment of metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin- 1 in the NB tumoral tissue, wherein said method comprises the following steps of: a) having a medium containing netrin- 1, or a fragment thereof, and a netrin- 1 receptor, or a fragment thereof, wherein: - said netrin-1, or a fragment thereof, and said netrin-1 receptor, or a fragment thereof, is able to specifically interact together to form a binding pair, and/or

- said netrin-1, or a fragment thereof, is able to induce the dimerization or multimerization of said netrin-1 receptor, or a fragment thereof, particularly the intracellular domain of said netrin-1 receptor; b) contacting said medium with the compound to be tested; c) - measuring the inhibition of the interaction between netrin-1, or a fragment thereof, and said netrin-1 receptor, or a fragment thereof, and/or

- determine whether said compound inhibit the dimerization or multimerization of said netrin-1 receptor, or a fragment thereof, particularly the dimerization of the intracellular domain of said netrin-1 receptor; and d) selecting said compound if:

- the measuring in step c) demonstrates a significantly inhibition of the interaction between netrin-1, or a fragment thereof, and netrin-1 receptor, or a fragment thereof, in presence of said compound, and/or

- the determination in step c) demonstrates a significantly inhibition of the dimerization or multimerization of said netrin-1 receptor, or a fragment thereof, in presence of said compound, particularly the dimerization of the intracellular domain of said netrin-1 receptor. By the terms interaction between netrin-1 and its netrin-1 receptor, it is intended to designate in the present application the interaction which result to the selective advantage for tumor cells to escape netrin-1 dependence receptors induced apoptosis, preferably due to elevated netrin-1 level.

So, the inhibition of this interaction can be obtained for example by the complete or partial inhibition of the binding of netrin-1 to its receptor, notably in presence of a competitive ligand (such as an antibody which is directed to this extracellular membrane domain of said netrin-1 receptor), or in presence of a compound able to form a specific complex with the netrin-1 (such as a soluble extracellular membrane domain of its netrin-1 receptor, or part thereof). In a preferred embodiment, at step a) said netrin-1 receptor is selected from the group of DCC and UNC5H (particularly UNC5H1, UNC5H2 or UNC5H3). In a preferred embodiment, at step a) said netrin-1 or/and said netrin-1 receptor are from mammal, particularly from mouse, rat or human, human being the more preferred.

In a preferred embodiment, at step c): - the measure of the inhibition of the interaction between netrin-1, or a fragment thereof, and said netrin-1 receptor, or a fragment thereof, is carried out by immunoassay (particularly by ELISA or by Immunoradiometric Assay (IRMA)), by Scintillation Proximity Assay (SPA) or by Fluorescence Resonance Energy Transfer (FRET); and/or - the dimerization or multimerization, or its inhibition, of said netrin-1 receptor, or fragment thereof, particularly the intracellular domain, is carried out by immunoprecipitation or FRET.

In a preferred embodiment, at step a) said medium contains cells which express at their surface membrane an endogenous netrin-1 receptor or a recombinant netrin-1 receptor, particularly at least the extracellular domain of a recombinant netrin-1 receptor.

In a preferred embodiment, at step a) said medium contains cells which express recombinant netrin-1 receptor.

In a preferred embodiment, at step a) said medium contains tumoral cells which express endogenously said netrin-1 receptor at their membrane surface and which express or overexpress netrin-1, and wherein at step c) the inhibition of the interaction between netrin-1 and its netrin-1 receptor in presence of the compound to be tested, is measured by the apoptosis or cells death induced by the presence of the compound to be tested.

In a preferred embodiment, at step a) said medium contains metastatic neuroblastoma cells, particularly cells selected from the group consisting of CLB-GEl and IMR32 cells.

The present invention also relates to a method for selecting a compound for the prevention or the treatment of metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the tumoral tissue, wherein said method comprises the following steps of: a) having a medium containing a mammal cell expressing an endogenous or a recombinant netrin-1 receptor, or a fragment thereof comprising at least its intracellular domain, preferably a tumor cell, more preferably a cell presenting dimerization or multimerization of its netrin-1 receptor intracellular domain or a cell wherein its netrin- 1 receptor intracellular domain is able to dimerize or multimerize in presence of netrin- i; b) contacting said medium with the compound to be tested, optionally the medium further containing netrin-1, or a fragment thereof able to interact with the extracellular domain of the netrin-1 receptor; c) determine whether the dimerization or multimerization of said netrin-1 receptor intracellular domain is inhibited in presence of said compound to be tested; d) optionally, determine whether the presence of the compound to be tested induces the cell death of said mammal cell; and e) selecting said compound if the determination in step c) demonstrates a significantly inhibition of the dimerization or multimerization of the intracellular domain of said netrin-1 receptor and/or if the determination in step d) demonstrates the cell death of said mammal cell.

In a forth aspect, the present invention is directed to a method for determining in vitro the efficiency of an anti-cancer treatment for a patient or for in vitro selecting patients who respond to a specific anti-cancer treatment and wherein said cancer is a metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the tumoral tissue, said method comprising the following step of:

(a) obtaining a primary tumor biopsy of said treated patient; and

(b) measuring of the netrin-1 expression level in said biopsy, wherein the efficiency of said anti-cancer treatment is correlated with the decrease of the amount of the netrin-1 expression level measured in said biopsy, or wherein the selected patients who respond to a specific anti-cancer treatment are patients where the amount of the netrin-1 expression level measured in their biopsy has been decreased after said specific treatment.

When administered to a patient, a compound of the present invention is preferably administered as component of a composition that optionally comprises a pharmaceutically acceptable vehicle. The composition can be administered orally, or by any other convenient route, and may be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer the selected compound of the present invention or pharmaceutically acceptable salts thereof.

The mode of administration is left to the discretion of the practitioner. In most instances, administration will result in the release of the compound into the bloodstream or directly in the primary tumor. These therapeutical compositions can additionally comprise a suitable amount of a pharmaceutically acceptable vehicle so as to provide the form for proper administration to the patient. The term "pharmaceutically acceptable" means approved by a regulatory agency or listed by a national or a recognized pharmacopeia for use in animals, mammals, and more particularly in humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is administered. Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical vehicles can be saline, gelatin, starch and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene, glycol, water and the like. Test compound compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions of the invention can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. Said composition is generally formulated in accordance with routine procedures as a pharmaceutical composition adapted to human beings for oral administration or for intravenous administration. The amount of the active compound that will be effective in the treatment can be determined by Standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for oral, intranasal, intradermal or intraveneous administration are generally about 0.01 milligram to about 75 milligrams per kilogram body weight per day, more preferably about 0.5 milligram to 5 milligrams per kilogram body weight per day.

It is to be understood that while the invention has been described in conjunction with the above embodiments, that the foregoing description and the following examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Figures Legend

Figures 1A-1I: Netrin-1 up-regulation is detected in aggressive neuroblastoma

A and B, Q-RT-PCR using specific netrin-1 and netrin-1 receptors primers was performed on total RNA from tumours from a total of 102 stage 4 NB patients. Figure IA, DCC and UNC5H level in the different stage 4 NB. Q-RT-PCR using DCC, UNC5H1-4 primers was performed. The average receptor level is indicated as: +/- barely detectable, + to +++: moderate to high expression. Figure IBb, an average of netrin-1 mRNA levels in stage 4S and stage 4 [lyr ^"] was respectively compared to the average netrin-1 detected in stage [lyr ⁺], using HPRT housekeeping genes as control. Error bars indicate s.e.m. Statistical treatment of the data: Mann- Whitney test compared to level of stage 4 [lyr ]. * indicates a p<0.05, ** indicates a p<0.01.

C, Representative netrin-1 immunohistochemistry on one stage 4 [lyr ] and one stage 4S tumour. Inset: Control without primary antibody is presented.

D, Netrin-1 mRNA levels in 102 stage 4S (n=24), stage 4 [lyr -] (n=12) and stage 4 [lyr +] (n=66) NB measured by Q-RT-PCR. HPRT housekeeping gene was used as a control. Average netrin-1 mRNA expression value for each subgroup is indicated by "m" value. Netrin-1 mRNA average levels in stage 4S and stage 4 [lyr -] were respectively compared to the average netrin-1 detected in stage [lyr +]. Statistical treatment of the data: Student t-test compared to level of stage 4 [lyr +]. * indicates a p<0.05, ** indicates a p<0.01. Each sample was assessed in two independent experiments. E, Representative netrin-1 immunohistochemistry on one stage 4 [lyr +] and one stage 4S tumor. Inset: Control without primary antibody is presented. Scale bar = 50 μm. T: tumor cells; S: stromal cells. Netrin-1 antibody specificity is further shown in Fig. 2 D and Figure 8B. Immunohistochemistry was performed on 4 stage 4 [lyr +] and 4 stage 4 S tumors. F, Average UNC5H mRNA levels in the different stage 4 NB. Q-RT-PCR using UNC5H1-4 specific primers was performed. UNC5H1-4 mRNA average levels in [lyr - ] stage 4 and in [lyr +] stage 4 were respectively compared to the average UNC5H1-4 levels detected in stage 4S. Error bars indicate s.e.m. Statistical treatment of the data: Student t-test compared to level of stage 4S. * indicates a p<0.05, ** indicates a p<0.01. Samples were analyzed in duplicates for each gene.

G, Representative UNC5H1 and UNC5H4 immunohistochemistries on [lyr+] stage 4 and stage 4S tumors. Inset: control without primary antibody is presented. Scale Bar = 50 μm. Immunohistochemistry was performed on 4 stage 4 [lyr +] and 4 stage 4S tumors H, Netrin-1 up-regulation is a marker of poor prognosis in stage 4S NB. Overall survival in a panel of 24 infants diagnosed with stage 4S NB with primary tumors showing either netrin-1 low (in grey) or netrin-1 high (in black) level. Statistical treatment of the data: Kaplan-Meier method, p value is indicated. I, Netrin-1 up-regulation is a marker of poor prognosis in infants with NB. As in E, with a panel of 30 infants bearing NB.

Figures 2A-2H: Netrin-l up-regulation is detected in neuroblastoma cell lines A, Netrin-1 expression measured by Q-RT-PCR in a panel of 28 NB cell lines. HPRT housekeeping gene was used as a control. The netrin-1 level is indicated as: -: not detectable; +/- barely detectable, + to ++++: moderate to very high expression. Mouse cell lines are in italics. Cell lines outlined and highlighted in grey are respectively netrin-1 low and netrin-1 high cell lines further used in the experiments. B, Netrin-1 receptors expression in IMR32 and CLB-Ge2 cell lines. DCC/UNC5H Q- RT-PCR was performed on netrin-1 expressing (IMR32) or netrin-1 low (CLB-Ge2) cells using specific primers. Ratio of netrin-1 and netrin-1 receptors expression to HPRT housekeeping gene is presented. C, Confocal analysis of UNC5H1, UNC5H3 and UNC5H4 receptors immunostaining in human IMR32 cells. Left and right panels correspond to IMR32 cells transfected with siRNA scr. and specific siRNA UNC5H respectively. Fluorescence intensity profile corresponding to the white dotted bar is presented under each panel. Scale bar: 10 μm.

D, Immunostaining on human IMR32 and CLB-Ge2 cell lines using netrin-1 antibody. Lower inset: Control without primary antibody is presented. Upper inset: Antibody specificity was tested by adding human recombinant netrin-1. Scale bar: 50 μm.

E, Confocal analysis of netrin-1 immunostaining on IMR32 cells. Fluorescence intensity profile corresponding to the white dotted bar is presented in the lower panel. Scale bar: 5 μm. F, Quantification of netrin-1 protein secreted in IMR32 and CLB-Ge2 cells conditioned medium by sandwich Elisa assay. Quantification in ng/mL was made according to a dose curve done with recombinant human netrin-1. Data are means of three independent experiments. Error bars indicate s.e.m. * indicates a p<0.05 using a two-sided Mann- Whitney test compared to level in IMR32 cells. G, Quantification of NTNl and MYCN genomic DNA compared to control NAGK genomic DNA by PCR, using genomic DNA specific primers for each gene, in IMR32 and CLB-Ge2 cells.

H, Quantification of NTNl promoter activity in IMR32 and CLB-Ge2 cells. Both cell lines were transfected with the vector pGL3-NetP-Luc encoding luciferase under the control of NTNl promoter. Data presented are normalized on luciferase activity in cells transfected with pGL3 empty vector. Data are means of four independent experiments. Error bars indicate s.e.m. * indicates a p<0.05 using a two-sided Mann- Whitney test compared to level in IMR32 cells. Figures 3A-3D: Downregulation of netrin-l autocrine loop by small interfering RNA triggers NB tumor cell death

A, Analysis of netrin-1 expression using Q-RT-PCR in IMR32 cell line not transfected (control) or 24h after transfection with scramble siRNA (siRNA scr.) or with netrin-1 siRNA (siRNA net.). Data are means of three independent experiments. Error bars indicate s.e.m. * indicates a p<0.05 using a two-sided Mann- Whitney test compared to level in control.

B, Immunostaining on IMR32 cell line using netrin-1 antibody in the absence of transfection (control) or 24h after transfection with scramble siRNA (siRNA scr.) or with netrin-1 siRNA (siRNA net.). Note that the general caspase inhibitor z-VAD-fmk was added to avoid cell death induced by netrin-1 siRNA. Inset: control without primary antibody is shown. Scale bar: 50 μm. C-D, Cell death induction in IMR32 and CLB-Ge2 cell lines was quantified in non transfected cells (control) or after transfection with either scramble siRNA (siRNA scr.) or netrin-1 siRNA (siRNA net.), using trypan blue exclusion assay (C) or relative caspase-3 activity assay (D). Data are means of four independent experiments. In C and D error bars indicate s.e.m.; * indicates a p<0.05 calculated using a two-sided Mann- Whitney compared to level of control. Figures 4A-4E: Disruption of netrin-1 autocrine loop by a decoy receptor fragment triggers NB tumor cell death

A, Scheme representing netrin-1 and its receptors DCC and UNC5H and the fifth fibronectin type III domain of DCC (DCC-5Fbn) used to induce cell death. The downstream effector DAP kinase (DAPK) implicated in UNC5H induced cell death is also represented.

B,C,D, Quantitative analysis of cell death in IMR32 and CLB-Ge2 cell lines treated with 1 μg/mL DCC-5Fbn, with or without addition of netrin-1 in excess (150 ng/niL) to reverse the effect of DCC-5Fbn. A negative control was also performed by adding an unrelated IL3R peptide produced in the same condition as DCC-5Fbn. Cell death was quantified by trypan blue exclusion assay (B) while apoptosis was monitored by measuring relative caspase-3 activity (C) or by TUNEL staining (D). Scale bar: 100 μm. In D, TUNEL staining was performed on 3 independent experiments. E, Effect of DCC-5Fbn on fresh [lyr +] stage 4 NB. Tumoral cells were directly resuspended from the surgical punction and were plated for 24H in presence of treatment. In B and C, data are means of six independent experiments. In E, Data are means of two independent experiments. Error bars indicate s.e.m.; ** indicates a p<0.01 calculated using a two-sided Mann- Whitney test compared to level of control. Figures 5A-5G: NB tumor cell death occurs via an UNC5H/DAPk pro-apoptotic signaling

A, Quantification of cell death in IMR32 cells transfected with either a dominant negative mutant for DCC pro-apoptotic activity (DN-DCC) or a dominant negative mutant for UNC5H pro-apoptotic activity (DN-UNC5H). Upper panels: DN-DCC and DN-UNC5H proteins expression were analysed by Western Blot. Lower panel: Cell death was quantified by measuring relative caspase-3 activity after scramble or netrin-1 siRNA transfection. s.e.m are indicated. Data are means of three independent experiments; * indicates a p<0.05 calculated using a two-sided Mann- Whitney test compared to level of control.

B, Quantification of cell death in IMR32 cells transfected with either a scramble siRNA or a netrin-1 siRNA together or not with a DCC siRNA. Upper panel: DCC siRNA efficiency was verified by Western Blot on HEK293T cells transfected with pCR-hDCC together with scramble or DCC siRNA. Lower panel: Cell death was quantified by measuring relative caspase-3 activity. Data are means of three independent experiments; s.e.m are indicated; * indicates a p<0.05 calculated using a two-sided Mann- Whitney test compared to level of control. Similar results were obtained when neogenin or MYCN were downregulated (see Supplementary Fig. 3 A). C, Analysis of specificity and efficiency of each UNC5H siRNA by Western Blot in HEK293T cells transfected with UNC5H1, H2, H3 or H4 encoding vector together with each UNC5H siRNA.

D,E, Quantification of cell death in IMR32 cells transfected with either a scramble siRNA or a netrin-1 siRNA together with various combination of UNC5H siRNA -i.e, one UNC5H (D) or two or three UNC5H (E) -. Apoptosis was monitored by measuring relative caspase-3 activity. The use of combined UNC5H1, UNC5H2, UNC5H3 and UNC5H4 or UNC5H1, UNC5H3 and UNC5H4 siRNAs leading to the absence of death induced by netrin-1 siRNA are presented in grey. Data are means of three independent experiments. Error bars indicate s e.m.; * indicates a p<0.05, calculated using a two- sided Mann- Whitney compared to level of control.

F,G, Immunodetection of phosphorylated DAPk (P-DAPK) in IMR32 cells either treated with DCC-5Fbn (F) or transfected with netrin-1 siRNA alone or with UNC5H1, H3 and H4 siRNAs (G). In F and G, immunodetection was performed on three independent experiments.

Figures 6A-6F: Disruption of netrin-1 autocrine loop inhibits NB progression and dissemination in a chick model A, Schematic representation of the experimental chick model. IMR32 or CLB-Ge2 cells were grafted in CAM at day 10 and DCC-5Fbn or PBS was injected on day 11 and day 14. Tumors and lungs were harvested on day 17. B,C,D, Effect of DCC-5Fbn on primary tumor growth and apoptosis.

B, Representative images of IMR32 (upper) or CLB-Ge2 (lower) primary tumors formed on CAM treated either with DCC-5Fbn (right) or PBS (left). Scale bar: 2 mm.

C, Quantitative analysis showing the average primary tumor size.

D, Caspase-3 activity was determined in the primary tumors lysates from DCC- 5Fbn/PBS treated IMR32 or CLB-Ge2 grafted embryos.

E, Effect of DCC-5Fbn on lung metastasis. Percentage of embryos with lungs invaded by human IMR32 or CLB-Ge2 cells after two injections (day 11 and say 14) of either

DCC-5Fbn or PBS was performed as described in the methods section.

F, Effect of DCC-5Fbn on lung metastasis regression. Quantification of lung metastasis in embryos CAM-grafted with IMR32 cells and treated after metastasis formation (day 14 and day 15) with DCC-5Fn or PBS. The number of embryos studied in each condition is indicated above the graphs and results from three independent experiments. In C,D,E,F, errors bars indicate s.e.m; C,D,F: * indicates a p<0.05, ** indicates a p<0.005 calculated using a two-sided Mann- Whitney test compared to level of control. E: ** indicates a p<0.005 calculated using a Khi-squared test. Figures 7A- 7D: Disruption of netrin-1 autocrine loop inhibits NB dissemination in a mouse model

A, Analysis of netrin-1 expression using Q-RT-PCR in IGR-N-91 cell line and IGR-N- 91 derived cell lines: PTX (Primary Tumor Xenograft), Bone marrow (BM), Blood and Myocardium (Myoc). Note that while PTX cells fail to express netrin-1, netrin-1 mRNA is highly expressed in Myoc cells. B, Immunostaining on IGR-N-91 cells and the different derived cell lines PTX, BM, Blood or Myoc using netrin-1 antibody. Scale bar: 50 μm. C, Cell death was quantified in IGR-N-91, PTX, BM, Blood or Myoc cell lines treated or not with DCC-5Fbn, with or without addition of netrin-1 in excess to reverse the effect of DCC-5Fbn. A negative control was also performed by adding an unrelated IL3R peptide produced in the same condition as DCC-5Fbn. Data are means of three independent experiments. Errors bars indicate s.e.m; * indicates a p<0.05, calculated using a two-sided Mann- Whitney test compared to level of control.

D, Quantification of lung colonization in PTX or Myoc cells-i.v injected mice treated with PBS or DCC-5Fbn for 22 days. Quantification was performed as described in the methods section. Large bars indicate the median value for both groups. P-value was calculated using a two-sided Mann- Whitney test compared to level of control.

Figures 8A-8D: Netrin-l upregulation in aggressive neuroblastoma and apoptosis / invasion molecular signatures

A, Quantification of netrin-1 signal detected by immunofluorescence on stage 4S and [lyr+] stage 4 NB sections. Data are presented as mean densitometric value measured in 8 distinct fields for each NB subgroup (4 different tumors in each group). Error bars indicate s.e.m. ** indicates a p < 0.01 using a Mann-Whitney test comparing both means.

B, Validation of netrin-1 antibody specificity by Western Blot. 25ng human recombinant netrin-1 was loaded on the separating gel (hrNTNl). Recombinant DCC- 5Fbn was used a control.

C, Quantification of DCC mRNA level by Q-RT-PCR in stage 4 and stage 4S neuroblastomas presented in Figure ID, using HPRT gene as a control. In black, mean UNC5H1 mRNA level in stage 4 and stage 4S NB is presented. Each sample was assessed in two independent experiments. D, Clustering of 9 neuroblastoma tumors (3 stage 4S and 6 stage 4 presented in Figure ID) according to apoptosis (left panel, 2346 genes) and invasion (right panel, 659 genes) molecular signatures, obtained on Affymetrix U95Av2 microarrays⁴¹.The similarity of features was computed by comprehensively comparing all features with each other in a pairwise manner using a modified Spearman coefficient of correlation as a distance metric as described. Color squares over the clusters indicate NTNl, DCC and UNC5H1 expression level (from green: low to red: high) and survival for each patient (white: survival at 10 years, black: death). Figures 9A-9D: Netrin-1 autocrine loop is a survival factor for neuroblastoma cell lines

A, Quantification of netrin-1 mRNA by Q-RT-PCR in a panel of 28 NB cell lines, using HPRT housekeeping gene as a control. Murine cell lines are in italics. B, Confocal analysis of netrin-1 (green) and N-Cadherin specific membrane staining (red) in IMR32 cells. Nuclei are stained with Hoescht (blue). Scale bar: 10 μm.

C, Detection of endogenous DCC and Neogenin proteins in IMR32 cells by Western Blot.

D, Quantitative analysis of apoptosis in netrin-1 positive CLB-VoIMo cells treated with DCC-5Fbn, with or without addition of netrin-1 in excess to reverse the effect of DCC-

5Fbn. A negative control was also performed by adding an unrelated IL3R peptide produced in the same condition as DCC-5Fbn. Apoptosis was monitored by measuring relative caspase-3 activity. Data are means of three independent experiments. Error bars indicate s.e.m. Figures 10A-10D: Disruption of netrin-1 autocrine loop triggers UNC5H pro- apoptotic activity in NB tumor cells.

A, Quantification of cell death induction in IMR32 cells by caspase-3 assay after transfection with either scramble siRNA (siRNA scr.) or netrin-1 siRNA (siRNA net.), together with MYCN siRNA (siRNA MYCN) or Neogenin siRNA (siRNA neog.) (right panel). Data are means of three independent experiments. Error bars indicate s.e.m. * indicates a p < 0.05 compared to level of control condition using a two-sided Mann- Whitney test. Efficiency of MYCN and Neogenin siRNAs was checked by Western Blot by detecting endogenous MYCN protein in IMR32 cells or neogenin in HEK293T cells transfected with pCDNA3 -Neogenin (left panels). B, Specificity and efficiency of siRNAs targeting each UNC5H receptor's expression was quantified by measuring UNC5H1-4 mRNA levels by Q-RT-PCR 48 hours after transfection. UNC5H mRNA subjected to Q-RT-PCR is indicated above. Data are given as a percentage of expression in non transfected cells. Data are means of two independent experiments. Error bars indicate s.e.m. C, Effect of each UNC5H siRNA and netrin-1 siRNA on netrin-1 protein expression was verified in Western Blot by detecting netrin-1 protein in HEK293T cells transfected with peak8-hNTNl-His together with each siRNA mentioned above. D, Impact of transfection of 5 different siRNAs on cell viability was measured by caspase-3 assay in CLB-Ge2 cells transfected with UNC5H1, H2, H3, H4 and netrin-1 siRNAs. Data are means of three independent experiments. Error bars indicate s.e.m.

EXAMPLES

Example 1: Materials and Methods

A) Cell line, transfection procedure, reagents:

Human neuroblastoma cell lines were from the tumor banks at Centre Leon Berard and at Institut Gustave Roussy. More specifically IMR32, CLB-GeI and CLB-Ge2 cell lines were cultured in RPMI 1640 Glutamax medium (Gibco®, Invitrogen, Inc, Carlsbad, CA) containing 10% fetal bovine serum. IGR-N-91 cell line and its derivatives as well as HEK293T cells were cultured in DME medium (Gibco®, Invitrogen) containing 10% fetal bovine serum. Cell lines were transfected using lipofectamine 2000 reagent (Invitrogen) for small interfering RNA (siRNA) or lipofectamine Plus reagent (Invitrogen) for plasmids. Netrin-1 was obtained from Apotech corp/Axxora (Lausanne, Switzerland) and was used at a concentration of 150 ng/ml in all in vitro assays.

B) Human NB tumours samples and biological annotations:

Following parents consents, surgical human neuroblastoma tumors material was immediately frozen. Material and annotations were obtained from the Biological Resources Centers of both national referent Institutions for NB treatment, i.e., Centre Leon Berard 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 described ³⁶. For immunohistochemistries, 5-μm sections were prepared and frozen at -80⁰C.

C) Plasmid constructs, siRNA and DCC-5Fbn production:

The dominant negative mutant for UNC5H and DCC (respectively pCR-UNC5H2-IC- D412N, pCR-DCC-IC-D1290N) and the plasmids encoding Neogenin (pCDNA3- Neogenin) and UNC5H1 (pCDNAS.l-UNCSHl-HA have been previously described ^{h 2}' ⁴⁰ . The plasmids encoding UNC5H2 (pCDNAS.l-UNCSB-HA), UNC5H3 (pCDNA3- UNC5C-HA) and UNC5H4 (pCAG3-hU5H4-His) were kind gifts of Dr. H. Arakawa, Dr. Tessier-Lavigne, K.L. Guan and Dr. Yamamoto. Human netrin-1 encoding plasmid (peak8-hNTNl-His) was obtained from Dr. Bredesen. Ps974-DCC-5Fbn allowing bacterial expression of the fifth fibronectin type III domain of DCC was obtained by inserting a Pstl/BamHl DNA fragment generated by PCR using pDCC-CMV-S as a template. DCC-5Fbn production was performed using a standard procedure. Briefly, 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, St Louis, MO). A peptide corresponding to the ectodomain of interleukin-3 receptor (IL3R) was produced in the same conditions and used as a control. For cell culture use, netrin-1, DCC and neogenin siRNAs were designed by Santa Cruz (CA) as a pool of 3 target- specific 20-25 nt siRNAs. UNC5H1, UNC5H2, UNCH3 and UNC5H4 siRNAs were designed by Sigma-Proligo (St Louis, MO). MYCN siRNA was designed by Dharmacon (Chicago, IL).

D) Cell death assays:

2 x 10⁵ cells were grown in serum-poor medium and were treated (or not) with DCC- 5Fbn or transfected with siRNA using Lipofectamine 2000 (Life Techn). Cell death was analysed 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 ^λ using ApoAlert CPP32 kit from Clontech (USA). For detection of DNA fragmentation, treated cells were cytospun and Terminal deoxynucleodityl transferase mediated dUTP-biotin Nick End Labelling (TUNEL) was performed with 300U/mL TUNEL enzyme (300 U/mL) and 6 μM biotinylated dUTP (Roche Diagnostics), as previously described ³⁷.

E) Quantitative RT-PCR:

To assay netrin-1, DCC, UNC5H receptors expression in neuroblastoma samples, total RNA was extracted from histologically qualified tumour biopsies (> 60% immature neuroblasts) using the Nucleospin RNAII kit (Macherey-Nagel) and 200ng were reverse-transcribed using IU Superscript II reverse transcriptase (Invitrogen), IU RNAse inhibitor (Roche Applied Science) and 250 ng random hexamer (Roche Applied Science). Total RNA was extracted from mouse and human cell lines using the Nucleospin RNAII kit (Macherey-Nagel) and 1 μg was reverse-transcribed using the iScript cDNA Synthesis kit (BioRad). Real-time quantitative RT-PCR was performed on a LightCycler 2.0 apparatus (Roche) using the Light Cycler FastStart DNA Master SYBERGreen I kit (Roche). Reaction conditions for all optimal amplification, as well as primer selection of 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 neuroblastoma was used as an internal control ³⁸. The sequences of the primers are following.

NTNl : 5'-TGCAAGAAGGACTATGCCGTC-S'

5'GCTCGTGCCCTGCTTATACAC-S'; UNC5H1 : 5'-CATCACCAAGGACACAAGGTTTGC-S'

5'GGCTGGAAATTATCTTCTGCCGAA-S';

UNC5H2: 5'-GGGCTGGAGGATTACTGGTG-S'

5'-TGCAGGAGAACCTCATGGTC-S';

UNC5H3: 5'-GCAAATTGCTGGCTAAATATCAGGAA-S'

5'-GCTCCACTGTGTTCAGGCTAAATCTT-S';

UNC5H4: 5'-GGTGAACCCAGCCTCCAGTCAG-S'

5'-CTTCCACTGACATCACTTCCTCCC-S';

DCC: 5'-AGCCAATGGGAAAATTACTGCTTAC-S'

5'-AGGTTGAGATCCATGATTTGATGAG-S'; and HPRT: 5'-TGACACTGGCAAAACAATGCA-S'

5'-GGTCCTTTTCACCAGCAAGCT-S'.

F) Immunohistochemistry and immunoblot:

1 x 10⁵ cells were centrifugated on cover slips with a cytospiner (Shandon Cytospin 3, Thermo Scientific, Waltham, MA). Tumor slides and cells were fixed in 4% paraformaldehyde. The slides were then incubated at room temperature for one hour with an antibody recognizing the human netrin-1 (1 :150, R&D systems, Minneapolis, MN), UNC5H1 (1 :100, Abeam), UNC5H3 (1 :100, R&D system) or UNC5H4 (1 :100, Santa Cruz). After rinsing in Phosphate Buffer Saline, the slides were incubated with an Alexa-488-Donkey anti-Rat antibody (Molecular Probes), an Alexa-488-Donkey anti- Rabbit antibody (Molecular Probes), a Cy3-Donkey anti-Mouse antibody (Jackson ImmunoResearch, Suffolk, UK) or an Alexa-488-Donkey anti-Goat antibody (Molecular Probes) respectively. For tumor slides, netrin-1 and UNC5H4 signals were amplified using biotinyl-tyramide (TSA, Pierce) and Alexa-488-Sreptavidine (Molecular Probes). Nuclei were visualized with Hoescht staining. Densitometric value corresponding to netrin-1 signal was quantified with Axio Vision Release 4.6 software. Immunoblots were performed as already described using anti-phospho-DAPk and anti- DAPk (38) from Sigma, anti-DCC (1 :500, Santa Cruz), anti-neogenin (1 :500, Santa Cruz), anti-HA (1 :7500, Sigma), anti-HIS (1 :1000, Quiagen), anti-MYCN (1:1000, BD- Pharmingen) or anti-α-actin (1 : 1000, Chemicon) antibodies.

G) Chicken model for NB progression and dissemination:

10⁷ neuroblastoma cells suspended in 40μL complete medium were seeded on 10-day- old (day 10) chick chorioallantoic membrane (CAM). lOμg DCC-5Fbn or same PBS volume were injected in the tumor on day 11 and day 14. For siRNA treatment, 4μg scramble or netrin-1 siRNA were injected in the same conditions as for DCC-5Fbn. On day 17, tumors were resected and area measured with Axio Vision Release 4.6 software. To test DCC-5Fbn effect on metastasis regression, 3μg DCC-5Fbn or PBS were injected on day 14 and day 15 in a chorioallantoic vessel. To assess metastasis, lungs were harvested from the tumor-bearing embryos and genomic DNA was extracted with NucleoSpin Tissue kit (Macherey Nagel). Metastasis was quantified by polymerase chain reaction (PCR)-based detection of the human AIu sequence using the primers 5' - ACGCCTGTAATCCCAGCACTT-3' (sense) and 5 '-TCGCCCAGGCTGGAGTGCA- 3' (antisense) with chick glyceraldehyde-3 -phosphate dehydrogenase-specific primers (sense, 5 '-GAGGAAAGGTCGCCTGGTGGATCG-S '; antisense, 5'-

GGTGAGGACAAGC AGTGAGGAACG-3') as controls. For both couple 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 were used to determine the threshold between neuroblastoma cells-invaded and -non invaded 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 Caspase 3/CPP32 Fluorimetric Assay Kit (Gentaur Biovision).

H) NB Metastasis in nude mice:

S even- week-old (20-22 g body weight) female athymic nu/nu mice were obtained from Charles River animal facility. 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 130μL of PBS into a tail vein (day 0). 20μg DCC-5Fbn or PBS with equal volume was injected daily, intra- peritoneally, during 22 days. Lungs were harvested on day 23. Lungs genomic DNA was extracted with NucleoSpin Tissue kit (Macherey Nagel) and quantification of human tumor cells in lungs was done by polymerase chain reaction (PCR)-based detection of the human AIu sequence using the primers 5' CACCTGT AATCCC AGC ACTTT-3' (sense) and 5'- CCCAGGCTGGAGTGCAGT-3 ' (antisense) using 25ng genomic DNA as already described (56). The PCR was performed under the following conditions: 95 ⁰C for 2 min, 30 cycles at 95 ⁰C for 30 sec, 65°C for 20 sec, 72 ⁰C for 20 sec. Quantification of human DNA in mice lungs was based on a standard curve using human genomic DNA isolated from PTX and Myoc cell lines.

I) Genomic DNA quantification:

Genomic DNA from IMR32 and CLB-Ge2 cells was extracted with NucleoSpin Tissue kit (Macherey Nagel). 50 ng genomic DNA was used to perform quantitative PCR using primers specific to NTNl and MYCN genomic sequences. Real-time quantitative PCR was performed on a LightCycler 2.0 apparatus (Roche) using the Light Cycler FastStart DNA Master SYBERGreen I kit (Roche). The N-acetylglucosamine kinase gene (NAGK), located on the 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 following. NTNl : 5'- CTGTGTCCCCCACTTGTTCT-3' / 5'-CCATGAACCCCACTGACTCT-S'; MYCN: 5'-GTGCTCTCCAATTCTCGCCT-S' / 5'-GATGGCCTAGAGGAGGGCT-S'; NAGK: 5'-TGGGCAGACACATCGTAGCA-S' / 5'-CACCTTCACTCCCACCTCAAC-S'.

J) Netrin-1 ELISA assay: Detection of netrin-1 protein in IMR32 and CLB-Ge2 cell culture medium was performed using a modified ELISA assay. Briefly, 96-well plates (Nunc-Immuno™ plate, maxisorp, Nalgene Nunc International, Denmark) were coated with 200ng/well 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% (w/v) BSA (Sigma- Aldrich) in PBS-Tween 0.05%. 3 mL of FBS free cell-culture medium were added sequentially (300μL/well) to coated 96-well plates and incubated for Ih at 37°C. After three washes with 0.5% BSA/PBS, 100 μl rat anti-netrin-1 antibody (diluted 1 :500 in blocking solution) were 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 (1:1000, Jackson ImmunoResearch) 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 (Pierce, Rockford, IL, U.S.A.). Luminescent signal was measured using Luminoskan Ascent apparatus (Thermo lab System, Dreieich, Germany).

K) 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 carried out 48h after transfection according to the manufacturer's protocol, using the Luminoskan Ascent apparatus (Thermo lab System). As an internal control of transfection efficiency, the renilla luciferase encoding plasmid (pRL-CMV, Promega) was co-transfected and for each sample firefly luciferase activity was normalized to the renilla luciferase activity.

Example 2: Clinical Aspects of Neuroblastoma (NB) stage, selected patient samples We focused on stage 4 NB with a specific interest in comparing netrin-1 and its receptors expression level between the 3 distinct clinical patterns of stage 4, based on disease distribution and age of the patients ²⁵. On the one hand, there are the neonates and infants (< 1 year of age) with stage 4S (2-5% of all NB) and the similarly young stage 4 without 4S features hereafter termed "[lyr ^"] stage 4" who make up 10% of the NB population. On the other hand there are the stage 4 children (> 1 year of age) comprising 45% of all NBs who will hereafter be termed "[lyr ⁺] stage 4". These 3 clinical aspects of stage 4 NB differ in their respective malignant behaviors and associated-prognosis: good for stage 4S and [lyr ^"] stage 4 (5 years-EFS > 80%), and dismal for [lyr ⁺] stage 4 (5 years-EFS of around 30%) despite intensive treatment including high-dose chemotherapy and hematopoietic stem-cell transplantation ²⁶' ²⁷.

Example 3: Analyzed the expression of netrin-1 and its dependence receptors in a panel of 102 stage 4 NB tumours: Netrin-1 is up-regulated in a large fraction of aggressive neuroblastoma. We first analyzed the expression of netrin-1 and its dependence receptors - i.e., DCC, UNC5H1, UNC5H2, UNC5H3, UNC5H4 - by Q-RT-PCR in a panel of 102 stage 4 NB tumors including 24 stage 4 S and 12 [lyr - ] stage 4. As shown in Figures IB and ID, netrin-1 is up-regulated in [lyr + ] stage 4 as compared to both stage 4S (p<0.05) and [lyr - ] stage 4 (p<0.01). Similar results were obtained when comparing netrin-1 protein level by immunohistochemistry (Figures 1C and IE and quantification in Figure 8A). Interestingly, netrin-1 is detected mainly in tumor cells and is barely detected in stroma cells (Figure IE). Conversely, netrin-1 dependence receptors' expression analysis showed that DCC was only weakly expressed in the different stage 4 NB (Figures IA and 8C) as reported ²⁸, while UNC5H1, UNC5H2, UNC5H3, and UNC5H4 expression showed no significant differences when comparing [lyr - ] versus [lyr + ] stage 4 (Figure 1 F). However, we observed that the different UNC5H receptors are up- regulated specifically in stage 4S (average increase in UNC5H expression in stage 4S versus other stages 4 NB: 2.98 fold, p<0.007) suggesting UNC5H receptors as hallmarks of stage 4S NB. The UNC5H1 and UNC5H4, that show the highest mRNA expression could also be seen at the protein level by immunohistochemistry (Figure IG). In an attempt to correlate netrin-1 up-regulation with the molecular signature of these tumors, we compared netrin-1 up-regulation and DCC/UNC5H1 level to the profile of gene expression performed in a small panel of 9 stage 4 NB⁴³. We failed to detect any correlation between netrin-1 up-regulation or DCC/UNC5H1 level with the molecular signature of apoptosis or invasion effectors (Figure 8D). Considering patients' outcomes, although 38% of [lyr + ] stage 4 NB have selected up-regulation of netrin-1, this event failed to be significantly associated with poor outcome in this aggressive form of the disease (not shown). Moreover no association between netrin-1 up-regulation and MYCN-amplification was detected (not shown). Thus, netrin-1 up-regulation may be considered as an additional component of the genetic complexity that these tumours display, and not an independent prognostic marker, following the example of MYCN amplification that lacks any prognostic value in [lyr ⁺] stage 4, in sharp contrast with infants stage 4 whose MYCN genomic content guides prognosis and therapy ²⁹' ³⁰.

Example 4: Netrin-l expression as a prognostic marker for the infants stage 4 NB Despite a largely favorable prognosis among infants with stage 4 NB - i.e, stage 4S and [lyr - ] stage 4 - with no MYCN-amplification (MNA), many succumb to the disease. Netrin-1 up-regulation may be considered as an additional component of the genetic complexity that these tumours display, and not an independent prognostic marker, following the example of MYCN amplification that lacks any prognostic value in [lyr ⁺] stage 4, in sharp contrast with infants stage 4 whose MYCN genomic content guides prognosis and therapy ²⁹' ³⁰. Thus, we assessed whether netrin-1 expression may serve as a prognostic marker for the infants with stage 4 NB. As shown in Figure IH, the overall survival of infants with stage 4S differed markedly based on whether the tumor displayed high level of netrin-1 expression ("netrin-1 high") or low level expression ("netrin-1 low"), netrin-1 expression threshold being its median expression value in the 102 cases. Indeed, while 100% of the infants survived after 10 years (including 1 MNA out of 17) when the NB 4S was netrin-1 low, the 5-year 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 [lyr - ] stage 4 -, a similar dichotomy was observed. Indeed, 5 -year overall survival was found to be 90% for the netrin-1 low infants, yet only 48% for netrin-1 high infants (p=0.032) (Figure II). 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 ³⁵.

Example 5: Netrin-l immunohistochemistry on IMR32 and CLB-GeI cells

Netrin-1 high expression is not only detected in 38% of [lyr + ] stage 4 and in poor outcome [lyr - ] stage 4 primary NB tumors but also in a fraction of NB cell lines mainly derived from stage 4 tumor material (Figures 2 A and 9A). Two human NB cell lines - i.e., 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 -i.e, UNC5H1, UNC5H3 and UNC5H4 show the highest expression (Figure 2B). Specifically, UNC5H1, UNC5H3 and UNC5H4 proteins could be detected at the plasma membrane by confocal analysis (Figure 2C). To test the hypothesis that the high netrin-1 mRNA level detected in IMR32 cells is associated with an autocrine netrin-1 production, we next performed netrin-1 immunohistochemistry on IMR32 and CLB-Ge2 cells. As shown in Figure IG, a netrin- 1 specific membrane staining was detected in a homogeneous pattern in IMR32 cells while no specific staining was detected for CLB-Ge2 cells. Confocal analysis further confirmed the presence of netrin-1 at the cell membrane (Figures 2E and 9B). 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 Figure 2F, 11.7 ng/mL of netrin-1 was recovered from the conditioned medium of IMR32 cells while no netrin-1 was detected from CLB-Ge2 cells conditioned medium. 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. As a first approach to apprehend the mechanisms leading to netrin-1 up-regulation in aggressive NB, we analyzed whether netrin-1 gene (NTNl) is amplified in IMR32 cells. As shown in Figure 2G, while MYCN was amplified both in IMR32 and CLB-Ge2 cells compared to the NAGK control gene, NTNl gene was not found to be amplified in these two cell lines. We then analyzed whether the increase in netrin-1 expression could be due to a differential netrin-1 promoter activation. A luciferase reporter gene fused to netrin-1 promoter⁴³ was then transfected into IMR32 or CLB-Ge2 cells, and luciferase activity was reported to an internal control in each cell line. As shown in Fig. 2 H, netrin-1 promoter activity was 13.8 fold higher in IMR32 cells than in CLB-Ge2 cells, thus supporting the view that netrin-1 up-regulation in NB is related to a gain in netrin-1 promoter activation.

Example 6: Netrin-l autocrine expression provides a selective advantage for 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 Figure 3 A, the addition of netrin-1 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 (Figure 3B). While scrambled siRNA failed to affect IMR32 cell survival, as measured by trypan blue exclusion, netrin-1 siRNA treatment was associated with IMR32 cell death (Figure 3C). In contrast, CLB-Ge2 cell survival was unaffected after netrin-1 siRNA treatment (Figure 3C). To determine whether this increase in cell death was in part due to an increase in apoptotic cell death, caspase-3 activity was measured in response to netrin- 1 siRNA treatment. As shown in Figure 3D, while 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 pro-apoptotic effect of the netrin-1 siRNA was observed in CLB-VoIMo cells, another netrin-1 high cell line (not shown). Example 7: Disruption of the netrin-1 autocrine loop is associated with NB cell death: Interference with netrin-1 triggers UNC5H-induced apoptosis in NB cells

As a second approach, we looked for a compound that could interfere with the netrin-1 ability to block DCC/UNC5H pro-apoptotic activity. We recently reported that the fifth fibronectin type III domain of DCC (DCC-5Fbn) (Figure 4A), located in the DCC ectodomain, interacts with netrin-1 and blocks the ability of netrin-1 to trigger multimerization of DCC and UNC5H receptors. Since multimerization inhibits DCC or UNC5H-induced cell death (Mille, F., F. Llambi, C. Guix, C, J. Fitamant, S. Castro- Obregon, D.E. Bredesen, C. Thibert and P. Mehlen. Unpublished), DCC-5Fbn antagonizes netrin-1 function, disrupting netrin-1 mediated inhibition of DCC/UNC5H pro-apoptotic activity. Thus, DCC-5Fbn acts as a trap for netrin-1 survival function. As shown in Figures 4B, 4C and 4D, the addition of DCC-5Fbn - but not the unrelated protein IL3R - triggered IMR32 apoptotic cell death as measured by trypan blue exclusion (Figure 4B), caspase-3 activity assay (Figure 4C) and TUNEL staining (Figure 4D). This effect was specific for netrin-1 inhibition, since DCC-5Fbn had no effect on CLB-Ge2 cells, and the addition of netrin-1 ultimately reversed the DCC- 5Fbn-induced IMR32 apoptotic cell death (Figures 4B, 4C and 4D). Similar results were obtained with the CLB-VoIMo cells (Suppl. Fig. 2 D). To determine whether the ability of DCC-5Fbn to kill NB cells is restricted to established NB cell lines, a surgical biopsy from a tumor with high netrin-1 level (not shown) was semi-dissociated and further incubated with DCC-5Fbn. As shown in Figure 4E, DCC-5Fbn triggered cell death as measured by caspase-3 activation, demonstrating that in vitro, disruption of the netrin-1 autocrine loop is associated with NB cell death.

Example 8: Disruption of the netrin-1 autocrine survival loop triggers UNC5H- induced cell death

We next investigated whether netrin-1 autocrine expression in NB cells acts as a general cell survival factor - i.e. such as Nerve Growth Factor- or whether it specifically inhibits death induced by netrin-1 dependence receptors. IMR32 cells were transfected with either a dominant negative mutant for DCC (DN-DCC) or for UNC5H (DN-UNC5H) pro-apoptotic activity. Cell death was then analysed in presence of DCC-5Fbn. While DNDCC expression fails to inhibit DCC-5Fbn-induced IMR32 cell death, expression of DNUNC5H renders IMR32 cells resistant to DCC-5Fbn (figure 5A). Thus, a netrin-1 autocrine loop blocks UNC5H-induced cell death in IMR32 cells. To more specifically address the identity of the UNC5H receptor(s) involved in this cell death induction, we down-regulated expression of each UNC5H receptor individually - i.e, UNC5H1, UNC5H2, UNC5H3 or UNC5H4- by a siRNA approach while inducing cell death using netrin-1 siRNA. None of single UNC5H siRNA was sufficient to inhibit netrin-1 siRNA-mediated IMR32 cell apoptosis, suggesting some redundancy in UNC5H- induced cell death (figure 5D). However, when combinations of siRNA were used, we observed that combined silencing of the 4 UNC5H receptors was sufficient to fully inhibit the death triggered by netrin-1 autocrine loop disruption (figure 5E). The respective importance of UNC5H receptors in these cells was then stressed by the different combination of two or three siRNA: only the combination of UNC5H1, UNC5H3 and UNC5H4 siRNA fully blocks cell death. Thus, in agreement with the level of UNC5H receptors expressed in IMR32 cells, it appears that disruption of the netrin-1 autocrine survival loop triggers UNC5H-induced cell death. Moreover, UNC5H1, UNC5H3 and UNC5H4 are the receptors involved in this pro-apoptotic effect. In fact, among IMR32 cells, the pro-apoptotic serine threonine kinase DAP kinase shown to be required for UNC5H-induced cell death exhibits a loss of its inhibitory autophosphorylation ³² upon DCC-5Fbn treatment (figure 5F) or netrin-1 siRNA transfection (figure 5G). Accordingly, autophosphorylation is restored by a treatment with an excess of netrin-1 or by a combination of UNC5H1, UNC5H3 and UNC5H4 siRNA. We next investigated whether netrin-1 autocrine expression in NB cells acts as a general cell survival factor - i.e., whether it has a trophic effect similar to that of neurotrophins - or whether it specifically inhibits death induced by netrin-1 dependence receptors. IMR32 cells were transfected with either a dominant negative mutant for DCC (DN- DCC) or for UNC5H (DN-UNC5H) pro-apoptotic activity. These dominant negative mutants of dependence receptors actually encode the intracellular domain of these receptors mutated in their caspase cleavage sites and these mutants have been shown both in vitro and in vivo to specifically block the pro-apoptotic activity of their wild- type counterparts³' ⁹. Cell death was then analyzed after netrin-1 inhibition by siRNA. While DN-DCC expression failed to inhibit netrin-1 siRNA- induced IMR32 cell death, expression of DN-UNC5H rendered IMR32 cells resistant to netrin-1 siRNA (Figure 5A). To more formally exclude the role in this death process of DCC or of altered form of DCC known to be expressed in IMR32 cells⁴⁴ and Figure 9C), DCC was down- regulated by a siRNA approach and cell death was induced via netrin-1 siRNA. As shown in Figure 5B, DCC siRNA had no effect on cell death per se and failed to inhibit netrin-1 siRNA- induced caspase-3 activation, strengthening that in these cells DCC is not pro-apoptotic. Neogenin, a DCC homolog has also been proposed to act as a receptor for netrin-1⁴⁵' ⁴⁶ even though this is still a matter of controversy⁵' ⁴⁷. We then investigated whether neogenin, which is expressed in IMR32 cells (Figure 9C), could be implicated in the IMR32 cell death observed here. Neogenin was down-regulated by a siRNA approach and, as shown in Suppl. Figure 1OA, this has no effect on netrin-1 siRNA mediated IMR32 cell death. Thus, the netrin-1 autocrine loop probably blocks UNC5H-induced IMR32 cell death. To more specifically address the identity of the UNC5H receptor(s) involved in this cell death induction, we down-regulated the expression of each UNC5H receptor individually - i.e, UNC5H1, UNC5H2, UNC5H3 or UNC5H4 - by a siRNA approach (Figure 5 C and Figure 10B), while inducing cell death using netrin-1 siRNA (Figure 10C). None of the single UNC5H siRNAs was sufficient to inhibit netrin-1 siRNA- mediated IMR32 cell apoptosis, suggesting some redundancy in UNC5H-induced cell death (Figure 5D). However, when combinations of siRNAs were used, we observed that combined silencing of the 4 UNC5H receptors was sufficient to fully inhibit the death triggered by netrin-1 autocrine loop disruption (Figure 5D) while the same combination of siRNAs had no effect on CLB-Ge2 cell survival (Figure 10D). The respective importance of UNC5H receptors in netrin-1 siRNA- induced cell death was then assessed by the different combination of two or three siRNAs: the combination of UNC5H1, UNC5H3 and UNC5H4 siRNAs was the only one to fully block cell death (Figure 5E). Thus, in agreement with the level of UNC5H receptors expressed in IMR32 cells, it appears that disruption of the netrin-1 autocrine survival loop triggers UNC5H-induced cell death. Moreover, UNC5H1, UNC5H3 and UNC5H4 are the receptors involved in this pro-apoptotic effect. Furthermore, in IMR32 cells, the pro-apoptotic serine threonine kinase DAP kinase, shown to be required for UNC5H-induced cell death, exhibited a loss of its inhibitory autophosphorylation³² upon DCC-5Fbn treatment (Figure 5F) or netrin-1 siRNA trans fection (Figure 5G). 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 anti-apoptotic protein BCL-2 was sufficient to inhibit netrin-1 siRNA induced-cell death but did not inhibit DAP kinase dephosphorylation hence suggesting that DAP kinase activation is not a result of cell death but specifically engaged by UNC5H after netrin-1 inhibition (data not shown).

Example 9: In vivo modulation of netrin-l is a relevant tool to limit/inhibit NB progression and dissemination and to induce regression of metastatic lesions: Interference with netrin-1 inhibits NB progression and dissemination We next assessed whether in vivo modulation of netrin-1 could be utilized 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- day-old chick embryos recapitulates both tumor growth at a primary site - i.e., within the CAM - as well as tumor invasion and dissemination at a secondary site - metastasis to the lung - (Figure 6A). In a first approach, IMR32 or CLB-Ge2 cells were loaded in 10-day-old CAM and embryos were treated on day 11 and day 14 with PBS or DCC- 5Fbn. 17-day-old chicks were then analyzed for primary tumor growth and metastasis to the lung. As shown in Fig. 6 BC, 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 (Figure 6D). More importantly, DCC-5Fbn also reduced lung metastasis formation, as shown in Figure 6E. Similar results were obtained when CAM-grafted embryos were treated with netrin-1 siRNA (not shown). 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, while pulmonary metastases are routinely detectable at day 13. As shown in Figure 6F, 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.

As a second in vivo approach, we used the IGR-N-91 model derived from a bone marrow metastasis of human MYCN-amplified stage 4 NB. When subcutaneous Iy xenografted into nude mice, IGR-N-91 cells gave rise to different tumor cell lines derived from the nude mouse primary tumor xenograft (PTX) or from disseminated metastatic foci into bone marrow (BM), blood (Blood) and myocardium (Myoc) of the animal³³. Of interest, while the parental IGR-N-91 and the cell line derived from the primary tumor xenograft (PTX) show no or very low netrin-1 expression, the different cell lines derived from secondary localizations showed a marked expression of netrin-1 both at the RNA level (Figure 7A) and at the protein level (Figure 7B). When cell death was investigated in these different cell lines upon treatment with DCC-5Fbn, a direct correlation was observed between the level of netrin-1 and cell susceptibility to DCC- 5Fbn (Figure 7C). Specifically, while PTX cells failed to undergo cell death upon DCC- 5Fbn treatment, this treatment triggered netrin-1 high Myoc cell death (Figure 7C). This observation supports the overall view that gaining netrin-1 dependence receptor resistance, via an autocrine netrin-1 expression in the case of the IGR-N-91 model, likely promotes NB tumor cell survival outside the primary tumor site. To test whether this netrin-1 expression may then be used as a tool to inhibit metastasis in vivo, Myoc and PTX cells were injected intravenously into nude mice and lung colonization was quantitated after daily intraperitoneal treatment with PBS or DCC-5Fbn. While lung colonization was not reduced upon DCC-5Fbn treatment in PTX-injected mice (not shown), a significant decrease in lung colonization was detected in DCC-5Fbn treated Myoc-injected mice (Figure 7D). Thus, in both chick and mouse models, disruption of netrin-1 autocrine loop inhibited or completely prevented the dissemination of netrin-1 - expressing NB cells.

Together the data obtained in the chick/mouse models described above, in NB cell lines as well as in the human pathology support the view that a fraction of NB shows an autocrine production of netrin-1. This elevated netrin-1 level confers a selective advantage acquired by the cancer cell to escape netrin-1 -dependence receptors induced apoptosis and, consequently, to survive in settings of environmental absence/limitation of netrin-1. From a mechanistic point of view, this autocrine expression of netrin-1 probably inhibits UNC5H-induced cell death. Yet, as DCC has been described to show a reduced expression in NB and this loss being associated with NB aggressiveness in the human pathology ²⁸, it is tempting to speculate that netrin-1 up-regulation can also, in some NB, inhibit DCC-induced apoptosis. Interestingly, this netrin-1 up-regulation appears to block a death signaling implicating the serine threonine DAP kinase which activity is known to be regulated via its autophosphorylation. It is then of interest to note that DAPk was described as a negative regulator of tumour progression and more specifically of metastasis ³⁴. Even though not described yet, it could be then tempting to speculate that a fraction of low netrin-1 expressing NB could have selected a loss of function of DAPk to survive. This would then fit with the recent finding that metastasis in NB is associated with a loss of caspase-8, a selective advantage that provides survival to NB cells by inhibiting the pro-apoptotic signaling of the dependence receptor α3βl ³⁵. The observation that low levels of netrin-1 in NB correlates with good outcome is of clinical interest in particular in NB bearing neonates and infants. Indeed, it predicts the survival of the infants (respectively 100% in 4S stage or 90% in infants in general) in a type of pathology in which therapeutic management is highly dependent on presentation and MYCN amplification ²⁹' ³⁰. This is particularly true with stage 4S infants who receive basically no (or alleviated) treatment based on the lack of MYCN amplification even though to date, one in ten of these infants will eventually die. Here, we propose that determination of low netrin-1 level would allow the confirmation of a good prognosis of the infants, while the infants with high netrin-1 level should be probably addressed for treatment. Regarding infants or children with high netrin-1 NB tumours an alternative/supplementary targeted treatment based on disruption of the netrin-1 autocrine survival loop may challenge regular high dose chemotherapy regimens. 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 positively affect a large fraction of the patients suffering from aggressive NB. Future preclinical and clinical studies should assess whether such therapeutic strategies, that could include chemical drugs, monoclonal antibodies or the DCC-5Fbn recombinant protein presented here, used alone or in combination with chemotherapy could be of benefit for NB bearing infant and child. These results demonstrate that a large fraction of aggressive NB have selected a gain of netrin-1 expression, and that high netrin-1 expression is associated with poor outcome especially for patients diagnosed before one year of age. We show that autocrine production of netrin-1 is a tumour growth and dissemination selective advantage in aggressive NB as it blocks the pro-apoptotic activity of the netrin-1 dependence receptors UNC5H. Therefore, netrin-1 up-regulation is a potent marker for poor prognosis in NB stage 4 diagnosed infants. Moreover, we propose that interference with the netrin-1 autocrine loop in NB cells could represent an alternative therapeutic strategy as disruption of this loop triggers NB cell death in vitro and leads to the prevention or regression of metastatic NB lesions in chicken and mouse models.

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Claims

1. An in vitro method for predicting whether a neuroblastoma (NB) identified in a patient is a metastatic or an aggressive neuroblastoma, or a neuroblastoma having a poor prognosis from a biological sample of said patient, said method comprising the following step of:

(a) obtaining an isolated biological sample from said patient wherein said isolated biological sample is selected from body fluid, from a tissue sample or a combination thereof; (b) measuring of the netrin-1 expression level in said biological sample.

2. The method according to claim 1, wherein an increase of the netrin-1 expression level in said biological sample, compared with expression of netrin-1 in a reference biological sample (non-metastatic primary NB biological sample or in a non-aggressive NB biological sample or in a biological sample from healthy patient), is significant of the presence of a metastatic NB or an aggressive NB, or a NB having a poor prognosis.

3. The method according to one of claims 1 and 2, wherein a ratio superior to 1.5, preferably to 2, 2.5 or 3.0 between netrin-1 expression in the biological sample to be tested and in the reference biological sample is significant of the presence of a metastatic NB or an aggressive NB, or a NB having a poor prognosis.

4. An in vitro method for predicting whether a neuroblastoma (NB) identified in a patient has a good prognosis from a biological sample of said patient, said method comprising the following step of:

(b) measuring of the netrin-1 receptor expression level in said biological sample.

5. The method according to claim 4, wherein the netrin-1 receptor is selected from the group consisting of DCC (deleted in Colorectal Cancer) or UNC5H1, UNC5H2, UNC5H3 and UNC5H4 UNC5H.

6. The method according to one of claims 1 to 5, wherein said biological sample is a body fluid selected from the group consisting of blood, serum, plasma, saliva, urine, milk, cerebrospinal fluid and, lymph, or a tissue sample or a biopsy susceptible to contain tumor cells.

7. The method according to one of claims 4 and 5, wherein the netrin-1 receptor expression level is determined at the membrane surface of cells of the tissue sample, preferably at the membrane surface of the tumor cells susceptible to be contained in the tissue sample, if the netrin-1 receptor protein expression level is determined, or in a body fluid, particularly from cell cytoplasm, blood, serum, plasma or urine whether the netrin-1 receptor mRNA level is determined.

8. The method according to one of claims 4, 5 and 7, wherein the netrin-1 receptor expression level to be evaluated in the biological sample, is compared in the same conditions with the expression of netrin-1 receptor in a biological sample from reference biological sample(from healthy patient or patient having non-metastatic primary NB or non-aggressive NB), an increase of the netrin-1 receptor expression level or a high level in said biological sample, compared to the reference sample, is significant of a good prognosis o f the NB .

9. The method for predicting according to one of claims 4, 5, 7 and 8, wherein a ratio superior to 1.5, 2, preferably to 2.5, to 3, to 3.5, to 4, to 4.5 and to 5, between netrin-1 receptor expression in the biological sample to be tested and in the reference biological sample is significant of a good prognosis of the NB.

10. A method according to one of claims 1 to 3 and a method according to one of claims 4 to 9, wherein the measured netrin-1 (for claims 1-3) or the netrin-1 (for claims

4-9) expression product is the RNA encoding netrin-1.

11. A method according to claim 4, wherein the RNA encoding netrin-1 or netrin-1 is measured by a quantitative real time reverse PCR method.

12. A method according to one claims 1 to 3 and a method according to one of one of claims 4 to 9, wherein the measured netrin-1 (for claims 1-3) or the netrin-1 (for claims 4-9) expression product is the netrin-1 protein level.

13. The method according to claim 12, wherein the netrin-1 protein level or the netrin-1 receptor protein level is measured by a method using specific antibodies able to specifically recognize said protein.

14. The method according to claim 7, wherein the netrin-1 protein level or the netrin-1 receptor protein level is measured by an ELISA method, by immunohistochemistry or immunoblot.

15. A method according to one of claims 1 to 14, wherein said patients are selected from patients having a stage 4 neuroblastoma.

16. A method according to claim 15, wherein said patients are patients having a stage 4S neuroblastoma.

17. A method according to one of claims 1 to 16, wherein said patients are infants.

18. A method for preventing and/or treating metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the

NB tumoral tissue, comprising administering an amount of a compound effective to inhibit the binding of netrin-1 to its dependence receptor or to inhibit the ability of netrin-1 to multimerize netrin-1 dependence receptor.

19. A method according to claim 18, wherein said dependence receptor is selected from the group consisting of DCC, UNC5H1, UNC5H2, UNC5H3 or UNC5H4.

20. A method for preventing and/or treating metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the NB biological sample, comprising administering an amount of a compound effective to inhibit the expression or the activity of the netrin-1 protein.

21. The method of claim 20, wherein said compound is selected from one or more of an anti-netrin-1 antibody, an UNC5H receptor, an UNC5H receptor ectodomain, a DCC ectodomain or an siRNA capable of inhibiting the expression of said netrin-1 by the tumors cells.

22. The method according to one of claims 18 to 21 wherein said compound is selected in the group consisting of the compound comprising the extracellular domain of a netrin-1 receptor or fragment thereof, said-1 receptors being selected from the group of DCC, UNC5H (particularly UNC5H1, UNC5H2 , UNC5H3 and UNC5H4).

23. The method according to one of claim 22, wherein said compound comprising an extracellular domain of netrin-1 receptor is from DCC, preferably said compound is DCC-EC-Fc or DCC-5Fbn.

24. A method for selecting a compound for the prevention or the treatment of metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the NB tumoral tissue, wherein said method comprises the following steps of: a) having a medium containing netrin-1, or a fragment thereof, and a netrin-1 receptor, or a fragment thereof, wherein: - said netrin-1, or a fragment thereof, and said netrin-1 receptor, or a fragment thereof, is able to specifically interact together to form a binding pair, and/or

- the measuring in step c) demonstrates a significant inhibition of the interaction between netrin-1, or a fragment thereof, and netrin-1 receptor, or a fragment thereof, in presence of said compound, and/or - the determination in step c) demonstrates a significantly inhibition of the dimerization or multimerization of said netrin-1 receptor, or a fragment thereof, in presence of said compound, particularly the dimerization of the intracellular domain of said netrin-1 receptor.

25. The method according to claim 24, wherein at step a) said netrin-1 receptor is selected from the group of DCC and UNC5H.

26. The method according to one of claims 24 and 25, wherein at step a) said netrin- 1 or/and said netrin 1 receptor are from mammal, particularly from human.

27. The method according to one of claims 24 to 26, wherein at step c):

- the measure of the inhibition of the interaction between netrin-1, or a fragment thereof, and said netrin-1 receptor, or a fragment thereof, is carried out by immunoassay

(particularly by ELISA or by Immunoradiometric Assay (IRMA)), by Scintillation Proximity Assay (SPA) or by Fluorescence Resonance Energy Transfer (FRET); and/or - the dimerization or multimerization, or its inhibition, of said netrin-1 receptor, or fragment thereof, particularly the intracellular domain, is carried out by immunoprecipitation or FRET.

28. The method according to one of claims 24 to 27, wherein at step a) said medium contains cells which express at their surface membrane an endogenous netrin-1 receptor or a recombinant netrin-1 receptor, particularly at least the extracellular domain of a recombinant netrin-1 receptor.

29. The method according to claim 28, wherein at step a) said medium contains cells which express recombinant netrin-1 receptor.

30. The method according to one of claims 28 and 29, wherein at step a) said medium contains tumoral cells which express endogenously said netrin-1 receptor at their membrane surface and which express or overexpress netrin-1, and wherein at step c) the inhibition of the interaction between netrin-1 and its netrin-1 receptor in presence of the compound to be tested, is measured by the apoptosis or cells death induced by the presence of the compound to be tested.

31. The method according to claim 30, wherein at step a) said medium contains metastatic neuroblastoma cells, particularly cells selected from the group consisting of CLB-GEl and IMR32 cells.

32. A method for selecting a compound for the prevention or the treatment of metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the tumoral tissue , wherein said method comprises the following steps of: a) having a medium containing a mammal cell expressing an endogenous or a recombinant netrin-1 receptor, or a fragment thereof comprising at least its intracellular domain, preferably a tumor cell, more preferably a cell presenting dimerization or multimerization of its netrin-1 receptor intracellular domain or a cell wherein its netrin- 1 receptor intracellular domain is able to dimerize or multimerize in presence of netrin- i; b) contacting said medium with the compound to be tested, optionally the medium further containing netrin-1, or a fragment thereof able to interact with the extracellular domain of the netrin-1 receptor; c) determine whether the dimerization or multimerization of said netrin-1 receptor intracellular domain is inhibited in presence of said compound to be tested; d) optionally, determine whether the presence of the compound to be tested induces the cell death of said mammal cell; and e) selecting said compound if the determination in step c) demonstrates a significantly inhibition of the dimerization or multimerization of the intracellular domain of said netrin-1 receptor and/or if the determination in step d) demonstrates the cell death of said mammal cell.

33. A method for determining in vitro the efficiency of an anti-cancer treatment for a patient or for in vitro selecting patients who respond to a specific anti-cancer treatment and wherein said cancer is a metastatic and/or aggressive NB and/or NB having poor prognosis related to the presence of an overexpression of netrin-1 in the tumoral tissue, said method comprising the following step of: (a) obtaining a primary tumor biopsy of said treated patient; and (b) measuring of the netrin-1 expression level in said biopsy, wherein the efficiency of said anti-cancer treatment is correlated with the decrease of the amount of the netrin-1 expression level measured in said biopsy, or wherein the selected patients who respond to a specific anti-cancer treatment are patients where the amount of the netrin-1 expression level measured in their biopsy has been decreased after said specific treatment.