WO2013061112A1 - Use of egfl7 modulators for promoting or inhibiting migration of immune cells across vascular endothelium - Google Patents

Abstract

The present invention relates to a method of modulating transmigration of immune cells across vascular epithelium. More particularly, the present invention addresses two different types of situations. In conditions where an upregulation of immune cells extravasation is needed, for example in cancers, Egfl7 antagonists are used. In conditions where a downregulation of leukocytes transendothelial migration is preferred, for example in pathological inflammation, Egfl7 or an agonist thereof is used.

Description

USE OF EGFL7 MODULATORS FOR PROMOTING OR INHIBITING MIGRATION OF IMMUNE CELLS ACROSS VASCULAR ENDOTHELIUM

The present invention relates to a method of modulating transmigration of immune cells across vascular epithelium. More particularly, the present invention addresses two different types of situations: conditions where an upregulation of immune cells extravasation is needed and conditions where a downregulation of leukocytes transendothelial migration is preferred. The present invention is based on the demonstration that elevated levels of Egfl7 prevent the normal activation of vascular endothelium.

One of the essential functions of the endothelial cell lining is to maintain the essentially impermeable nature of the blood vessels controlling the passage of solutes and immune cells from the circulation to the tissues.

The transmigration of leukocytes through the endothelium is a normal process which, during inflammation, results in the infiltration of circulating cells from the blood stream into tissues. Leukocyte migration across the endothelium is a complex multistep process involving tethering, rolling, firm adhesion and finally extravasation. The underlying mechanisms that regulate these processes have been the basis for many recent studies. Traditionally, leucocyte extravasation had been believed to occur through a paracellular route, which involves localized disruption of endothelial cell junctions. However, more recently, a transcellular route has been described involving the passage through the endothelial cell body. Leucocytes are also able to migrate through epithelium to monitor mucosal tissues and microenvironments. A number of molecules are known to regulate transmigration of leucocytes through epithelial and endothelial layers. On the endothelial side, extravasation involves at least E- and P-selectin, ICAM-1 (intracellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1 ), PECAM/CD31 (platelet-endothelial cell adhesion molecule), CD99, VE-cadherin (vascular endothelial cadherin) and JAM (junctional adhesion molecule) proteins (Garrido-Urbani et al., 2008; Muller, 2009).

The traversal of immune cells across the endothelial barrier and into the tissue space is an integral component of an immune response induced by infection or injury. Some conditions are characterized by aberrant or otherwise unwanted endothelial transmigration.

In the particular case of cancer, new blood vessels, formed mostly by endothelial cells, sustain the nutriment and oxygen supplies of the developing solid tumor (Chung et al., 2010). The tumor endothelium forms an imperfect, tortuous, and leaky bamer (Jain, 2005). Metastatic cells enter the blood circulation through this endothelium (intravasation) and spread across the organism as an alternative route to lymphatic metastasis (Alitalo and Carmeliet, 2002; Hashizume et al., 2000; Mazzone et al., 2009). On the other hand, infiltration of circulating immune cells, such as natural killer (NK) cells and CD8+ cytotoxic T-lymphocytes, into the tumor mass plays a part in the control of tumor growth through interferon-γ (IFNg) and cytotoxic-based mechanisms (Castermans and Griffioen, 2007; Herberman et al., 1975; Koebel et al., 2007; Shrikant and Mescher, 1999). In order to destroy tumor cells, circulating immune cells must first infiltrate the tumor mass. The tumor blood vessel endothelium, although imperfectly tight, actively protects tumor cells from the immune system through its barrier function. Tumor escape from immunity may be achieved by preventing infiltration of effector immune cells (Wu et al., 1992) through the down-regulation of these endothelial adhesion molecules (Dirkx et al., 2003; Griffioen et al., 1996; Kuzu et al., 1993; Piali et al., 1995).

The inventors originally characterized Egfl7 (VE-statin) as a gene specifically expressed by blood vessel endothelial cells in normal organs during development and in the adult (Lelievre et al., 2008; Soncin et al., 2003, included herein by reference). In human cancer, this specificity of expression is lost as Egfl7 has been detected in tumor cells themselves, in addition to endothelial cells. Its expression levels correlate with a higher tumor grade in glioma (Huang et al., 2010) and colon cancer patients (Diaz et al., 2008), and with a poorer prognosis and higher metastatic score in hepatocarcinoma patients (Wu et al., 2009). Although these observations suggested a role for Egfl7 in cancer progression, its direct role in tumor development had not been studied before the study disclosed in the experimental part below.

Based on a supposed role of Egfl7 in cancer angiogenesis, contradictory strategies have been described, such as the use of anti-Egfl7 antibodies for inhibiting angiogenesis (US 2010/0285009), or the use of an agonist of Egfl7, including Egfl7 itself, for decreasing angiogenesis (US 2008/01 1391 1).

In another research field, Badiwala et al. have shown that incubation of cells submitted to hypoxia or to cyclosporine with Egfl7 inhibited ICAM-1 upregulation (Badiwala et al., 201 1 ; Badiwala et al., 2010). They also showed that Egfl7 inhibits neutrophil adhesion to human coronary endothelial cells subsequent to calcineurin- inhibition-induced injury (Badiwala et al, 201 1). They deduced that Egfl7 could be protective against inflammation processed following arterial injury. However, these authors did not describe a downregulation of leukocytes transendothelial migration in the presence of Egfl7.

In a comprehensive study reported below, the inventors of the present application show that Egfl7 is an endogenous regulator of endothelial cell activation which, when expressed by tumor cells, inliibits the recruitment of immune cells, thereby protecting the tumor from the host immunity. The present invention hence pertains to a modulator of Egfl7, for use as an agent for promoting or inhibiting migration of immune cells across vascular endothelium.

In the present application, "immune cells" designate leukocytes, including lymphocytes, granulocytes (including neutrophils), and monocytes.

In particular, the present invention relates to the use of a modulator of Egfl7 for modulating the migration of lymphocytes across the epithelium, including the extravasation step.

In the present text, the term "modulator" designates any molecule which upregulates or downregulates the activity of Egfl7. Such modulation may be achieved by any suitable means and includes:

(i) Modulating absolute levels of the active or inactive forms of Egfl7 (for example increasing or decreasing Egfl7 concentrations) such that either more or less Egfl7 is available for interacting with its downstream targets. This can be achieved by acting either at the transcription level (for example, by introducing a siRNA targeting the gene of Egfl7 to decrease its expression, or by introducing into a cell a nucleic acid molecule encoding Egfl7 or functional equivalent, derivative or analogue thereof in order to upregulate the capacity of said cell to express Egfl7, to increase its expression), at the translation level, or at the post-translational level (for example by modulating the half life of Egfl7 by any means such as modifying its ability to be ubiquitinated). Addition of Egfl7 itself can also be performed to increase its concentration and hence, its activity. The mouse Egfl7 protein sequence is available in the database Swiss-Prot (Genbank) under the reference Q9QXT5, whereas the human sequence is available, in the same database, under the reference Q9UHF1. The sequences are also enclosed as SEQ ID No: 1 (mouse Egfl7) and SEQ ID No: 2 (human EGfl7).

(ii) Agonising or antagonising Egfl7 such that the functional effectiveness of any given Egfl7 molecule is either increased or decreased. Examples of antagonists of Egfl7 activity which can be used to that purpose are competitors such as fragments of the protein, as well as antibodies, fragments of antibodies, aptamers and the like.

Of course, and as appears in what precedes, a modulator according to the present invention can be a proteinaceous or a non-proteinaceous molecule. The proteinaceous molecules described above may be derived from any suitable source such as natural, recombinant or synthetic sources and include fusion proteins or molecules which have been identified following, for example, natural product screening. The reference to non-proteinaceous molecules may be, for example, a reference to a nucleic acid molecule or it may be a molecule derived from natural sources, such as for example natural product screening, or may be a chemically synthesised molecule. The present invention contemplates analogues of Egfl7 or small molecules capable of acting as agonists or antagonists. Chemical agonists may not necessarily be derived from the Egfl7 protein, but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to meet certain physiochemical properties. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing Egfl7 from carrying out its normal biological function.

Screening for the modulatory agents hereinbefore defined can be achieved by any one of several suitable methods including, but in no way limited to, contacting a cell comprising the Egfl7 gene or functional equivalent or derivative thereof with an agent and screening for the modulation of Egfl7 protein production or functional activity, modulation of the expression of a nucleic acid molecule encoding Egfl7 or modulation of the activity or expression of a downstream Egfl7 cellular target (such as ICAM-1). Detecting such modulation can be achieved utilising techniques such as Western blotting, electrophoretic mobility shift assays and/or the readout of reporters of Egfl7 activity.

The modulators which are used according to the present invention may take any suitable form. For example, proteinaceous agents may be glycosylated or unglycosylated, phosphorylated or dephosphorylated to various degrees and/or may contain a range of other molecules used, linked, bound or otherwise associated with the proteins such as amino acids, lipid, carbohydrates or other peptides, polypeptides or proteins. Similarly, the subject non-proteinaceous molecules may also take any suitable form. Both the proteinaceous and non-proteinaceous agents herein described may be linked, bound otherwise associated with any other proteinaceous or non-proteinaceous molecules. For example, in one embodiment of the present invention, said agent is associated with a molecule which increases its solubility, availability and/or half-life, and/or which permits its targeting to a localised region and/or its entry to a cell, and/or which provides it with a further activity, such as cytotoxicity.

In what follows, absent any further indication, the term "antagonist of Egfl7" will indifferently designate any molecule inhibiting Egfl7 expression or promoting its degradation, as recited in point (i) above, or any molecule antagonizing Egfl7 activity, such as those described in the above point (ii). In the same manner, the term "agonist of Egfl7" will indifferently designate Egfl7 itself, any molecule promoting its expression or inhibiting its degradation, or any molecule agonizing its activity.

The method of the present invention contemplates the modulation of transendothelial cell migration in both in vitro and in vivo. Although the preferred method is to treat an individual in vivo, it should nevertheless be understood that the method of the invention can be applied in an in vitro environment, for example to provide an in vitro model of leukocyte extravasation. According to a particular embodiment of the present invention, the modulator is an antagonist of Egfl7, which promotes migration of immune cells across vascular endothelium. In particular, the antagonist of Egfl7 according to this embodiment favors the migration of lymphocytes from the blood vessels to the perfused tissues, across the epithelium.

The present invention is particularly useful in the area of cancer treatment. Indeed, an antagonist of Egfl7 according to the present invention will advantageously be used as an agent for increasing tumor infiltration by immune cells, and in particular by lymphocytes, especially T lymphocytes, as well as NK cells and dendritic cells. According to a particular embodiment, the antagonist of Egfl7 is used as an agent for preventing tumor escape from immunity.

The inventors have observed that Egfl7 not only represses tumor endothelium activation, but also decreases expression of Tie-2, thereby affecting endothelium integrity and increasing tumor vessel permeability to tumor cells. Hence, inhibiting Egfl7 in tumors, especially in tumors which over-express it, leads not only to a better infiltration of the tumor mass by immune cells, but also to a decreased migration of tumor cells into vascular vessels. The present invention hence also pertains to an antagonist of Egfl7 for use as an agent for preventing tumor cells intravasation, in particular for preventing metastasis.

Another object of present invention is a method for the treatment of a solid tumor in a patient in need thereof, comprising a step of administrating to said patient an antagonist of Egfl7. An antagonist of Egfl7, for use as an agent for treating a solid tumor, is hence part of the present invention. Non-limitative examples of solid tumors which can be treated by this method comprise breast cancer, prostate cancer, colon cancer, rectal cancer, bladder cancer, lung cancer, melanoma, non-Hodgkin lymphoma, endometrial cancer, pancreatic cancer, kidney cancer, thyroid cancer, brain tumor and head and neck cancer. The treatment will be mostly effective in tumors over-expressing Egfl7. Accordingly, the method can comprise an initial step of measuring, for example from a biopsy of a tumor, the level of expression of Egfl7 in said tumor. Treatment with an antagonist of Egfl7 according to the present invention will be decided if the tumor expresses Egfl7, especially if the expression level is higher than in normal controls (expression level in normal tissues or in the same organ of individuals without cancer).

The terms "patient", "individual" and "subject" as used herein include humans, primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals (e.g., foxes, kangaroos, deer). Preferably, the individual is human or a laboratory test animal. Even more preferably, the patient is a human. In a particular embodiment, the antagonist of Egfl7 is formulated for intravenous administration. Other routes of administration include, but are not limited to, intratumoral, respiratory, intratracheal, nasopharyngeal, intraperitoneal, subcutaneous, intracranial, intradermal, intramuscular, intraoccular, intranasal, infusion, oral, rectal, and via an implant. Viral vectors as well as encapsulated forms can advantageously be used.

Of course, the present invention can be combined with another antitumor or anti-cancer treatment. In particular, it can be combined with anti-tumor immunotherapy, especially for treating tumors which over-express Egfl7. The anti-tumor immunotherapy treatments which can be combined with Egfl7 antagonists according to the invention include stimulatory treatments with interleukins, as well as cellular or peptide- based cancer vaccines. The combination will lead to a far better efficiency of said antitumor vaccine, since the immune cells generated by said vaccine will more efficiently infiltrated the tumor mass. Non-limitative examples of anti-tumor therapeutic vaccines which can be combined with Egfl7 antagonists according to the present invention are Sipuleucel-T (Provenge™, Dendreon Corporation), as well as the single-peptide-based vaccines disclosed in Table 1 below.

Table 1: Single peptide vaccines currently in clinical development According to another embodiment, the Egfl7 antagonist according to the present invention is combined to chemotherapy and/or radiotherapy, especially those which target metastases.

Although the main indication of Egfl7 antagonists is the presence of a solid tumor and/or metastases, there may be different circumstances in which it is desirable to upregulate transendothelial cell migration, such as, for example, infections by a pathogen, especially by a bacterium, a fungus, a virus or a multicellular parasite.

As already mentioned above, the antagonist of Egfl7 used according to the present invention can be a fragment of Egfl7, possibly fused to another moiety. Other molecules, such as antibodies, fragments of antibodies, aptamers (all targeting Egfl7), as well as oligonucleotides inhibiting Egfl7 expression can be used according to the present invention. Examples of anti-Egfl7 antibodies are disclosed in US 2009/0297512 Al, US 2010/0285009 Al, WO 2007/106915 A2 and WO 2005/1 17968 A2. Expression vectors encoding an inactive fragment of Egfl7 or an antibody, fragment of antibody or aptamer targeting Egfl7 can also be used according to the present invention. Such expression vectors, as well as viral vectors or transformed cells comprising them, are also considered as "Egfl7 antagonists" in the present text. A pharmaceutical composition, comprising an inhibitor of Egfl7 as described above, is also part of the present invention.

Another aspect of the present invention pertains to the use of Egfl7 or agonists thereof, for inhibiting endothelium activation and/or migration of immune cells across vascular endothelium. This can be helpful for preventing and/or treating pathologies characterized by aberrant, unwanted or otherwise excessive cellular transendothelial cell migration in an individual. This is of particular significance in the context of conditions such as atheromas, rheumatoid arthritis and inflammatory bowel disease. This aspect of the present invention is also particularly advantageous in situations where isolation of a particular organ or area from the immune system is needed, for example in the context of grafts or auto-immune diseases such as Type I diabetes.

The use of Egfl7 or an agonist thereof, for inhibiting migration of immune cells across vascular endothelium, is hence part of the present invention.

Badiwala et al. suggested that Egfl7 could be used to attenuate the endothelial injury which occurs during hypoxia/reoxygenation during transplantation (Badiwala et al., 2010). They also suggested that Egfl7 could attenuate endothelial injury and arteriopathy resulting from chronic cyclosporine treatment of grafted people (Badiwala et al., 201 1). However, these authors did not envision any effect of Egfl7 different from preventing tissue injury due to inflammation at the level of vascular endothelium. In particular, they did not envision to isolate the grafted organ from immune cells, to protect it from the host immune response. In the experimental part which follows, the inventors have shown that Egfl 7 produced by tumor cells (and not endothelial cells) had an effect on tumor vascular vessels, by preventing their activation and thereby inhibiting transmigration of immune cells towards the tumor. This effect, which is negative in the case of tumors, could be beneficial in other circumstances, such as in the case of grafted organs. Indeed, as a foreign body in their host, grafted organs are naturally attacked by the host's immune system, including lymphocytes and NK cells. Except in rare cases such as grafts between homozygotic twins, this renders immunosuppressant treatments compulsory, in spite of all the adverse effects of such treatments. By isolating the grafted organ from immune cells, Egfl7 could reduce the need for immunosuppressive drugs. Accordingly, the present invention also pertains to the use of Egfl7 or an agonist thereof, as an agent for protecting a graft against immune cells of the host, especially against lymphocytes. A method for preventing graft rejection in an individual, comprising a step of administering Egfl7 or an agonist thereof to said individual, is also part of the present invention. In this method, Egfl7 or its agonist is preferentially administered locally, at the tumor site. This can be performed by any appropriate means, such as, for example, an injection pump continuously delivering Egfl7 in (or nearby) the grafted organ. This could also be achieved by grafting, in the grafted organ or in its near vicinity, organoids made of genetically transformed cells (preferably autologous cells) over-expressing Egfl7. Egfl7 can be administered in soluble form, as well as in encapsulated form.

According to a particular embodiment of the invention, Egfl7 or an agonist thereof is used as an agent for protecting a graft against immune cells of the host, in combination with an immunosuppressant drug. Non-limitative examples of drugs which can be used in this context are azathioprine, ICAM-1 , corticosteroids such as dexamethasone, anti-inflammatory molecules, calcineurin inhibitors (cyclosporine, tacrolimus), mTOR inhibitors (Sirolimus, Everolimus and rapamycine analogs), mycophenolic acid, and anti-CD20 or anti-IL2-receptor antibodies.

Another aspect of the present invention is the use of Egfi7 or an agonist thereof, as an agent for preventing or treating a chronic inflammatory disease, and/or an auto-immune disease in which the patient's own immune cells destroy a particular tissue or organ. For example, type-1 diabetes is due to the destruction of islets of Langherans, located in the pancreas, by T lymphocytes infiltrating these islets. This destruction could be prevented by locally providing Egfl7 or an agonist thereof, to inhibit T lymphocytes infiltration of said islets. In cases where the prevention failed, transplantation of islets of Langerhans is an emerging treatment procedure for patients with type 1 diabetes. However, the procedure is associated with massive tissue loss caused by the immune system. In the context of the present invention, the transplanted islets are transformed to express Egfl7 or an agonist thereof, or associated with cells expressing Egfl7 or agonist thereof. Other examples of auto-immune diseases and chronic inflammatory diseases which can be treated or attenuated by Egfl7 or an agonist thereof according to the present invention include as lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, Reynaud's syndrome, rheumatoid arthritis, psoriasis etc.

Egfl7 or an agonist thereof can also be administered to a patient to prevent or suppress delayed-type hypersensitivity reactions.

The invention further includes the above-described methods for suppressing an inflammatory response of the specific defense system in which an immunosuppressive agent is additionally provided to the subject. Such an agent is preferably provided at a dose lower (i.e. a "sub-optimal" dose) than that at which it would normally be required. The use of a sub-optimal dose is possible because of the combined effect of the agent of the present invention. Examples of suitable immunosuppressive agents are listed above.

Other conditions related to inflammatory reactions can also benefit from the present invention. Indeed, since Egfl7 and its agonists can prevent extravasation of leukocytes, they can prevent or attenuate non-specific inflammations. Such inflammatory reactions are due to reactions of the "non-specific defense system" which are mediated by leukocytes incapable of immunological memory. Such cells include granulocytes and macrophages. As used herein, inflammation is said to result from a response of the nonspecific defense system, if the inflammation is caused by, mediated by, or associated with a reaction of the non-specific defense system. Examples of inflammation which result, at least in part, from a reaction of the non-specific defense system include inflammation associated with conditions, such as adult respiratory distress syndrome (ARDS) or multiple organ injury syndromes secondary to septicemia or trauma, reperfusion injury of myocardial or other tissues, acute glomerulonephritis, reactivearthritis, dermatoses with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory disorders, thermal injury, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocyte transfusion associated syndromes and cytokine-induced toxicity.

Immune responses to therapeutic or diagnostic agents such as, for example, bovine insulin, interferon, tissue-type plasminogen activator or murine monoclonal antibodies substantially impair the therapeutic or diagnostic value of such agents, and can, in fact, cause diseases such as serum sickness. Such a situation can be remedied through the use of Egfl7 or agonists thereof. In this embodiment of the present invention, Egfl7 or its agonist would be administered in combination with the therapeutic or diagnostic agent. The Egfl7 or agonist thereof prevents the recipient from recognizing the agent, and therefore prevents the recipient from initiating an immune response against it. The absence of such an immune response results in the ability of the patient to receive additional administrations of the therapeutic or diagnostic agent. The agonist of Egfl7 according to the present invention (which can be Egfl7 itself) can be delivered by any route and under any pharmacological form. Examples of routes of administration include, but are not limited to, intravenous, intratumoral, respiratory, intratracheal, nasopharyngeal, intraperitoneal, subcutaneous, intracranial, intradermal, intramuscular, intraoccular, intranasal, infusion, oral, rectal, and via an implant. Examples of pharmaceutical formulations for delivering Egfl7 or an agonist thereof include, but are not limited to, particles comprising an expression vector encoding Egfl7 or agonist thereof (such as viral vectors, naked DNA, DNA encapsulated in a liposome or in any other encapsulating means, etc.), proteins (possibly encapsulated, possibly coupled to another moiety), etc. Accordingly, a further aspect of the present invention is a pharmaceutical composition comprising, as an active ingredient, an expression vector encoding Egfl7 or an agonist thereof. Non-limitative examples of such compositions include viral vectors expressing Egfl7, transformed cells, for example transformed epithelial cells, over-expressing Egfl7, and organoids expressing Egfl7.

Other characteristics of the invention will also become apparent in the course of the description which follows of the biological assays which have been performed in the framework of the invention and which provide it with the required experimental support, without limiting its scope.

EXAMPLES

Figure legends

Figure 1. Egfl7 promotes tumor growth and metastasis. A. 4T1-Ctrl and 4T1-Egfl7 cells were injected into the mammary fat-pad of Balb/c mice, tumors were dissected after 27 days. Tumor extracts (75μg) were analyzed by Western-blotting using anti-HA and anti-actin antibodies in order to visualize the expression of exogenous Egfl7 in reference to actin in two representative samples of each condition. B. Tumor sections were immunostained using a specific anti-Egfl7 antibody (brown) in order to visualize the higher levels of expression in 4T1-Egfl7 tumors, bar represents ΙΟΟμιη. C. 4T1-Ctrl (o) and 4T1-Egfl7 (■) cells were implanted into the mammary fat-pad of Balb/c mice at day 0 and growing tumors measured over time, insert, average final weights of 4T1-Ctrl and 4T1-Egfl7 tumors. D. left, Lungs of mice bearing 4T1-Ctrl and 4T1-Egfl7 primary tumors were dissected and photographed, arrows indicate macro-metastases, right, average numbers of lung surface macro-metastases counted in each animal group. E. LLC-Ctrl (o) or LLC-Egfl7 (■) were injected in the skin of C57B1/6 mice and tumors measured at regular times after injection, insert, average weights of LLC-Ctrl and LLC-Egfl7 tumors after 25 days. Data are representative of a set of 4 and 3 experiments performed in similar conditions using 4T1 and LLC1 models, respectively. *; p<0.05.

Figure 2. A. 4T1-Ctii (o) and 4T1-Egfl7 (■) cells were plated at 1 ,250 cells/cm² in culture dishes and counted every day. Insert: over-expression of Egfl7 in 4T1- Egfl7 cells assessed by SDS-PAGE analysis of cell extracts and Western-blotting using antibodies directed against Egfl7-HA (top) and actin (bottom). B. 4T1-Ctrl and 4T1-Egfl7 cells (5x10⁴ cells) were plated onto 8 μπι-pore diameter membranes (0.3 cm , Falcon), allowed to migrate for 24h at 37°C, trypsinized and counted. Data are expressed as percentage of migrated cells over the added numbers of cells counted in both chambers. C. 4T1-Ctrl and 4T1 -Egfl7 cells (250 cells/cm²) were seeded at low density in 0.45% agar, and cultured for 14 days, colonies were individually counted. D. LLC-Ctrl (o) and LLCEgfl7 (■) cells were plated at 2,500 cells/cm² in culture dishes and counted every day. Insert: over-expression of Egfl7 in LLC-Egfl7 cells assessed by SDS-PAGE analysis of cell extracts and Western-blotting using antibodies directed against Egfl7-HA (top) and actin (bottom). E. LLC-Ctrl and LLC-Egfl7 cells (5x10⁴ cells) were plated onto 8 μιη-pore diameter membranes (0.3 cm², Falcon), allowed to migrate for 24h at 37°C, trypsinized and counted. Data are expressed as percentage of migrated cells over the added numbers of cells counted in both chambers. F. LLC-Ctrl and LLC-Egfl7 cells (250 cells/cm ) were seeded at low density in 0.45% agar, and cultured for 10 days, colonies were individually counted. The numbers of colonies were evaluated using the ImageJ vl .42q software. Figure 3. Increased necrosis, hypoxia, angiogenesis, and permeability in 4T1-Egfl7 tumors. A. left, 4T1-Ctrl and 4T1-Egfl7 tumor sections were stained with hematoxylin/eosin and necrosis identified as unstained areas, bars represent 1mm, right, necrotic areas were quantified as a percentage of total tumor area in the 4T1 and the LLC1 models,. B. 4T1-Ctrl and 4T1-Egfl7 tumors were analyzed for hypoxia in 15 non- overlapping sections of 4T1 -Ctrl and 4T1-Egfl7 tumors. C. left, Sections of 4T1-Ctrl and 4T1 -Egfl7 tumors immunostained for CD31/PECAM (brown) in order to visualize the tumor endothelium, bar represents Ι ΟΟμηι, right, quantification of CD31⁺ vessels in angiogenic hot-spots in the 4T1 and LLC1 tumors, the area fraction represents the percentage of stained area in 20 non-overlapping 2.4 mm² fields. D. Mice bearing 4T1-Ctrl or 4T1-Egfl7 tumors were injected with Lycopersicon esculentum-FYTC lectin and Dextran-Texas Red. Tumors were dissected and analyzed by fluorescent microscopy. Red staining outside FITC-stained vessels indicates blood leakage, bar represents 100 μηι. Histogram on the right indicates the mean percentage of leaky vessels counted in 25 independent 0.15mm² fields of 4T1-Ctrl and 4T1 -Egfl7 tumors. *; p<0.05.

Figure 4. 4T1-Ctrl and 4T1-Egfl7 tumors were analyzed for apoptosis after TUNEL staining and counting positive cells in 30 non-contiguous sections. Cleaved caspase-3 levels were determined after SDS-PAGE analysis of tumor extracts (n=6 per condition), western-blotting using a specific antibody and luminescence measurement of band intensities. Ki67 proliferation index quantified by image analysis of 14 non- overlapping immunostained 4T1 and LLC tumor sections. Data are representative of 2 experiments performed in similar conditions.

Figure 5. Egfl7 prevents the infiltration of immune cells into 4T1- Egfl7 tumors. A. Hematoxylin/eosin staining of 27-day tumors shows numerous small nucleated cells accumulated in the lumens of 4T1-Egfl7 tumor blood vessels (right, arrows), such cells are rare in 4T1-Ctrl tumor blood vessels (left, arrows), bar represents ΙΟΟμιη. B. CD3s immunostaining of tumor sections (brown) shows a large infiltration of T-lymphocytes in 4T1-Ctrl tumors (left, arrows pointing to some). Most of the CD3s⁺ cells remained in the vessel lumens of 4T1-Egfl7 tumors (right, arrows), bar represents Ι ΟΟμηι. right, average numbers of infiltrated CD3s⁺ cells (outside of blood vessels) counted in 30 independent 0.15mm fields. C. Average numbers of cells positive for the indicated molecule counted within the tumor masses in 25 independent 0.15mm² fields. Data are representative of 2 experiments performed in similar conditions. **; p<0.0l , * * * ; / θ.001

Figure 6. Immunostainings of 4T1-Ctrl (left) and 4T1-Egfl7 (right) tumor sections with the T-lymphocyte markers CD4 (A, red) and CD8 (B, red), B- lymphocyte marker CD 19 (C, confocal microscopy, red), macrophage marker CD68 (D, red), natural killer cells marker NKp46 (E, brown) and dendritic cell marker CDl l c (F, confocal microscopy, red) antibodies and DAPI (blue) counterstaining illustrate the depletion of immune cells within the 4T1-Egfl7 tumors. Data are representative of 2 experiments performed in similar conditions.

Figure 7. Spleens of Balb/c animals bearing 4T1-Ctrl (white bars) and 4T1-Egfl7 tumors (black bars) were analyzed by flow cytometry for measuring the proportion of T-lymphocytes (CD3e, TCR, CD4, CD8), B-lymphocytes (CD19), and Natural Killer cells (NKp46). Material and methods: FITC-conjugated monoclonal Abs against mouse CD4, CD8, CD45, PE-conjugated anti-CD4, -CD45, -NKP46, APC- conjugated anti-CD3, -CD19, purified anti-CD16/32, PE-Cy7 conjugated anti-CD8, and isotype controls were from BD Pharmingen, 7-AAD was from eBioscience. Spleens were harvested and mechanically homogenized. After washes, red blood cells were removed with lysis buffer (Sigma) and spleen cells counted using Trypan blue (Sigma). For flow cytometry analysis, 2 to 5xl0⁵ cells/well were plated in 96- well plates and resuspended in 50 μΐ of PBS, 2% fetal bovine serum containing a rat anti-mouse CD16/CD32 mAb (Fc receptor blocking) for 15 min on ice. After washes, cells were resuspended in 50 μΐ of PBS, 2% fetal bovine serum containing FITC or PE-conjugated anti-CD45 on ice for 30min, washed twice, and then incubated for 30 min on ice with the appropriate combination of Abs. After the last wash, cells were resuspended in PBS and 5 μΐ of 7-AAD were added to each tube. Mononuclear CD45+/7-AAD- cells were then analyzed on a LSR Fortessa using the FACSDiva software (Becton Dickinson).

Figure 8: Egfl7 has no effects in the absence of a functional immune system. A. 4T1 -Ctrl (o) and 4T1-Egfl7 (■) cells were injected into the mammary fat pad of immunosuppressed SCID-beige mice and the developing tumors were measured overtime. Overlays of growth curves of 4T1-Ctrl (dashed) and 4T1-Egfl7 tumors (dotted) implanted in Balb/c mice in similar conditions are plotted at the same scale for comparison, insert: average final weights of 4T1-Ctrl and 4T1-Egfl7 tumors. B. left, Lungs were dissected and photographed, arrows indicate macro-metastases, right, average numbers of macro- metastases counted at the surface of lungs of each animal group.

Figure 9. A. Primary aortic smooth muscle cells (5xl0⁴ cells/well) were placed in the upper chamber of cell culture inserts (8 μιτι pore size, Becton-Dickinson) in medium containing the indicated amounts of rEgfl7 and in the presence (+) or not (-) of 40 ng/ml porcine platelet-derived growth factor-BB (R&D). After 6hr, the cells that had migrated to the lower compartment were stained using hemalun and counted under a microscope. Full inhibition of cell migration was achieved in the presence of 50 ng/ml rVE-statin. B. Dendritic cells (lxlO⁵ cells/well) were incubated in the presence of LPS (1 μg/ml) and rEgfl7 (150 ng/ml) when indicated (+) for 24hr and the supernatants tested for the presence of IL12p40 by ELISA. T-lymphocytes were incubated on anti-CD3 -coated wells (5x10⁵ cells/well) in the presence of an anti- CD28 and in the presence of rEgfl7 (150 ng/ml) where indicated (+) for 5 days. Cell proliferation was evaluated by incubating the cells with Alamar blue for 24hr and followed by a spectrophotometric dosage.

Figure 10. Egfl7 alters the tumor endothelium characteristics. A. dendritic cells (lxl O⁵, left) were cultured for 24h in the presence of LPS and rEgfl7 where indicated (+) and IL6 was quantified in the supernatants. NK cells (5x10⁴, middle) were incubated for 48h with IL12 and IL18 with and rEgfl7 where indicated (+) and IFNg was quantified in the supernatants. Purified splenic T-lymphocytes (5xl 0⁵, right) were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of rEgfl7 where indicated (+) for 48h and production of IFNg in the supernatants was quantified. B. 4T1 -Ctrl and 4T1-Egfl7 tumors were sectioned and immunostained for ICAM- 1 (left, green) or VCAM-1 (right, red), and counterstained with DAPI (blue), bars represent Ι ΟΟμηι. C. Relative quantities of ICAM- 1 and VCAM-1 transcripts measured by RT-qPCR in endothelial cells isolated from 4T1 -Egfl7 and 4T1 -Ctrl tumors. For each sample, levels were normalized to the actin expression levels. Data are expressed at 2^"AACt, taking 4T1 - Ctrl values as reference. D. Expression levels of the indicated genes in endothelial cells isolated from 4T1-Ctrl (white bars) and 4T1 -Egfl7 (black bars) tumors as assessed by RT- qPCR as above. Data are representative of 2 experiments performed in similar conditions.

Figure 11 : Egfl7 prevents the adhesion of T-lymphocytes on endothelial cells. A. left, Dil-labelled Jurkat T-lymphocytes were seeded onto a monolayer of confluent endothelial HUVEC cells which had been treated for 24hr with a medium conditioned by 4T1 -Ctrl (CM-Ctrl) or 4T1 -Egfl7 (CM-Egfl7) cells, bar represents Ι ΟΟμηι, right, values represent the average numbers of adhering Jurkat cells counted in 15 independent 2.4mm² fields. B. left, Dil-labelled Jurkat T-lymphocytes were seeded onto a monolayer of confluent HUVEC which had been transfected with a control siRNA (si-Ctrl) or a siRNA targeting Egfl7 (si-Egfl7), bar represents Ι ΟΟμηι, right, values represent the average numbers of adhering Jurkat cells counted as in A. These experiments are representative of 3 experiments performed in similar conditions. C. Expression levels of E- selectin, P-selectin, vcam- 1 , icam-1 , and CD31/PECAM measured by RT-qPCR in HUVEC transfected with a control siRNA (si-Ctrl) or a siRNA targeting Egfl7 (si-Egfl7). Levels are expressed at 2^"AACt, taking si-Ctrl values as reference. D. Dil-labelled Jurkat T- lymphocytes were seeded onto a monolayer of confluent HUVEC which had been previously transfected with a control siRNA (si-Ctrl) or siRNAs targeting Egfl7 (si-Egf!7), icam-1 (si-ICAM-1), or vcam-1 (si-VCAM- 1) and quantified, values represent the average numbers of adhering Jurkat cells counted in 9 independent 2.4 mm fields, *; p<0.05.

Figure 12. A. Analysis of the expression levels of Egfl7 transcripts in HUVEC transfected with a siRNA targeting Egfl7 (si-Egfl7) or control (si-Ctrl) as assessed by RT-qPCR, B. Transfection of HUVEC cells with si-Egfl7 increased icam- 1 expression; this effect was counteracted by the cotransfection of a siRNA targeting Icam-1. C. Transfection of HUVEC cells with si-Egfl7 increased Vcam-1 expression; this effect was counteracted by the co-transfection of a siRNA targeting Vcam-1. Expression levels of Icam-1 and Vcam-1 were measured by RT-qPCR in HUVEC cells. Levels are expressed at 2^"AACt taking si-Ctrl values as reference.

Figure 13: High expression of Egfl7 correlates with low VCAM-1, ICAM-1 and IFNg in human tumors. A. Expression of Egfl7 (top, brown) and ICAM-1 (bottom, brown) was analyzed by immunohistochemistry in a human breast ductal invasive carcinoma (noted SBR II, RE+, RP+, HER2 0). The tumor tissue which expresses large amounts of Egfl7 within tumor cells (top left, *), shows low levels of ICAM-1 in adjacent blood vessels (bottom left, arrows). The peritumoral region which expresses low levels of Egfl7, mostly in blood vessels (top right, *), shows strong expression of ICAM-1 in blood vessel endothelium (bottom right, arrows). B. Expression of ICAM-1 and VCAM- 1 in blood vessels was quantified as the mean percentage of positive vessels in 25 independent 0.15mm² fields in tumors expressing low- (0+, n=5) and high- (3+, n=5) levels of Egfl7, *; p<0.05. C. Human breast carcinomas (n=12 per group) were analyzed for expression of Egfl7 (black bars) and IFNg (grey bars) by qRT-PCR. For each sample, data were normalized to the p2-microglobulin expression levels. Data are expressed at 2^{" AACt}, taking the lowest average value as reference for each gene.

Material and Methods

Cells. Mouse mammary carcinoma 4T1 (ATCC CRL-2539), lung adenocarcinoma LLC1 (ATCC CRL-1642) and Jurkat (ATCC TIB- 152) cells were obtained from ATCC and were not further tested or authenticated. 4T1 and Jurkat cells were cultured in RPMI, 10% fetal bovine serum (FBS), lOOU/ml penicillin, 100μg/ml streptomycin. LLC1 were cultured in DMEM, 10% FBS, lOOU/ml penicillin, 100μg/ml streptomycin. Human primary umbilical vein endothelial cells (HUVEC, Lonza) were cultured in EGM-2 and used between passage 1 and 5. Conditioned medium was produced by incubating 4T1-Ctrl or 4T1-Egfl7 cells (2xl04/cm²) in EBM-2 (Lonza), 0.2% BSA for 24 hr. Medium was filtered (0.22μηι) before use. All cells were cultured in a humidified 95% air/5% C0₂ incubator at 37°C.

Cloning. The mouse Egfl7 cDNA (Soncin et al., 2003) was cloned in frame with a C-terminal influenza hemagglutinin (HA)-coding sequence in the pMSCV plasmid (Clontech), allowing the production of retrovirus after transfection in HEKGP cells. 4T1 and LLC1 cells were infected with control or Egfl7-coding viruses and whole cell populations were selected for puromycin resistance (4μ§ ηιι) for 7 days.

Transfection. HUVEC were plated in 2cm² well-plates (25000 cells/cm²) and transfected the next day with lOnM siRNA (Dharmacon) in Primefect siRNA reagent (Lonza) mixed with EGM-2. After 24hr, EGM-2 was added and cells cultured for 24hr or 48hr.

Tumor models. Eight week-old female Balb/c, C57B1/6 and SCID-beige mice were from Charles River (7 mice per experimental group in each experiment unless otherwise stated). 4T1 and LLC1 cells (5xl0⁵/50 μΐ) were injected either in the mammary fat pad (Balb/c, SCID-beige) or subcutaneously (C57B1/6). Tumors were measured using an electronic caliper and volume calculated as volume=width x width x length x id 6 (Tomayko and Reynolds, 1989). Mice were housed according to European legislation; all protocols were approved by the local ethics committee (#CEEA02/2009).

Tumor vessel perfusion. Blood vessel leakage was assessed by injecting Ι ΟΟμΙ of PBS, 0.5mg/ml Lycopersicon esculentum lectin-FITC (Vector Laboratories) and 2.5mg/ml Dextran (70kD)-Texas Red (Molecular Probes) in the tail vein. Mice were euthanized after l Omin, and tumors were collected and processed for cryosection. Hypoxia was estimated after peritoneal injection of 0.15M NaCl, 60mg/kg pimonidazole-HCl (Hypoxyprobe, HPI). Mice were sacrificed after 30min, tumors were collected, proceeded for paraffin inclusion, and hypoxyprobe detected by immunohistochemistry.

Immunohistocliemistry. Tumors were either fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (7μη ) or frozen in OCT compound, sectioned at Ι Ομηι and post-fixed with 1% paraformaldehyde (5min). Immunostainings were performed using antibodies as listed in Table 2. For necrosis analysis, sections were stained with hematoxylin/eosin and necrotic areas identified as unstained regions. Apoptotic cells were visualized using the Terminal Transferase recombinant kit (Roche). Proliferating cells were detected by staining with a Ki67 antibody (Roche). For optical microscopy, sections were counterstained with hematoxylin. For immunofluorescence microscopy, cell nuclei were labeled by incubating in DAPI solution (1 μg/ml, Sigma) for l min at room temperature. Slides were analyzed using an Axioplan2 or an AxioImagerZl microscope (Zeiss) and the AxioVs V4.8.2.0 software or a confocal LSM710 microscope (Zeiss) using the ZEN2008 software (MICPaL, Lille). Angiogenic hotspots were analyzed by image analysis of CD31 -stained sections using the ImageJ vl .42q software (Abramoff et al, 2004). Antigen (mouse) Dilution Reference

Egfl7 1/500 AbyD

CD31/PECAM 1/100 BD-Pharmingen, 550274

ICAM-1 1/1000 Abeam, ab25375

VCAM-1 1/250 BD-Pharmingen, 550547

CD3s 1/100 Abeam, ab5690

CD4 1/100 BD- Pharmingen, 553649

CD8 1/200 Pharmingen, 550281

CD19 1/250 Pharmingen, 561738

CD68 1/100 Abeam, ac53444

NKp46 1/100 R&D, AF2225

CDl lc 1/250 Pharmingen, 550261

Table 2: Antibodies used in immunochemistry

Tumor endothelial cell isolation. Freshly collected tumors (n=6/group) were minced and dissociated in DMEM, lmg/ml collagenase-I, 10μg/ml DNase-I for 30min at 37°C and filtered (90μm and 40μηι mesh). Cells from each tumor were treated separately. Cells were incubated in red blood cell lysis buffer (Sigma) for 5min at room temperature. Washed cells were incubated in DMEM, 0.2% FBS containing a rat anti- mouse CD16/CD32 (Fc-block, BD-Pharmingen, 553141, ^g/10⁶ cells) for lh at 4°C, then with anti-rat IgG coated magnetic beads (Dynabeads, Invitrogen) which had been incubated with a rat anti-mouse CD45 antibody (BD-Pharmingen, 550539) for 20min at 4°C. CD45^" cells were collected and incubated with magnetic beads pre-incubated with a rat anti-mouse CD31/PECAM antibody (BD-Pharmingen, 553370) for 20 min at 4°C. CD457CD31⁺ and CD457CD3 r cells were separated and lysed in TRIzol. Enrichment was evaluated by measuring the expression levels of CD31.

Quantitative RT-PCR. Cells or tissues were homogenized in Trizol (Life

Technologies). Total RNA were extracted and reverse transcribed using a high capacity cDNA reverse transcription kit (Life Technologies). Quantitative PCR (qPCR) were performed using TaqMan gene expression assays, reagents and conditions (Life Technologies).

Western-blotting. Proteins were extracted in RIPA buffer, analyzed by

12% SDS-PAGE and blotted onto Immobilon-P (Millipore). Egfl7-HA, cleaved-caspase-3, and actin were detected using specific antibodies from Covance (HA.1 1 Clone 16B12, 1/1500), Cell signaling (9664S, 1/1000), and Santa Cruz Biotechnology (sc-1615, 1/1000), respectively. Chemiluminescence was measured using a Luminescent Image System (LAS3000, Fujifilm).

Adhesion assay. For T- lymphocytes adhesion assays, Jurkat cells (lxlO⁶) were incubated with Dil (2 μΜ, Molecular Probes) for l Omin at 37°C and allowed to adhere (10⁵ cells/cm₂) onto a monolayer of confluent HUVEC for 20min at 22°C. Fluorescent Jurkat cells were counted under a UV-microscope.

Immune cell activation. To generate dendritic cells, bone marrow- derived cells (BM-DC) were cultured in IMDM medium supplemented with 10% FBS and 1% of supernatant from GM-CSF-expressing J558-GM-CSF cells for 14 days. Cells (10⁵ cells/well) were stimulated or not with ^g/ml LPS for 24h, in the presence or not of 150ng/ml mouse recombinant (r)Egfl7 (Caetano et al., 2006). IL6 and IL12p40 were quantified in the supernatants by ELISA (BD-Biosciences). For NK cell function, mouse liver mononuclear cells were labeled with APC-conjugated anti-CD5 and PE-conjugated anti-NKl . l mAbs. CD57NK1.1⁺ cells were sorted using a FACSAria (BD Biosciences), cultured in RPMI, 5% FCS (10⁴ cells/well) and stimulated with lOng/ml IL12 and lOng/ml IL18 in the presence or not of 150ng/ml rEgfl7 for 48h. IFNg production was analyzed in supernatants by ELISA (eBiosciences, Paris, France). T-cells were purified from spleens by negative selection using a Dynal T-cell isolation kit (Life Technologies). T-cells (10⁵ cells/well) were cultured in RPMI, 5% FCS and stimulated or not with plate-bound anti- CD3 ^g/ml) and soluble anti-CD28 (^g/ml), in the presence or not of 150ng/ml rEgfl7 for 48h. IFNg production was analyzed in supernatants by ELISA. Proliferation was assessed 96h later, using AlamarBlue (Life technologies).

Human tumor samples. Human tumors were collected, processed and stored at the anatomo-pathology laboratory of the Centre Oscar Lambret, Lille, France. Tissues were fixed in 4% paraformaldehyde (Prolabo) and embedded in paraffin. Sections (4μπι) were stained using a polyclonal anti-hEgfl7 antibody (1/30, R&D, AF3638) in a Discovery automat (not shown). Other immunostainings were performed using specific antibodies against ICAM-1 (1/50, Abeam Ab53013) and VCAM-1 (1/500, Abeam Ab98954). Slides were analyzed by two independent observers and compared to corresponding hematoxylin/phloxin safran-stained slides for identification of the tumor sub-regions. Tumors were classified from 0+ (no Egfl7 staining) to 3+ (intense Egfl7 staining).

Statistics. In each experiment, data points have been collected at least in triplicate. The Mann- Whitney- Wilcoxon test was used to compare the mean values between groups. * p<0.05, ** pO.Ol, *** pO.001. Error bars in graphs represent the calculated standard error.

RESULTS

Egfl7 promotes tumor growth and metastasis

In order to study the potential role of Egfl7 in tumor development, mouse 4T1 breast cancer cells were infected with a retrovirus encoding the full length, HA- tagged, mouse Egfl7 (4T1-Egfl7) or with a control virus (4T1-Ctrl). When implanted into the mammary fat pad of syngeneic Balb/c mice, 4T1-Egfl7 cells formed tumors which showed a marked accumulation of Egfl7 (Fig. 1A, B). These tumors grew much faster in volume than 4T1-Ctrl tumors and, accordingly, the mean final weight of 4T1 -Egfl7 tumors was twice as high as that of 4T1-Ctrl tumors (Fig. 1 C). Mice bearing 4T1-Egfl7 tumors also had a 3.6-times higher frequency of lung metastasis when compared to 4T1-Ctrl tumors (Fig. ID). Since in vivo experiments can vary with the cell lines and animal models used, similar experiments were performed using the LLC1 lung carcinoma cell line implanted in syngeneic C57BL/6 mice. LLC-Egfl7 cells also produced tumors that grew faster than LLC-Ctrl (Fig. IE). The effects of Egfl7 on tumor growth and metastasis in vivo were not due to intrinsic modifications of the tumor cell properties since expression of Egfl7 had no effects on 4T1 and LLC1 proliferation, migration, or clone formation in anchorage-independent conditions in vitro (Fig. 2).

In vivo, 4T1-Egfl7 tumors displayed larger necrotic areas than 4T1-Ctrl tumors (Fig. 3A) and a similar tendency was observed in LLC1 tumors. Hypoxia was higher in 4T1-Egfl7 tumors than controls (Fig. 3B) whereas the apoptosis, cleaved caspase- 3 levels and Ki67 proliferation indexes were comparable between tumors (Fig. 4). Microvessel density in angiogenic hot-spots was slightly increased in 4T1 -Egfl7 tumors when compared to controls, but was not significantly different between LLC1 tumors (Fig. 3C). Interestingly, 4T1-Egfl7 tumor blood vessels were much more permeable than 4T1- Ctrl vessels (Fig. 3D), and although excessive edema was not observed, blood lakes were more frequent in LLC-Egfl7 tumors than in LLC-Ctrl tumors (2.7% +/- 0.93 and 1.1% +/- 0.4 of the tumor area, respectively).

Egfl7 -expressing tumors are less infiltrated by immune cells.

Interestingly, a detailed observation revealed the presence of numerous small round cells in the lumen of 4T1 -Egfl7 tumor blood vessels, whereas these cells were less frequently observed within the tumor tissue itself. In contrast, 4T1 -Ctrl tumors had very few cells accumulated in their blood vessels and the tumor tissue appeared more granular than 4T1-Egfl7 tumors (Fig. 5A). Since these cells resembled circulating or infiltrated immune cells, staining for CD3e; a global marker of T-lymphocytes was performed. The 4T1-Egfl7 tumor tissue contained 69% fewer CD3s cells than 4T1-Ctrl tumors, most of the CD3s⁺ cells remaining in the lumens of the blood vessels (Fig. 5B). Staining for CD4⁺ and CD8⁺ cells showed that both T-cell populations were affected, with a 42% and 45% decrease in the respective number of cells infiltrated in 4T1-Egfl7 tumors, when compared to 4T1-Ctrl (Fig. 5C, Fig. 6). These effects were not restricted to the T-cell lineage as the numbers of infiltrated B-lymphocytes (CD19⁺), macrophages (CD68⁺), NK cells (NKp46⁺) and dendritic cells (CDl lc⁺) were decreased by more than half in 4T1- Egfl7 tumors when compared to 4T1-Ctrl tumors (Fig. 5C, Fig. 6). This correlated with large differences in the transcript levels of immune-stimulating cytokines IFNg and IL12b, and, to a lower extent, ILla, and ILlb, which levels were strongly reduced in 4T1-Egfl7 tumors when compared to controls (Table 3). Similar results were obtained using the LLC- C57BL/6 model; LLC-Egfl7 tumors expressed much less CD3E and IFNg transcripts than LLC-Ctrl tumors (Table 4). On the other hand, the spleens of Balb/c mice carrying 4T1- Ctrl or 4T1-Egfl7 tumors showed no significant differences in the relative numbers of T- (CD3s⁺, TCR⁺) and B-lymphocytes (CD 19⁺), or of NK cells (NKp46⁺), suggesting that the immune depletion was not systemic in mice carrying 4T1-Egfl7 tumors but was locally restricted to the tumor tissue (Fig. 7).

Table 3: RT-qPCR analysis of expression levels of cytokines in 4T1- Ctrl and 4T1-Egfl7 tumors. Expression levels were analyzed by RT-qPCR using specific Taqman assays. Data are expressed as 2^"AACt where ACt =Ct of gene - Ct of actin for the same sample, and AACt=ACt 4T1-Egfl7 - ACt 4T1-Ctrl.

Table 4. Expression levels of the T-lymphocytes marker CD3s in LLC- Ctrl and LLC-Egfl7 tumors were assessed by SyBR-Green RT-qPCR using the primers 5'- aac acg tac ttg tac ctg aaa get c (SEQ ID No: 1) and 5 '-gat gat tat ggc tac tgc tgt ca (SEQ ID No: 2). Expression levels of IFNg transcripts were assessed by RT-qPCR using specific Taqman assays. Data are expressed as 2^~AACt where ACt =Ct of gene - Ct of actin for the same sample, and AACt=ACt LLC-Egfl7 - ACt LLC-Ctrl.

Altogether, these results show that 4T1 -Egfl7 tumors formed a local immune-deficient environment. The effects of Egfl7 on tumor growth depend on the immune system

In order to directly assess the importance of the host immune system on the effects of Egfl7, 4T1-Ctrl and 4T1-Egfl7 cells were injected in the mammary gland of immuno suppressed SCID-beige mice, which lack functional T-, B-, and NK cells. As expected, 4T1-Ctrl tumors grew much faster and induced a higher rate of metastasis in SCID-beige mice when compared to Balb/c immunocompetent mice (Fig. 8), thus confirming the repressing effects of immune cells on tumor development. On the other hand, expression of Egfl7 in tumor cells failed to accelerate tumor growth and metastasis in SCID-beige mice so that no differences between the growth rates of 4T1-Ctrl and 4T1- Egfl7 tumors could be seen.

Thus, the effects of Egfl7 on tumor growth and metastasis depend on an active host immune system.

Egfl7 represses leukocyte adhesion molecules in tumor endothelial cells

The inventors next addressed the potential effects of Egfl7 on the functions of immune cells. rEgfl7, which was active as an inhibitor of PDGF-BB-induced smooth muscle migration (Fig 9A, (Shioi et al., 2006; Soncin et al., 2003)), had no effect on the LPS-induced release of IL6 (Fig. 10A) and IL12p40 (Fig. 9B) by dendritic cells. Similarly, rEgfl7 did not affect IFNg production by NK cells stimulated with IL12 and IL18 and failed to modulate the anti-CD3/anti-CD28-stimulated production of IFNg by T- lymphocytes (Fig. 10A) as well as their proliferation (Fig. 9B).

The inventors thus investigated whether Egfl7 could alter the recruitment of immune cells within the tumors. Expression of cell adhesion molecules such as E- and P-selectins, ICAM-1 , VCAM-1 and CD31/PECAM by endothelial cells plays a crucial role in leukocyte rolling and adhesion before trans-endothelial migration (Muller, 2009). They detected high levels of expression of ICAM-1 and VCAM-1 in 4T1-Ctrl tumor blood vessels whereas expression was weak in 4T1-Egfl7 tumors (Fig. 10B). To confirm this, CD457CD31⁺ endothelial cells from tumors were isolated. In agreement with the immunostainings, the expression levels of ICAM-1 and VCAM-1 in endothelial cells purified from 4T1-Egfl7 tumors were reduced by 57% and 70%, respectively, when compared to controls (Fig. IOC). Endothelial cells isolated from 4T1-Egfl7 tumors also expressed much lower levels of E-selectin (Fig. 10D). These results provide a likely explanation for the observed deficit of immune cells and cytokines within 4T1-Egfi7 tumors. In addition, endothelial genes involved in promoting vessel integrity, maturation, and quiescence such as Tie-2 and PAI-1 were repressed by more than 80% in endothelial cells of 4T1-Egfl7 tumors whereas expression of uPA was increased in these cells (Fig. 10D). Expression of D114 was strongly repressed, in agreement with the observed increased vascular density and hypoxia, but in apparent contradiction with the enhanced tumor development (Kuhnert et al., 201 1). Of note, fit- 1 expression was strongly increased in 4T1-Egfl7 tumor endothelial cells and the expression levels of CD31/PECAM, P-selectin, VE-cadherin, eNOS, and of the integrin sub-units αν, 3, βΐ and β3 were not modified (not shown).

Altogether, these results suggest that the decreased frequency of immune cells within 4T1-Egfl7 tumors is rather due to the inhibition of their recruitment rather than to the repression of their functions, and corresponds to phenotypic modifications of the endothelium.

Egfl7 directly regulates leukocyte adhesion on endothelial cells

The possibility that Egfl7 directly affects the expression of leukocyte adhesion molecules by endothelial cells was studied in vitro using the Jurkat T-cell lymphoma model of immune cell adhesion on human primary HUVEC endothelial cells, a model known to depend on ICAM-1 and VCAM-1 (Chan et al., 2000). Treatment of HUVEC with medium conditioned by 4T1 -Egfl7 cells reduced the number of T- lymphocytes adhering onto the endothelial monolayer by half when compared to cells incubated with a medium conditioned by 4T1-Ctrl cells (Fig. 1 1A). Repressing the endogenous egfl7 gene in endothelial cells using RNA interference (Fig. 12 A) doubled the number of T-lymphocytes adhering to HUVEC when compared to control (Fig. 1 IB). This correlated with a large increase in expression of E-selectin, Vcam-1 , and Icam-1 transcripts while expression of P-selectin and CD31/PECAM was not affected (Fig. 11C). Further, repressing either Icam-1 or Vcam-1 (Fig. 12B, C) in endothelial cells treated with a siRNA targeting Egfl7 reduced the effects of this latter siRNA on T-cell adhesion (Fig. 1 1D), suggesting that the repressing effects of Egfl7 on depend directly on the repression of Icam-1 and Vcam-1.

Expression of ICAM-1, VCAM-1 and IFNg is repressed in human tumors expressing Egfl7

In order to validate our observations in human cancers, expression of ICAM-1, VCAM-1 and IFNg was analyzed in a series of human breast carcinomas which were selected on the basis of their expression levels of Egfl7 in tumor cells (GLP, FS, personal communication). Within the same lesion, there was a 23% and 13% decrease in the numbers of blood vessels expressing ICAM-1 and VCAM-1 , respectively, when these vessels were in close vicinity to tumor cells expressing high levels of EgfI7, when compared to areas where expression of Egfl7 was low (peritumoral, Fig. 13 A, B). Furthermore, the levels of expression of IFNg were inversely correlated to the levels of expression of egfl7 measured in a series of human breast tumor samples (Fig. 13C).

Discussion

The inventors show here that Egfl7 is a natural repressor of endothelial cell activation. It inhibits the expression of endothelial adhesion molecules, and consequently reduces the adhesion of lymphocytes onto the endothelium. When placed in a tumor context, these effects result in an increased escape from immunity and a more rapid tumor growth.

The few previous studies that have described the expression of Egfl7 in human cancers all suggested that Egfl7 could promote tumor growth and metastasis (Diaz et al., 2008; Huang et ah, 2010; Wu et al., 2009) but no experimental study had addressed the direct role of Egfl7 in tumor development. Here, the results obtained using two independent tumor models and mouse genetic backgrounds validate this initial hypothesis made on clinical observations. Furthermore, over-expressing Egfl7 in experimental tumors allowed the inventors to understand its functions in more details. Egfl7 is not an oncogene since it does not confer er se intrinsic proliferative or invasive properties to lung or breast tumor cells in vitro. In vivo, Egfl7 shows no effects on tumor growth when compared to controls, in the absence of a functional immune system. The above data indicate that the effects of Egfl7 on tumor growth and metastasis are rather indirect: Egfl7 promotes tumor escape from immunity which, in turn, promotes tumor progression. Interestingly, Egfl7 has no effect on the immune cells themselves. Indeed, it does not directly activate dendritic cells, NK cells or T-lymphocytes and does not affect their activation status upon stimulation. The main effect of Egfl7 is to repress the tumor endothelium activation so that immune cells remain sequestered in the blood circulation, thus preventing their infiltration within the tumor mass. This explains why all immune cells analyzed were under- represented in tumors expressing Egfl7, regardless of the cell type. The in vitro adhesion assays indicate that this diminished recruitment of immune cells is directly mediated by Egfl7 through the down-regulation of endothelial adhesion molecules. The down- regulation of endothelial adhesion molecules in tumors (Dirkx et al., 2003; Griffioen et al., 1996; Kuzu et al., 1993; Piali et al., 1995) and in human cancers (Griffioen et al., 1996; Piali et al., 1995; Shioi et al., 2006) had already been observed. Egfl7 favors tumor escape from immunity by downregulating the expression of endothelial adhesion molecules through mechanisms which are still elusive.. Of note, Egfl7 was recently reported to down- regulate the NF-KB pathway in human coronary artery endothelial cells after an ischemia/reoxygenation treatment (Badiwala et al., 2010). Based on this observation and the above results, it is thus possible that a direct repression of the NF-kB pathway by Egfl7 in endothelial cells contributes to the repression of ICAM-1 and, possibly, that of VCAM-1 and E-selectin.

The inventors also observed that expression of Egfl7 in tumors increases blood vessel permeability and decreases expression of Tie-2 when compared to controls, suggesting that the endothelium integrity is altered in the presence of Egfl7. Since Egfl7 was shown to inhibit PDGF-BB-induced smooth muscle cell migration (Soncin et ah, 2003), it is likely that its expression prevents the recruitment of perivascular cells to newly formed tumor blood vessels, thus decreasing vascular tightness. Such a lack of vessel integrity is commonly observed in tumors (Jain, 2005) and is proposed to favor tumor spreading through metastasis (Mazzone et al., 2009; Rolny et al., 201 1). Interestingly, overexpression of angiopoietin-2 in tumors also increases vessel permeability but correlates with a higher rate of immune cell infiltration within the primary tumors, and with a reduced tumor growth rate (Fagiani et al., 201 1). Altogether, these observations suggest that, depending on the conditions, the disruption of the endothelium might lead both to a reduced primary tumor and to an increase in metastasis which could explain the observed dual anti-primary tumor/pro-metastatic effects of anti-angiogenic therapies (Ebos et al., 2009; Paez-Ribes et al., 2009).

In human breast tumor cells, the inventors observed that expression of Egfl7 corresponds to a local decrease in ICAM-1 and VCAM-1 expression in adjacent blood vessels, whereas more distant vessels are not affected. This suggests that Egfl7 has similar local effects on blood vessels in human tumors to those observed in experimental tumors in mice. The inverse correlation between the expression levels of Egfl7 in human tumors and those of IFNg further validates their hypothesis and suggests that Egfl7 produces an immune-deficient environment within human breast cancer tissues.

Tumor escape from immunity is undoubtedly an interesting process to consider for the design of therapeutic tools aimed at preventing cancer progression and metastasis. Egfl7 therefore represents a new target for interfering with this process.

REFERENCES

Abramoff, M.D., Magalhaes, P.J. and Ram, S.J. (2004) Image Processing with Image. J. Biophotonics International, 1 1, 36-42.

Alitalo, K. and Carmeliet, P. (2002) Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell, 1, 219-227.

Badiwala, M.V., Guha, D., Tumiati, L., Joseph, J., Ghashghai, A., Ross, H.J., Delgado, D.H. and Rao, V. (2011) Epidermal growth factor-like domain 7 is a novel inhibitor of neutrophil adhesion to coronary artery endothelial cells injured by calcineurin inhibition. Circulation, 124, S I 97-203.

Badiwala, M.V., Tumiati, L.C., Joseph, J.M., Sheshgiri, R., Ross, H.J., Delgado, D.H. and Rao, V. (2010) Epidermal growth factor-like domain 7 suppresses intercellular adhesion molecule 1 expression in response to hypoxia/reoxygenation injury in human coronary artery endothelial cells. Circulation, 122, S I 56-161.

Caetano, B., Drobecq, H. and Soncin, F. (2006) Expression and purification of recombinant vascular endothelial-statin. Protein Expr Purif, 46, 136-142.

Castermans, K. and Griffioen, A.W. (2007) Tumor blood vessels, a difficult hurdle for infiltrating leukocytes. Biochim Biophys Acta, 1776, 160-174.

Chan, J.R., Hyduk, S.J. and Cybulsky, M.I. (2000) Alpha 4 beta 1 integrin/VCAM-1 interaction activates alpha L beta 2 integrin-mediated adhesion to ICAM-1 in human T cells. J Immunol, 164, 746-753.

Chung, A.S., Lee, J. and Ferrara, N. (2010) Targeting the tumour vasculature: insights from physiological angiogenesis. Nat Rev Cancer, 10, 505-514.

Diaz, R., Silva, J., Garcia, J.M., Lorenzo, Y., Garcia, V., Pena, C., Rodriguez, R., Munoz, C, Garcia, F., Bonilla, F. and Dominguez, G. (2008) Deregulated expression of miR-106a predicts survival in human colon cancer patients. Genes Chromosomes Cancer, 47, 794-802.

Dirkx, A.E., Oude Egbrink, M.G., Kuijpers, M.J., van der Niet, S.T., Heijnen, V.V., Bouma-ter Steege, J.C., Wagstaff, J. and Griffioen, A.W. (2003) Tumor angiogenesis modulates leukocyte-vessel wall interactions in vivo by reducing endothelial adhesion molecule expression. Cancer Res, 63, 2322-2329.

Ebos, J.M., Lee, C.R., Cruz-Munoz, W., Bjarnason, G.A., Christensen, J.G. and Kerbel, R.S. (2009) Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell, 15, 232-239.

Fagiani, E., Lorentz, P., Kopfstein, L. and Christofori, G. (201 1) Angiopoietin- 1 and -2 Exert Antagonistic Functions in Tumor Angiogenesis, yet Both Induce Lymphangiogenesis. Cancer Res, 71 , 5717-5727. Garrido-Urbani, S., Bradfield, P.F., Lee, B.P. and Imhof, B.A. (2008) Vascular and epithelial junctions: a barrier for leucocyte migration. Biochem Soc Trans, 36, 203-211.

Griffioen, A.W., Damen, C.A., Martinotti, S., Blijham, G.H. and Groenewegen, G. (1996) Endothelial intercellular adhesion molecule- 1 expression is suppressed in human malignancies: the role of angiogenic factors. Cancer Res, 56, 1 1 1 1- 1 1 17.

Hashizume, H., Baluk, P., Morikawa, S., McLean, J.W., Thurston, G., Roberge, S., Jain, R.K. and McDonald, D.M. (2000) Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol, 156, 1363-1380.

Herberman, R.B., Nunn, M.E., Holden, H.T. and Lavrin, D.H. (1975) Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells. Int J Cancer, 16, 230-239.

Huang, C.H., Li, X.J., Zhou, Y.Z., Luo, Y., Li, C. and Yuan, X.R. (2010) Expression and clinical significance of EGFL7 in malignant glioma. J Cancer Res Clin Oncol, 136, 1737-1743.

Jain, R.K. (2005) Normalization of tumor vasculature: an emerging concept in anti angiogenic therapy. Science, 307, 58-62.

Koebel, CM., Vermi, W., Swann, J.B., Zerafa, N., Rodig, S.J., Old, L.J., Smyth, M.J. and Schreiber, R.D. (2007) Adaptive immunity maintains occult cancer in an equilibrium state. Nature, 450, 903-907.

Kuhnert, F., Kirshner, J.R. and Thurston, G. (201 1) D114-Notch signaling as a therapeutic target in tumor angiogenesis. Vase Cell, 3, 20.

Kuzu, I., Bicknell, R., Fletcher, CD. and Gatter, K.C (1993) Expression of adhesion molecules on the endothelium of normal tissue vessels and vascular tumors. Lab Invest, 69, 322-328.

Lelievre, E., Hinek, A., Lupu, F., Buquet, C, Soncin, F. and Mattot, V. (2008) VE-statin/egfl7 regulates vascular elastogenesis by interacting with lysyl oxidases. Embo J, 27, 1658-1670.

Mazzone, M., Dettori, D., Leite de Oliveira, R., Loges, S., Schmidt, T., Jonckx, B., Tian, Y.M., Lanahan, A. A., Pollard, P., Ruiz de Almodovar, C, De Smet, F., Vinckier, S., Aragones, J., Debackere, K., Luttun, A., Wyns, S., Jordan, B., Pisacane, A., Gallez, B., Lampugnani, M.G., Dejana, E., Simons, M., Ratcliffe, P., Maxwell, P. and Carmeliet, P. (2009) Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization. Cell, 136, 839-851.

Muller, W.A. (2009) Mechanisms of transendothelial migration of leukocytes. Circ Res, 105, 223-230. Paez-Ribes, M., Allen, E., Hudock, J., Takeda, T., Okuyama, H., Vinals,

F., Inoue, M., Bergers, G., Hanahan, D. and Casanovas, O. (2009) Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis.

Cancer Cell, 15, 220-231.

Piali, L., Fichtel, A., Terpe, H.J., Imhof, B.A. and Gisler, R.H. (1995)

Endothelial vascular cell adhesion molecule 1 expression is suppressed by melanoma and carcinoma. J Exp Med, 181, 81 1-816.

Rolny, C, Mazzone, M., Tugues, S., Laoui, D., Johansson, I., Coulon, C,

Squadrito, M.L., Segura, I., Li, X., Knevels, E., Costa, S., Vinckier, S., Dresselaer, T., Akerud, P., De Mol, M., Salomaki, PL, Phillipson, M., Wyns, S., Larsson, E., Buysschaert,

I., Botling, J., Himmelreich, U., Van Ginderachter, J. A., De Palma, M., Dewerchin, M.,

Claesson- Welsh, L. and Carnieliet, P. (201 1) HRG inliibits tumor growth and metastasis by inducing macrophage polarization and vessel normalization through downregulation of

P1GF. Cancer Cell, 19, 31-44.

Shioi, K., Komiya, A., Hattori, K., Huang, Y., Sano, F., Murakami, T.,

Nakaigawa, N., Kishida, T., Kubota, Y., Nagashima, Y. and Yao, M. (2006) Vascular cell adhesion molecule 1 predicts cancer-free survival in clear cell renal carcinoma patients.

Clin Cancer Res, 12, 7339-7346.

Shrikant, P. and Mescher, M.F. (1999) Control of syngeneic tumor growth by activation of CD8+ T cells: efficacy is limited by migration away from the site and induction of nonresponsiveness. J Immunol, 162, 2858-2866.

Soncin, F., Mattot, V., Lionneton, F., Spruyt, N., Lepretre, F., Begue, A. and Stehelin, D. (2003) VE-statin, an endothelial repressor of smooth muscle cell migration. Embo J, 22, 5700-571 1.

Tomayko, M.M. and Reynolds, CP. (1989) Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol, 24, 148-

154.

Wu, F., Yang, L.Y., Li, Y.F., Ou, D.P., Chen, D.P. and Fan, C. (2009) Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma. Hepatology, 50, 1839-1850.

Wu, N.Z., Klitzman, B., Dodge, R. and Dewhirst, M.W. (1992) Diminished leukocyte-endothelium interaction in tumor microvessels. Cancer Res, 52, 4265-4268.

Claims

1. A modulator of Egfl7, for use as an agent for promoting or inhibiting migration of lymphocytes across vascular endothelium.

2. The modulator of claim 1, which is an antagonist of Egfi7, wherein said antagonist of Egfl7 promotes migration of lymphocytes across vascular endothelium.

3. The antagonist of Egfl7 according to claim 2, for use as an agent for increasing tumor infiltration by immune cells.

4. The antagonist of Egfl7 according to claim 2 or claim 3, for use as an agent for preventing tumor escape from immunity.

5. The antagonist of Egfl7 according to claim 2, for use as an agent for preventing tumor cells intravasation.

6. The antagonist of claim 5, for use as an agent for preventing metastasis.

7. The antagonist according to any of claims 2 to 6, for use as an agent for treating a solid tumor.

8. The antagonist according to any of claims 2 to 7, which is formulated for intravenous administration.

9. The antagonist according to any of claims 2 to 8, for use in combination with another anti-tumor treatment.

10. The antagonist according to claim 9, for use in combination with an anti -tumor immunotherapy.

1 1. The antagonist according to any of claims 2 to 10, which is a fragment of Egfl7.

12. The antagonist according to any of claims 2 to 10, which is selected in the group consisting of antibodies, fragments of antibodies, aptamers and oligonucleotides inhibiting Egfl7 expression.

13. The modulator of claim 1, which is Egfi7 or an agonist thereof, wherein said modulator inhibits migration of immune cells across vascular endothelium.

14. Egfl7 or its agonist according to claim 13, for use as an agent for protecting a graft against immune cells of the host.

15. Egfl7 or its agonist according to claim 14, for use in combination with an immunosuppressant drug.

16. Egfl7 or its agonist according to claim 13, for use as an agent for preventing and/or treating a disease associated with an excessive activation of the vascular epithelium.

17. The agonist according to any of claims 13 to 16, which comprises an expression vector encoding Egfl7.