PREVENTION OF TUMOUR METASTASIS BY INHIBITION OF NECROPTOSIS

Abstract

The present invention relates to an inhibitor of necroptosis for use in preventing the metastasis of tumours. Further, the present invention relates to a method of preventing the metastasis of tumours by inhibiting necroptosis and to a method for modulating the transmigration of metastasising tumour cells through endothelium by modulating necroptosis as well as to an in-vitro method of identifying an inhibitor of necroptosis suitable as a lead compound and/or as a medicament for the prevention of tumour metastasis. Moreover, the present invention also relates to a method of identifying necroptotic and necrotic cells.

Claims

1. (canceled)

2. A method of preventing the metastasis of tumours by inhibiting necroptosis comprising administering a pharmaceutically effective amount of an inhibitor of necroptosis to a subject in need thereof.

3. A method for modulating the transmigration of metastasising tumour cells through endothelium by modulating necroptosis, comprising the steps of: a) contacting endothelium with a modulator of necroptosis; and b) providing metastasising tumour cells and allowing said metastasising tumour cells to transmigrate through the endothelium.

4. An in vitro method of identifying an inhibitor of necroptosis suitable as a lead compound and/or as a medicament for the prevention of tumour metastasis, comprising the steps of: a) allowing metastasizing tumour cells to transmigrate the endothelium i) in the presence of a test agent and ii) in the absence of said test agent; b) determining the level of tumour cell transmigration through the endothelium and/or the level of endothelial cell necroptosis for a)i) and a)ii); c) comparing the level(s) determined in step b) for a)i) with the level(s) determined in step b) for a)ii), wherein a decrease in the level(s) for a)i) as compared to the level(s) of a)ii) is indicative for the test agent to be an inhibitor of necroptosis suitable as a lead compound and/or as a medicament for the prevention of tumour metastasis.

5. An inhibitor or modulator of necroptosis that is selected from the group consisting of an inhibitor of receptor interacting protein 1 (RIPK1), an inhibitor of receptor interacting prtein 3 (RIPK3), an inhibitor of mixed lineage kinase domain like protein (MLKL) or a combination thereof.

6. The methods of claim 2, wherein the subject or the endothelium is mammalian.

7. The inhibitor or modulator according to claim 5, wherein the inhibitor or modulator prevents or moduklates the formation of a necrosome andor a necroptosis-inducing activity of the necrosome.

8. The inhibitor or modulator according to claim 5, wherein the inhibitor or modulator is an antibody, an antibody mimetic, a dominant negative protein, a siRNA, a shRNA, a miRNA, a ribozyme, an aptamer, anucleic acid molecule, an antisense nucleic acid molecule, a small molecue or modified version of these.

9. The inhibitor or modulator accrding to claim 5, wherein the inhibitor or modulator is an inhibitor of RIPK3.

10. The inhibitor or modulator according to claim 5, wherein the inhibitor or the modulator is selected from the group consisting of necrostatin-1 (Nec-1; 5-(1H-indol-3-ylmethyl)-3 -methyl-2-thioxo-4-imidazolidinone, 5-iIndo1-3 -ylmethyl)-3 -methyl-2-thio-hydantoin), necrostatin-1 stable (5- ((7-chloro -1H-indo1-3 -yl)methyl)-3 -methyl-2,4-imidazolidinedione, 5- ((7-chloro- 1H-indo1-3 - yl)methyl)-3 -methylimidazolidine-2,4-dione) , necrostatin-1 inactive (5- ((1H-indo1-3 -yl)methyl)-2-thioxoimidazolidin-4-one), Necrosulfonamide (NSA; (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide), an anti-RIPK3 siRNA, an anti-MLKL siRNA or a combination thereof.

11. The method of claim 3,wherein the modulator is an inhibitor of TGF-beta-activating kinase 1 (TAK1).

12. The inhibitor or modulator acccording to claim 5, that is an inhibitor of death receptor 6 (DR6).

13. The inhibitor or modulator according to claim 5 having admixed thereto or being associated in a separate container with a further pharmaceutically active agent.

14. A method of identifying necroptotic and necrotic cells comprising: a) contacting cells with a marker for plasma membrane breakdown; b) contacting said cells with a marker for chromatin condensation and/or chromatin fragmentation; and c) determining whether plasma membrane breakdown and chromatin condensation and/or chromatin fragmentation occurs, wherein it is indicative that said cells are necroptotic or necrotic, if plasma membrane breakdown is determined in step c) in the absence of chromatin condensation and/or chromatin fragmentation as occurring in apoptotic cells.

15. The method of claim 14, wherein the marker for plasma membrane breakdown in step a) is selected from the group consisting of a membrane-impermeable DNA-binding dye and/or the marker for chromatin condensation and/or chromatin fragmentation is selected from the group consisting of membrane-permeable DNA-binding dyes.

16. The method of claim 2, wherein the inhibitor or modulator is selected from the group consisting of an inhibitor of receptor interacting protein 1 (RIPK1), an inhibitor of receptor interacting protein 3 (RIPK3), an inhibitor of mixed lineage kinase domain like protein (MLKL) or a combination thereof.

17. The method of claim 2, wherein the inhibitor or modulator is selected from the group consisting of necrostatin-1 (Nec-1; 5-(1H-indol-3-ylmethyl)-3-methyl-2-thioxo-4-imidazolidinone, 5-iIndo1-3-ylmethyl)-3-methyl-2-thio-hydantoin), necrostatin-1 stable (5-((7-chloro-1H-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione, 5-((7-chloro-1H-indol-3-yl)methyl)-3-methylimidazolidine-2,4-dione), necrostatin-1 inactive (5-((1H-indol-3-yl)methyl)-2-thioxoimidazolidin-4-one), Necro sulfonamide (NSA; (E)-N-(4-(N-(3-methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2- yl)acrylamide), an anti-RIPK3 siRNA, an anti-MLKL siRNA or a combination thereof.

18. The method of claim 3, wherein the inhibitor or modulator is selected from the group consisting of an inhibitor of receptor interacting protein 1 (RIPK1), an inhibitor of receptor interacting protein 3 (RIPK3), an inhibitor of mixed lineage kinase domain like protein (MLKL) or a combination thereof.

19. The method of claim 3, wherein the inhibitor or the modulator is selected from the group consisting of necrostatin-1 (Nec-1; 5-(1H-indol-3-ylmethyl)-3-methyl-2-thioxo-4-imidazolidinone, 5-iIndol-3-ylmethyl)-3-methyl-2-thio-hydantoin), necrostatin-1 stable (5-((7-chloro-1H-indol-3-yl)methyl)-3-methyl-2,4-imidazolidinedione, 5-((7-chloro- 1H-indol-3-yl)methyl)-3-methylimidazolidine-2,4-dione), necrostatin-1 inactive (5-((1H-indol-3-yl)methyl)-2-thioxoimidazolidin-4-one), Necrosulfonamide (NSA; (E)-N-(4-(N-(3 -methoxypyrazin-2-yl)sulfamoyl)phenyl)-3-(5-nitrothiophene-2-yl)acrylamide), an anti-RIPK3 siRNA, an anti-MLKL siRNA or a combination thereof.

Description

[0159] The figures show:

[0160] FIG. 1: Schematic overview on interactions of tumour, immune and endothelial cells during metastasis (adopted from Labelle and Hynes, 2012).

[0161] FIG. 2: Method to discriminate apoptotic from necrotic cell death A: Human Umbilical Vein Endothelial Cells (HUVEC) were treated with the indicated stimuli and analysed by staining with Hoechst 33342 and EtDlll. TRAIL and Staurosporine induced apoptosis whereas cells treated with H.sub.2O.sub.2 or O.sub.2 showed a necrotic phenotype. B: TNF treatment resulted in a necrotic phenotype in L929 cells which could be blocked by addition of necrostatin-1 as shown by staining with Hoechst 33342 and EtDlll.

[0162] FIG. 3: Tumour cells (TCs) induce necroptosis in endothelial cells (ECs) in vitro A: CFPac and MDA-MB-231 tumour cells induce endothelial cell necroptotic cell death in HUVEC in vitro as shown by staining with Hoechst 33342 and EtDlll. B: Quantification of necroptotic HUVEC after treatment with different TCs. C: Quantification of necroptotic HUVEC after treatment with increasing numbers of MDA-MB-231 TCs. D: Intravenously injected TCs (B16 or LLC1) induce death of endothelial cells of the mouse lung in vivo. This death is necroptotic as shown by positive staining with EtDlll in the absence of nuclear condensation or fragmentation as shown by DAPI staining. Further, cleaved caspase could not be detected. E: CD31 and ERG served as endothelial specific markers.

[0163] FIG. 4: Increased necroptosis in endothelial cells (ECs) leads to increased tumour cell (TC)-induced endothelial cell death and metastasis. A: Deletion of TAK1 increases TC-induced necroptosis in HUVEC. B: Necroptosis induced by TCs (B16) in endothelial cells in vivo is increased in mice lacking TAK1 in endothelial cells)(TAK1.sup.ECKO as compared to wild type (wt) mice. C: Quantification of necroptotic ECs induced by TCs (B16 or LLC1) in wt and TAK1.sup.ECKO mice D: Silencing of TAK1 increases the number of transmigrated TCs. E: Increased metastasis formation by TCs (B16) in TAK1.sup.ECKO mice as compared to wt mice.

[0164] FIG. 5: Pharmacological inhibition of tumour cell (TC)-induced endothelial cell (EC) death results in reduced metastasis formation. A: Necrostatin 1(Nec-1) reduces the number of TC-induced necroptotic HUVEC in vitro. B: Nec-1 reduces TC (B16) induced necroptotic ECs and metastasis formation by TCs (B16 or LLC1) in vivo. C: Quantification of the effect of Nec-1 on the number of TC (B16)-induced necroptotic ECs in vivo. D: Quantification of B16 metastasis in the presence and absence of Nec-1 in vivo. E: Quantification of LLC1 metastasis in the presence and absence of Nec-1 in vivo.

[0165] FIG. 6: DR6 is required for tumour cell (TC) induced endothelial cell (EC) death and TC transmigration A: siRNA screen to identify potential receptors that mediate TC-induced necroptotic EC death (RIPK3 and MAP3K7 (=TAK1) served as internal controls) B: confirmation of the screening hit (DR6) with different siRNAs C: reduced transendothelial migration of TC through a HUVEC monolayer with silenced DR6 expression (knockdown efficiency >70%, data not shown) D: effect of DR6-Fc fusion proteins (R&D Systems, #144-DR) on TC-induced necroptotic EC death E: effect of DR6-Fc fusion proteins on TC transendothelial migration through a HUVEC monolayer.

[0166] FIG. 7: Tumor cell-induced endothelial necroptosis and metastasis formation are regulated by endothelial RIPK3 and raspase-8 (A and B) Endothelial cells (HUVECs) were transfected with control siRNA (siCTRL) or with siRNAs directed against the messenger RNAs encoding RIPK3 (siRIPK3), MLKL (siMLKL) or caspase-8 (siCasp8), and (A) endothelial cell death was determined in the absence of tumor cells (−TC, white bar) or presence of MDA-MB-231 tumor cells (+TC, black bars). The number of transmigrated MDA-MB-231 tumor cells over a HUVEC monolayer after siRNA-mediated gene knockdowns as indicated was determined in (B) (n=6 wells per condition). (C) Effects of endothelial-specific deletion of RIPK3(RIPK3.sup.ECKO) on tumor cell-induced endothelial cell death (6 h, left panel) and metastasis formation in vivo (12d, right panel).

[0167] Schematic on the top right shows the experimental design. 6 h: representative confocal images of lung sections 6 h after i.v. injection of B16 tumor cells into animals of the indicated genotype and stained for cleaved caspase 3 (providing a green colour), EthD-III (providing a yellow colour), CD31 (providing a red colour) and cell nuclei (DAPI, providing a blue colour). 12d: representative images of lungs 12d after i.v. injection of B16 tumor cells into animals of the respective genotype. Cre-negative littermates served as control. Bar length: 50 μm. (D-F) Quantification of (D) EthD-III-positive endothelial cells at 6 h, (E) number of extravasated tumor cells at 6 h or (F) lung metastases at 12d after i.v. injection of B16 tumor cells into RIPK3.sup.EcKQ mice. Quantifications in (D) and (F) are based on images as shown in (C). The white bar in (D) represents mice that received PBS instead of tumor cells (n=4-6 animals per condition). All panels are representative results of three or more independent experiments. Shown are mean values ±SEM (A, D, E) or ±SD (B, F); *p<0.05; **p <0.01; ***p<0.001 (one-way ANOVA and Bonferroni's post hoc test).

[0168] FIG. 8: DR6 is expressed in endothelial cells of various human organs Representative confocal images of human tissues of the indicated origin stained for CD31 (providing a red colour), DR6 (providing a green colour) and cell nuclei (DAPI, providing a blue colour) (left panel). HUVECs in which DR6 was knocked-down (siDR6) served as control for antibody-specificity (lower panel). No perrneabilization agents were used for the stainings. Control-IgG antibody and donkey anti-rabbit secondary antibody coupled to AF488 served as negative controls (right panels). Bar length: 5 μm.

[0169] FIG. 9: Ligand binding to DR6 induces endothelial necroptosis and promotes metastasis formation (A) Effects of (mouse) IgG.sub.2 -Fc or DR6-Fc fusion proteins (0.2 pgig per dose) on tumor cell-induced endothelial cell death (6 h, left panel) and metastasis formation in vivo (12d, right panel). Schematic on the top right shows the experimental design. 6 h: representative confocal images of lung sections 6 h after i.v. injection of B16 tumor cells and treatment as indicated. Control animals received PBS instead of tumor cells. Tissue sections were stained for cleaved caspase 3 (green), EthD-III (yellow), CD31 (red) and cell nuclei (DAPI, blue). 12d: representative images of lungs 12d after i.v. injection of B16 tumor cells and the respective treatment. Bar length: 50 μm. (B-D) Quantification of (B) EthD-III-positive endothelial cells at 6 h, (C) number of extravasated tumor cells at 6 h or (D) lung metastases at 12d after i.v. injection of B16 or LLC1 tumor cells into mice treated as indicated. Quantifications in (B) and (D) are based on images as shown in (A). The white bar in (B) represents mice that received PBS instead of tumor cells (n=4-6 animals per condition).

[0170] All panels are representative results of three or more independent experiments. Shown are mean values ±SEM (B, C) or ±SD (D); *p<0.05; **p<0.01; ***p<0.001; n.s., not significant (one-way (B) ANOVA and Bonferroni's post hoc test or unpaired, two-tailed Student's t-test (C, D)).

[0171] FIG. 10: Direct tumor cell-endothelial cell contact is required for the induction of endothelial necroptosis Quantification of cell death (necrosis) in HUVECs alone (white bars) or cultured in the presence of different types of tumor cells as indicated (black bars, direct contact) or stimulated with conditioned medium from overnight co-cultures from endothelial cells with tumor cells (grey bars, condit. medium) Shown are mean values ±SEM; **p<0.01; ***p<0.001; n.s. not significant (one-way ANOVA and Bonferroni's post hoc test).

[0172] FIG. 11: APP-deficiency in tumor cells has no effect on tumor cell proliferation, cell death or migration (A) Analysis of knockdown efficiency in B16 or LLC1 tumor cells using siRNAs against APP (siAPP). Scrambled siRNA (siCTRL) served as control. Shown is the relative mRNA expression normalized to GAPDH levels and to the level detected in scramble siRNA-treated samples. (B-D) Quantitative evaluation of (B) cell numbers, (C) cell death or (D) the migratory ability of B16 or 1.1C1 tumor cells transfected with siRNA against APP (siAPP) as compared to tumor cells transfected with scramble siRNA (siCTRL) (n=3-6 wells per condition).

[0173] All panels are representative results of three or more independent experiments. Shown are mean values ±SD; ***p<0.001; n.s., not significant (unpaired, two-tailed Student's t-test).

[0174] FIG. 12: APP expressed by tumor cells induces endothelial necroptosis and promotes metastases formation (A and B) Quantification of (A) cell death (necrosis) in HUVECs in the absence of tumor cells (-TC, white bar) or exposed to MDA-MB-231 tumor cells transfected with siRNA against mRNA encoding APP (.sub.231 sip,: or control siRNA (231.sup.scIRL) (+TC, black bars) Transendothelial migration of 231.sup.siCTRL or 231.sup.siAPP tumor cells over a HUVEC monolayer was determined in (B) (n=6 wells per condition). (C) Effects of B16 tumor cells with silenced APP expression (B16.sup.siAPP) on endothelial cell death (6 h, left panel) and metastasis formation in vivo (12d, right panel). Schematic on the top right shows the experimental design. 6 h: representative confocal images of lung sections 6 h after i.v. injection of B16.sup.siAPP or B16.sup.siCTRL tumor cells and stained for cleaved caspase 3 (providing a green colour), EthD-III (providing a yellow colour), CD31 (providing a red colour) and cell nuclei (DAPI, providing a blue colour). 12d: representative images of lungs 12d after i.v. injection of the respective tumor cells. Bar length: 50 μm. (D-F) Quantification of (D) EthD-III-positive endothelial cells at 6 h, (E) number of extravasated tumor cells at 6 h or (F) lung metastases at 12d after i.v. injection of APP-silenced (B16.sup.siAPP and LLC1.sup.siAPP) or control B16 tumor cells (B16.sup.siCTRL) and control LLC1 tumor cells (LLC1.sup.CTRL). Quantifications in (D) and (F) are based on images as shown in (C). The white bar in (D) represents mice that received PBS instead of tumor cells (n=4-6 animals per condition).

[0175] Shown are mean values ±SEM (A, D, E) or ±SD (B, F); **p<0.01; ***p<0.001; n.s., not significant (one-way ANOVA and Bonferroni's post hoc test (A, B, D) or unpaired, two-tailed Student's t-test (E, F)).

[0176] FIG. 13:

[0177] Representative images and quantification of metastases formed in the lungs of animals upon i.v. injection of DMSO, 1-methyltryptophan (1-MT, 1.65 μg/g per dose) or the stable isoform of Nec-1 (Nec-ls, 1.65 μg/g per dose) shortly before and at 3 h and 6h after B16 tumor cell injection into the tail vein. Formation of lung metastases was determined 12d thereafter (n=4-5 animals per condition).

[0178] All panels are representative results of three or more independent experiments. Shown are mean values ±SD; **p<0.01; n.s., not significant (one-way ANOVA and Bonferroni's post hoc test).

[0179] The examples illustrate the invention:

[0180] Example 1: Methods

[0181] In vitro:

[0182] Endothelial cells were grown to reach 80-90% confluency before addition of Ethidium homodimer III (EtDIII) alone or together with substances or with tumour cells together with substances or inhibitors as indicated in the figures. After 6-18 h, cells were stained with Hoechst33342 and images were acquired. In order to identify the mode of cell death, all images were analyzed with FIJI. The total number of nuclei was determined through a low threshold over all Hoechst positive nuclei. On the same channel a second, separate threshold was used to determine the number of condensed nuclei. A third threshold on the second channel was used in order to determine the number of nuclei that were stained positive for EtDlll. Knockdown in cells was achieved by double-transfection with siRNA using Lipofectamie RNiMAX.

[0183] In vivo: Tumour cells and substances or inhibitors were injected i.v. as depicted in the scheme in FIG. 5B into wildtype animals or animals of the respective genotype. To identify EtDIII-positive cells in vivo, animals were injected with EtDIII i.v. 10 min before sacrifice. Organs were isolated and processed for immunohistochemical analysis. For determining the number of metastasis, animals were sacrificed, lungs were isolated and macroscopically analyzed. All reagents used for these studies (incl. DR6-Fc) are commercially available (e.g. Lonza, CellSignaling, Sigma, R&D, etc.).

[0184] Cell Death Assays: HUVECs, HMVECs-L or L929 cells (1.5×10.sup.4 at seeding in 100 μl) were cultured for 24 hours in 96-well plates. To induce cell death, cells were stimulated overnight with either rhTRAIL (100 ng/ml, Peprotech), Staurosporine (0.5 pM, Jena BioScience), H.sub.2O.sub.2 (1 mM, AppliChem) or rmTNFa (100 ng/ml, Peprotech) or cultured under hypoxic conditions (1% O.sub.2, Coy Laboratory Inc). Alternatively, for co-culture experiments, 1.5×10.sup.3 GFP-expressing tumor cells, Calcein-AM-labeled tumor cells, COS-1 or HEK cells (for more details see supplementary experimental procedues), or freshly isolated human peripheral blood mononuclear cells (PBMC) stained with Calcein-AM containing 20 times the number of platelets were added alone, in combination with each other or in the presence of the indicated substances onto the endothelial cell monolayer and cultured overnight: Nec-1 (30 μM), z-VAD-fmk (100 μM), 1-MT (30 μM), DR6-Fc or IgG1-Fc (0.1-1 μg/ml). PBMCs were isolated using standard protocols with Ficoll density gradient centrifugation. For supernatant experiments, HUVEC monolayers grown to confluency were cultured with conditioned medium obtained from HUVECs co-cultured in the presence of tumor cells for 18 h. For knockdown experiments, 1.5×10.sup.4 HUVECs were transfected using Lipofectamine RNAiMAX (Life Technologies) with different sets of siRNA (Sigma or Qiagen) and cultured on 96-well plates. Knockdown efficiencies were determined by Western Blotting using antibodies against RIPK3 (Abcam), Caspase-8 (ProSci) and a-tubulin (Sigma) upon lysis with Laemmli buffer or by quantitative PCR (Roche). In cases where siRNA-mediated knockdown was performed on tumor cells, cells were transfected using Lipofectamine RNAiMAX with different sets of siRNA (Sigma) and seeded 48h after transfection on confluent monolayers of HUVECs. Knockdown efficiencies were determined using quantitative PCR (Roche). Tumor cell number upon knockdown was determined by counting Hoechst33342-positive cells, and tumor cell death was determined by counting condensed and/or EthD-III-positive nuclei (see below). Cell migration was determined by a scratch assay Wang et al., 2007).

[0185] Cell Death Analysis: For all conditions, EthD-111 (1.6 μM, Biotium) was added to the medium before overnight culture, and Hoechst33342 (2 μM, Thermo Scientific) was added shortly before automated image acquisition in an atmosphere-controlled chamber (37° C., 5% CO2) using an Olympus IX81 microscope. Based on cells cultured under defined apoptotic, necrotic or necroptotic conditions and stained with Hoechst (a cellpermeable nuclear dye) and EthD-III (a membrane-impermeant nuclear dye), morphological criteria for discriminating apoptotic from necrotic (or necroptotic) cells as compared to living cells were defined as follows: a living cell appears with a normal round to kidney-shaped nucleus (as visualized by Hoechst) and is negative for EthD-III. An apoptotic cell appears with a strong condensed and frequently fragmented nucleus and is negative for EthD-III. A necrotic or necroptotic cell appears with a normal round to kidney-shaped nucleus or with a minor degree of nuclear shrinkage (no condensation and no fragmentation) and is positive for EthDIII.

[0186] A late apoptotic cell is positive for EthD-III but can be discriminated from a necrotidnecroptotic cell because of its strong condensation (and frequent fragmentation) of the nucleus. To all images to be analyzed, a Gaussian blur with a radius of 3 pixels was applied to prevent repeatedly counting of fragmented parts of apoptotic nuclei. Endothelial cells were defined as GFP- or Calcein-AM-negative cells. The total number of all endothelial nuclei was determined through a low threshold (TH1) and application of a watershed on the resulting binary image over all Hoechst positive nuclei (minus the nuclei from tumor cells). When possible, a second separate threshold (TH2) was used to determine the number of condensed nuclei.

[0187] The number of EthD-III-positive cells was determined through an independent second low threshold only. In cases where this automated analysis failed, the mode of cell death was determined manually for each individual endothelial cell by application of the criteria summarized above. All images were analyzed in lmageJ (NIH). Each experiment was performed at least three times with a minimum of six wells per condition and four independent images acquired per well.

[0188] Metastasis Models: 50 μl containing 5x10.sup.4 unlabeled or CFSE-labeled tumor cells (B16F10 melanoma or L.L.C1 lung carcinoma cells) or tumor cells with silenced APP expression or fluorescent microspheres (15 μm, Life Technologies) in PBS were injected to the lateral tail vein of mice. For inhibitory experiments, two doses of 50 μl Nec-1 (1.65 μg/g), Nec-1 s (1.65 μg/g), 1-MT (1.65 μg/g), z-VAD(OMe)-fmk (4 μg/g) or two doses of 25 μl of rmDR6-Fc or rmigG.sub.2A.-Fc (each at 0.2 μg/g) were injected into the tail vein shortly before and 3 hours after tumor cell injection. In all cases, for evaluation of tumor cell-induced endothelial cell death, six hours after injection of tumor cells, 50 pi EthD-III (300 μM in PBS) were injected i.v. and after 10 minutes animals were sacrificed and perfused with PBS and 4% paraformaldehyde and directly processed for immunohistochemical analysis. For evaluation of the number of extravasated tumor cells, CFSE-labeled B16 tumor cells were injected i.v. and 6h later non-perfused lungs were isolated and fixed in 4% paraformaldehyde, Cryosections of tissues were stained for cleaved caspase 3 (CellSignal), Annexin V (Santa Cruz Biotechnology), ERG (Abcam) and CD31 or CD45 (BD Biosciences). DAPI (Life Technologies) was used to visualize nuclei.

[0189] TUNEL staining kit was from Roche. Sections were analyzed in XYZ views on a Leica SPS confocal microscope. The number of EthD-III-positive endothelial cells was determined by manual counting of EthD-III/ERG- or EthD-III/CD31-positive cells on a minimum of four random tissue sections per organ. For quantification of extravasating tumor cells, cryosections were analyzed by two criteria: tumor cells directly surrounded by CD31 staining (i.e. blood vessel) and with a noninvasive phenotype (i.e. round cell shape) were scored as intravascular, while cells outside blood vessels with an invasive phenotype (i.e. irregular cell shape with protrusions) were scored as extravascular. For evaluation of lung metastases, an additional (third) treatment with the aforementioned substances was performed at 6 hours after tumor cell injection, and lung metastases were analyzed macroscopically twelve days thereafter. A minimum of three animals per group was used. All experimental animal precedures were approved by the Hessian Regional Board.

[0190] Statistical Analysis: If not stated otherwise, one representative of at least 3 independently performed experiments is shown. In all studies, comparison of mean values was conducted with unpaired, two-tailed Student's f-test or one-way or two-way ANOVA with Bonferroni's post hoc test. In all analyses, statistical significance was determined at the 5% level (p<0.05). Depicted are mean values ±SD or ±SEM as indicated in the figure legends.

[0191] Materials: Media and supplements were from Life Technologies. Nec-1 was from Enzo Life Sciences. Nec-1s was from BioVision. Z-VAD-fmk was from Alexis and z-VAD(OMe)-fmk was from Cayman. 1-MT was from Sigma. rhDR6-Fc, rhlgG.sub.1-Fc, rmDR6-Fc and rmIgG.sub.2A-Fc were from R&D Systems. Calcein-AM was from AAT Bioquest, CFSE was from Alexis. Anti-DR6 antibody for Western Blot and immunohistochemical stainings was from Bioss.

[0192] Cells: Human primary endothelial cells and media were from Lonza. MDA-MB-231-GFP tumor cells were from AntiCancer. THP-1, A549, PC3, MeWo and SK-MEL-28 cells were from CLS. B16F10 and LLC1 were from ATCC. L929 cells were a kind gift from Jan Wiegers (Biocenter Innsbruck, Austria), U-87 MG cells were from Stefan Rieken (University Hospital, Heidelberg, Germany), MIA PaCa-2 and CFPAC-1 cells were from Nathalia Giese (University Hospital, Heidelberg, Germany), Sh-SY5Y, HeLa and HT1080 were from Michael Bahr (DKFZ, Germany) and MOLT-4 cells were from Jacek Witkowski (Medical

[0193] University of Gdansk, Poland). COS-1 cells were from ATCC. All cells were incubated at 37° C. and 5% CO.sub.2. Human umbilical vein endothelial cells (HUVECs) and human microvascular vein endothelial cells from lung (1-IMVECs-L) were cultured in EGM2 or EGM2-MV medium, respectively, and passages <P6 were used for all experiments. All other cell lines were cultured in either RPMI or DMEM supplemented with 10% FBS, penicillinistreptomycin (100 unitsiml) and glutamine (2 mM). Primary mouse lung endotheliai cells were isolated and cultured as described previously (Sivaraj et al., 2013).

[0194] Transwell Assays: For a detailed description see (Schumacher et al., 2013). Briefly, for inhibitory experiments, HUVECs (1.5×10.sup.4 at seeding in 50 μl) were cultured for 2 days or, for knockdown experiments, 8×10.sup.3 HUVECs were transfected using Lipofectamine RNAIMAX with different sets of siRNA (Sigma or Qiagen) and cultured on 96-transwell plates with polyester membranes of 8-μm pore size (Corning) with daily medium changes until reaching confluency. For transmigration, the medium from the upper compartment was removed and 7.5×10.sup.3 GFP-expressing or Calcein-AMIabeled tumor cells were added in 50 μl EGM-2 medium alone or in the presence of different substances (see above). For all experiments, transmigrated tumor cells on the lower side of the filter were imaged (Zeiss Axio Observer.Z1 or Olympus IX81) and quantified with ImageJ. For permeability assays, EGM-2 medium containing 70 kDa FITC-Dextran (2 mg/ml) was added on top of the endothelial monolayer in the upper compartment. After 90 min, the amount of passed FITC-Dextran to the lower compartment containing EGM-2 only was measured (FlexStation3, Molecular Devices). Each experiment was performed at least three times with a minimum of five wells per condition.

[0195] Genetic Mouse Models: Control C57BI/6 animals were obtained from Charles River. To generate RIPK3 conditional knock-out animals, an 880-bp fragment containing loxPflanked exon 2 and 3 from ripk3 as well as the 5′ homology arm and the 3′ homology arm was amplified from BAC RPCI-23-237G18 (Children's Hospital Oakland Research Institute) and cloned into the pKOII targeting vector additionally containing a Frt-flanked neomycin resistance gene (neo.sup.R) and the Diphtheria toxin A gene (dta) as negative selection marker. The targeting vector was linearized with Notl and introduced into V6.5 ES cells by electroporation. Upon treatment with 400 μg/ml G418, DNA from 400 clones was isolated, and screened for correct recombination by Southern Blot. Two independent ES cell clones were injected into C5761/6 blastocysts, which were subsequently transferred to pseudopregnant females to generate chimeric offspring. Male chimeras were bred with C57BI/6 female mice to produce heterozygotes. The germ line transmission was confirmed in the F1 generation using PCR genotyping strategy. Mice were then crossed to Flp-deletes mice to remove the neomycin cassette and thereafter crossed with Tie2CreER.sup.T2 animals to obtain endothelial cell-specific knock-out animals (Tie2-CreER.sup.T2;RIPK3.sup.IoxP/IoxP=RIPK3.sup.ECKO). A IoxP-PCR reaction was used for detection of the wt allele (+) and the flexed (fl) allele. To induce recombination, animals were treated with 5×1 mg/d tamoxifen (Sigma) and 7-9 days later experiments were started. RIPK3 deletion in endothelial cells was confirmed by comparing protein levels on isolated endothelial cells from lungs of knock-out animals (Tie2-CreER.sup.T2;RIPK3.sup.IoxP/IoxP(−/−)) with endothelial cells from lungs of control animals (RIPK3.sup.rvfl (+/+)) using Western blot. Quantification of in vivo permeability was performed using the Miles assay. Briefly, eleven days after induction of the knockout by tamoxifen, mice received a 100 μtail vein injection of 0.5% Evans blue dye in PBS. After 30 minutes mice were killed, and extravasated blue dye was eluted from the lungs with formamide at 56° C. and measured by spectrometry at 620 nm.

[0196] Human Samples: Frozen human tissue samples were obtained from Zyagen and stained for CD31 (Acris) and DR6 (Bioss). Experiments with human samples were performed according to the regulations of the local ethics committee of the Hessian Regional Medical Board, and informed consent was obtained from all subjects.

[0197] Example 2: Method to discriminate apoptotic from necrotic cell death

[0198] An assay to discriminate apoptotic from necroticinecroptotic cell death was established. To test the method HUVEC were used under control conditions or treated with various known stimuli for apoptosis (TRAIL and staurosporine) or necrosis (H.sub.2O.sub.2 and low oxygen concentrations). Cells were stained with EtDIII and Hoechst and nuclear morphology was analyzed. Control-treated cells showed kidney-like shaped nuclei with no signs of chromatin condensation or fragmentation and no EtDIII staining. In contrast, nuclei from apoptotic cells (TRAIL or staurosporine) were condensed and fragmented, some of which were also positive for EtDIII (Le. late apoptosis). Nuclei from necrotic cells (H.sub.2O.sub.2 or low oxygen concentrations) showed only minor changes in nuclear morphology without chromatin condensation or fragmentation but were positive for EtDIII (FIG. 2A). Results were validated by applying the same staining methods to mouse embryonic fibroblast L929 cells. These cells are commonly used as standard in vitro model system to study necroptosis, because stimulation of these cells with TNFα results in necroptotic cell death and can be blocked by the presence of the RIPK1 inhibitor Necrostatin-1 (Nec-1). L929 cells treated with INFα (necroptotic) showed a similar staining pattern as HUVECs treated with H.sub.2O.sub.2 or low oxygen, and this effect could be blocked by the addition of Nec-1 (FIG. 2B).

[0199] Example 3: Tumour cells induce necroptosis in endothelial cells in vitro

[0200] Based on the assay for to discriminating apoptotic from necroptotic cell death described herein above, it was shown that various tumour cells are able to induce endothelial cell necroptotic cell death (FIGS. 3A and 3B). Furthermore, the degree of endothelial cell necroptosis depends on the tumour cell number (FIG. 3C), i.e. the more tumour cells are added, the higher the percentage of endothelial cell necroptotic cell death. Applying the same method for visualising necrotidnecroptotic endothelial cells, it was shown that tumour cells are also able to induce endothelial cell death in vivo. Upon intravenous injection of tumour cells, endothelial cells of the mouse lung were positive for EtDIII without signs of nuclear condensation or fragmentation as visualized by DAPI staining. Furthermore, apoptotic markers such as cleaved caspase-3 could not be detected (FIGS. 3D). EtDIII-positive cells co-localised with ERG, a marker for endothelial cells (FIG. 3E).

[0201] Example 4: Increased tumour cell-induced endothelial cell death leads to increased metastasis

[0202] TGF-beta-activating kinase 1 (TAK1) is a signal molecule acting as molecular switch between cell survival and cell death. It was previously show that absence or inhibition of TAK1 results in increased necroptosis in endothelial cells (Morioka et al., 2012). Importantly, it was found herein that endothelial cells in which TAK1 was silenced using sRNA showed increased necroptosis upon stimulation with tumour cells (FIG. 4A).

[0203] Furthermore, mice that lack TAK1 specifically in endothelial cells (TAK.sup.en also showed increased necroptotic cell death in endothelial cells which coincided with increased tumour metastasis (FIGS. 4B to 4E).

[0204] Example 5: Pharmacological inhibition of tumour cell-induced endothelial cell death results in reduced metastasis formation

[0205] Endothelial cells treated with the necroptosis inhibitor necrostatin-1 (Nec-1) in vitro showed reduced tumour cell-induced necroptosis (FIG. 5A). Furthermore, animals treated with Nec-1 also showed reduced tumour cell-induced endothelial necroptosis and reduced metastasis formation in vivo (FIGS. 5A to 5E).

[0206] Example DR6 is required for tumour cell (TC) induced endothelial cell (EC) death and TC transmigration

[0207] An siRNA screen was performed to identify potential receptors that mediate tumour cell-induced endothelial cell necroptotic cell death. siRNAs specific for RIPK3 and TAK1 (MAP3K7) were used as a positive and negative control, respectively. The screen revealed that knockdown of DR6 (TNFRSF21) reduced endothelial cell necroptosis to an extent comparable to the reduction achieved by knockdown of RIPK3 (FIG. 6A). In order to confirm the data obtained in the siRNA screen with regard to DR6, different siRNAs specific for DR6 (siDR6#1, siDR6#2 and siDR6#3) were used to knock down DR6 in endothelial cells. This reduced the number of endothelial cells which died by necroptosis upon treatment with tumour cells (TCs) as compared to endothelial cells which had been treated with a control siRNA (siCTRL) (FIG. 6B). Knockdown of DR6 using the different siRNAs resulted in a reduction of DR6 expression by >70% (data not shown) and also reduced the transmigration of tumour cells through a HUVEC monolayer (FIG. 6C). Endothelial cell death and tumour cell transmigration were also reduced by a DR6-Fc fusion protein (DR6-Fc) in a concentration dependent manner. IgG1-Fc was used as a negative control (FIGS. 6D and 6E).

[0208] Example 7: Knockdown of RIPK3, MLKL and Caspase-8

[0209] RIPK3 and MLKL are considered to be specific regulators of necroptosis (Newton et al., 2014; Wang et al., 2014). Knockdown of RIPK3 or MLKL in endothelial cells in vitro blocked tumor cell-induced endothelial necroptotic cell death (FIG. 7A). In contrast, knockdown of caspase-8, one of the major negative regulators of necroptosis (Gunther et al., 2011; Oberst et al., 2011), resulted in an increase in tumor-cell induced endothelial necroptosis (Ag. 7A). Again, the degree of tumor cell-induced endothelial cell necroptosis correlated with the ability of tumor cells to migrate over an endothelial cell layer (compare FIG. 7A with FIG. 7B).

[0210] To further test whether loss of RIPK3 affects tumor cell-induced endothelial cell death, tumor cell extravasation and metastasis formation in vivo, endothelial cell-specific knock-out mice were generated by crossing tamoxifen-inducible Tie2-CreER.sup.T2 animals (Korhonen et al., 2009) with mice carrying foxed alleles of RIPK3 (Tie2-CreER.sup.T2;RIPK3.sup.IoxP/IoxP, henceforth termed)RIPK3.sup.ECKO). However, animals with endothelial cell-specific loss of RIPK3 showed reduced numbers of EthD-III-positive endothelial cells as well as reduced numbers of extravasated tumor cells 6 hours after tumor cell injection, and these animals developed fewer metastases (FIG. 7C-F).

[0211] Example 8: DR6 is expressed in different human organs

[0212] DR6, which was also found to be expressed in mouse and human endothelial cells of different vascular beds (FIG. 8), belongs to a subgroup of the TNF receptor family containing a cytosolic death domain that enables cell death signaling (Lavrik et al., 2005; Pan et al., 1998).

[0213] Example 9: Induction of necroptosis by DR6 ligand binding

[0214] Given that DR6 promotes tumor cell-induced endothelial necroptosis and metastasis, it was tested whether ligand binding to DR6 is required for these effects. To compete with the endogenous DR6 for a putative ligand, the DR6 ectodomain fused to an Fc fragment (DR6-Fc) was employed which functions as decoy receptor. Treatment of animals with DR6-Fc was sufficient to reduce endothelial necroptosis and tumor cell extravasation, and these animals also developed less metastases (FIG. 9A-D).

[0215] Example 10: Further functional characterization of the DR6 ligand

[0216] A previously identified ligand of DR6 is amyloid precursor protein (APP) (Nikolaev et al., 2009), that is highly expressed in the nervous system (Aydin et al., 2012; Muller and Zheng, 2012) as well as in several cell types including tumor cells (Hansel et al., 2003; Krause et al., 2008; Meng et al., 2001; Takagi et al., 2013; Woods and Padmanabhan, 2013) and can induce programmed cell death (Nikolaev et al., 2009). It was found that tumor cells in which APP mRNA expression levels were reduced to less than 3% using siRNA-mediated knockdown had lost the ability to induce endothelial necroptosis and showed a strongly reduced ability to transmigrate an endothelial cell layer when compared to tumor cells transfected with control-siRNA (FIG. 12A and B).

[0217] After intravenous injection of tumor cells with transiently reduced APP expression (FIG. 11A) into wild-type animals, we found significantly reduced numbers of necroptotic endothelial cells and extravasated tumor cells in the lungs of these animals (FIG. 12C-F). Importantly, tumor cells with strongly reduced APP expression showed normal proliferation, cell survival and basal migratory activity (FIG. 11B-D) but almost completely lost the ability to form metastases (FIG. 12C-F). This shows that APP expressed by tumor cells and endothelial DR6 are required for tumor cell-induced endothelial necroptosis and that this activity promotes tumor cell extravasation and metastasis formation.

Further References

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