Abstract
The present invention relates to an N-terminal fragment of an angiopoietin like 4 (ANGPTL4) polypeptide, or a therapeutically active variant thereof, for use in the treatment of cancer in a subject. Moreover, the present invention provides an agent which increases the amount of an N-terminal fragment of an angiopoietin like 4 (ANGPTL4) polypeptide for use in treating cancer. Further, encompassed by the present invention is a method for identifying a candidate compound for the treatment of cancer.
Claims
1. A method for the treatment of cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an N-terminal fragment of an angiopoietin like 4 (ANGPTL4) polypeptide, or a therapeutically active variant thereof.
2. The method of claim 1, wherein the treatment of cancer is the inhibition of metastasis development.
3. The method of claim 2, wherein the inhibition of metastasis development is the inhibition of metastasis development after surgical removal of a tumor.
4. The method of claim 3, wherein the N-terminal fragment, or therapeutically active variant thereof, is administered perioperatively.
5. The method of claim 3, wherein the N-terminal fragment, or therapeutically active variant thereof, is administered before and/or after surgical removal of the tumor.
6. The method of claim 3, wherein the tumor is a primary tumor and wherein the subject has not developed metastases at the time of the surgical removal of the tumor.
7. The method of claim 1, wherein the N-terminal fragment, or therapeutically active variant thereof, is administered intravenously or intraperitoneally.
8. The method of claim 1, wherein the subject is a human subject and wherein the angiopoietin like 4 (ANGPTL4) polypeptide is human ANGPTL4.
9. The method of claim 1, wherein the N-terminal fragment, or therapeutically active variant thereof, is selected from a) a polypeptide comprising a sequence as shown in SEQ ID NO: 1, SEQ ID NO 2, SEQ ID NO 7, or SEQ ID NO 8, b) a subfragment of the polypeptide of a), wherein the subfragment has a length of at least 50 amino acids, and c) a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to the polypeptide of a) or the subfragment of b).
10. The method of claim 1, wherein the N-terminal fragment, or therapeutically active variant thereof, is capable of i. decreasing the activity of lipoprotein lipase, ii. binding to Syndecan-4, and/or iii. forming oligomers.
11. The method of claim 1, wherein the cancer is selected from melanoma, breast cancer, colorectal cancer, ovarian cancer, renal cancer, gastrointestinal cancer, lung cancer, primary cutaneous lymphomas and hepatocellular carcinoma.
12. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent which increases the amount of an N-terminal fragment of an angiopoietin like 4 (ANGPTL4) polypeptide.
13. A method for identifying a candidate compound for the treatment of cancer, comprising a) determining the amount of an N-terminal fragment of an angiopoietin like 4 (ANGPTL4) polypeptide in a first sample from a subject, wherein said first sample has been obtained from the subject prior contacting the subject with the candidate compound, b) determining the amount of the N-terminal fragment of an angiopoietin like 4 (ANGPTL4) polypeptide in a first sample from a subject, wherein said second sample has been obtained after contacting the subject with the candidate compound, and c) comparing the amount of the N-terminal fragment of an angiopoietin like 4 (ANGPTL4) polypeptide in the second sample to the amount in the first sample, wherein an increased amount in the second sample as compared to the amount in the first sample is indicative for a candidate compound for the treatment of cancer.
14. The method of claim 13, wherein the sample is blood, serum or plasma sample.
15. The method of claim 13, wherein the method is an in vitro method.
Description
[0172] The Figures show:
[0173] FIG. 1: Identifying ANGPTL-4 and its cleavage parts in human primary tumors and serum. A Analysis of open source Oncomine data, including 49 studies with 13.139 patients with different tumor entities, identified ANGPTL-4 as one of the most upregulated genes. B Immunohistochemistry staining using a C-terminal-specific antibody in TMAs of lung cancer, breast cancer, SCC and melanoma with the corresponding healthy tissue. C Immunofluorescence staining using an N-terminal-specific antibody in TMAs of colon cancer, SCC and melanoma with the corresponding healthy tissue. D Percentage of tumor samples expressing ANGPTL-4 in TMAs (colon cancer n=30 in all entities, healthy skin n=7, compound nevus n=7, actinic keratosis n=120, SCC n=120, melanoma n=150). E Western blot of human melanoma tissue with C-terminal specific antibody. F Analysis of Western blots detecting flANGPTL-4 or cleavage parts in melanoma (n=6) and colon cancer (n=9) G Western blot of human melanoma tissue with N-terminal specific antibody. H Analysis of Western blots detecting flANGPTL-4 or cleavage parts in melanoma (n=6), colon cancer (n=9), breast cancer (n=9) and HCC (n=9). I Western blot of human melanoma patient serum (n=13) using a C-terminal specific antibody. J Western blot of human melanoma patient serum (n=13) using a N-terminal specific antibody.
[0174] FIG. 2: ANGPTL-4 and its cleavage parts in primary tumor models. A Macroscopic image and IF for CD31 of A375 xenograft primary tumor model with downregulation of ANGPTL-4 (shRNA) compared to control (shcontrol). B Microvessel density of A375 primary tumors downregulating ANGPTL-4 (sh1 and sh2; n=6-7) compared to control (shcontrol; n=5)). C Macroscopic image and IF for CD31 LLC syngenic primary tumor model with upregulation of flANGPTL-4 (LLC Angptl-4) compared to control (LLC Lenticontrol). D Microvessel density of LLC primary tumors upregulating ANGPTL-4 (LLC Angptl-4; n=9) compared to control (LLC Lenticontrol; n=13). E Ex vivo aortic ring assay in which rings were stimulated with either PBS (n=12), VEGF low (n=12), Angptl-4 (n=14) or the combination of VEGF low and Angptl-4 (n=17). F Quantification as number of sprouting vessels per aortic ring. G Representative pictures of sprouting assay; HUVEC were stimulated with either PBS, VEGF low, Angptl-4 or the combination of VEGF low and Angptl-4. Additionally, inhibition with integrin αvβ3-antibody. H Quantification of sprout number per spheroid in all conditions. I B16F10 primary tumor model with tumor cells lentivirally overexpressing flANGPTL-4, nANGPTL-4, cANGPTL-4 in comparison to control tumors. Representative images of CD31 vessel staining in primary tumors. J Quantification of vascularized area in primary tumors. K Cornea pocket assay using flANGPTL-4 (n=9), cANGPTL-4 (n=4), nANGPTL-4 (n=4) in comparison to control (PBS; n=4) and all conditions in combination with VEGF low (n=4; flANGPTL-4 n=8, cANGPTL-4 n=5; nANGPTL-4 n=5), representative images of new vessels infiltrating the cornea upon stimulation. L Quantification of vascularized area in the cornea with or without subcritical dose of VEGF (VEGF low). Data are presented as mean±SD; *p<0.05; **p<0.01, ***p<0,001 by 2-tailed Student's t-Test for in vitro and ex vivo data and as mean±SEM; *p<0.05; **p<0.01, ***p<0,001 by 2-tailed Mann-Whitney U Test for in vivo experiments
[0175] FIG. 3: Analyzing the role of different cleavage fragments of ANGPTL-4 in metastasis models. A Schematic of the modified B16F10 experimental metastasis model. Mice were injected with 1×10.sup.6 B16 overexpressing or control cells s.c. and five days later with 2.5×10.sup.5B16F10 WT cells i.v. All mice were sacrificed at day 19. B Quantification of lung metastasis. C Representative images of metastatic lungs. D Mass spectrometric analysis to assess the flANGPTL-4/nANGPTL-4 ratio in control and flANGPTL-4 mice (control group n=9 mice, flANGPTL-4 group n=5 mice). E PET-CT of three representative mice from the LLC spontaneous metastasis model. F Tumor weights at time of resection of the primary tumor (all groups n=10 mice). G Survival curve (based on animal protocol termination criteria) of mice with tumors overexpressing control construct, flANGPTL-4 or nANGPTL-4 (all groups n=10 mice). H Schematic of the B16F10 WT experimental metastasis model and treatment with recombinant mouse nANGPTL-4. I Quantification of lung metastasis and representative images of each group (PBS group n=11 mice, nANGPTL-4 group n=9 mice). J Schematic of the LLC spontaneous metastasis model and perioperative treatment with recombinant mouse nANGPTL-4. K Survival curve of LLC experiment (PBS group n=9 mice, nANGPTL-4 group n=9 mice). Data are presented as mean±SEM; *p<0.05; **p<0.01, ***p<0,001 by 2-tailed Mann-Whitney U Test.
[0176] FIG. 4: Effect of nANGPTL-4 on tumor and endothelial cells. A Annexin V staining of overexpressing B16F10 tumor cells and quantitative FACS analysis of live cells (n=4 in all conditions). B Schematic of the treatment of mice used for microarray analysis (n=4 mice per group). C Geneset enrichment analysis of treated mouse lung endothelial cells [denatured nANGPTL-4 compared to functional nANGPTL-4]. D Confirmation of nANGPTL-4 effect on human endothelial cells (HUVEC) stimulated with PMA, mouse nANGPTL-4 or human nANGPTL-4 or both (n=2) E Electron microscopic pictures of HUVEC stimulated with PBS or human nANGPTL-4. Data are presented as mean±SD; *p<0.05; **p<0.01, ***p<0,001 by 2-tailed Student's t-Test
[0177] FIG. 5: nANGPTL-4 inhibits metastasis via SDC4-dependent sprouting angiogenesis. A Microarray of sorted mouse lung endothelial cells stimulted with either denatured nANGPTL-4 or functional nANGPTL-4. B Cornea pocket assay using control, flANGPTL-4 and its cleavage products with or w/o VEGFlow. C Sprouting Data of HUVEC stimulated with human recombinant protein (n=3-5). D HUVEC stimulated with nANGPTL-4 and expression levels of Pfkfb3 and SDC4 were assessed via RT-qPCR.
EXAMPLES
[0178] The invention will be merely illustrated by the following Examples. The said Examples shall, whatsoever, not be construed in a manner limiting the scope of the invention.
Example 1: Identifying ANGPTL-4 and its Cleavage Parts in Human Primary Tumors and Serum
[0179] High ANGPTL-4 protein expression revealed in a meta-analyses of 49 different studies with 15 different tumor entities with 13.139 patient samples with a dichotomous read-out for twenty angiogenic factors indicated in most studies (35/49) a poor outcome for the patients (FIG. 1A). Investigating the expression patterns of ANGPTL-4 in different human tumors, we screened its expression in Tissue Microarrays (TMA) of melanoma, cutaneous squamous cell carcinoma (cSCC), colon cancer, breast cancer and lung cancer and compared it with healthy control tissue. Using immunofluorescence (IF) and immunohistochemistry (IHC) with different antibodies, we observed widespread, elevated levels of ANGPTL-4 expression in the majority of the tumor samples compared to the corresponding healthy tissues (FIG. 1A-C). Comparative clinical studies of the IF studies in melanoma, cSCC and colon cancer samples showed an increase in ANGPTL-4 expression levels with tumor progression (FIG. 1D). Extending experiments were aimed to assess which form of ANGPTL-4 (flANGPTL-4, cANGPTL-4 or nANGPTL4) is mainly found in the tumor tissues. To address this question, we performed Western blot analysis using human anti-c- and anti-n-specific ANGPTL-4 antibodies on melanoma, colon cancer, hepatocellular carcinoma (HCC), as well as breast cancer tissues (exemplary of melanoma FIGS. 1E & 1G). flANGPTL-4 was detectable in all samples with the c-specific antibody (FIG. 1F) as well as with the n-specific antibody (FIG. 11I). In no samples was only cleaved ANGPTL-4 form detectable. FlANGPTL-4 and the cleavage products (cANGPLT-4 or nANGPTL-4) were expressed in a minority of samples. We further analyzed the levels of n-, c- and flANGPTL-4 in serum samples of melanoma patients (FIGS. 1 I & 1J, longer exposure time vs. 1I). Mainly nANGPTL-4 was detectable in the serum of the patients. flANGPTL-4 was significantly less compared to nANGPTL-4 (FIG. 1I) and cANGPLT-4 was less than flANGPTL-4 (FIG. 1J).
Example 2: ANGPTL-4 and its Cleavage Parts in Primary Tumor Models
[0180] To study the contribution of ANGPTL-4 in tumor progression, we modified the expression of ANGPTL-4 expression in different established human and mouse cell line models. In a xenograft melanoma mouse model with A375 tumors with intrinsically high levels of ANGPTL-4, loss of ANGPTL-4 expression with a lentiviral approach resulted in reduced vascularity of the tumors (FIG. 2A) with reduced CD31-expressing vessels (FIG. 2B). In a syngeneic lung carcinoma model with LLC tumor cells, increased expression of ANGPLT-4 led to macroscopically better vascularized tumors (FIG. 2C) with increased CD31-expressing vessels in the tumors (FIG. 2D). In order to investigate, how flANGPTL-4 enhances angiogenesis, the aortic ring assay as well the spheroid sprouting assays were performed. FlANGPTL-4 mono-stimulation did not alter angiogenesis in the ex vivo aortic ring assay. Yet, flANGPTL-4 enhanced low dose VEGF-induced angiogenesis (FIGS. 2E and F). Similarly, mono-stimulation of the EC with flANGPTL-4 did not alter sprouting angiogenesis in the in vitro sprouting assay. FlANGPTL-4 enhanced VEGF low dose sprouting angiogenesis in the spheroid sprouting assay, too. Pretreatment of the EC with an integrin αvß3 blocking antibody diminished this effect (FIGS. 2G and H), suggesting that the pro-angiogenic effect of flANGPTL-4 requires VEGF and is integrin-dependent. Next, we investigated the role of the different ANGPTL-4 forms (nANGPTL-4, cANGPTL-4 and flANGPTL4) in another syngeneic mouse melanoma tumor model. Growth of the different primary tumors was observed and plotted as growth curve (data not shown). Here, we observed a strong growth advantage of B16F10 cells overexpressing cANGPTL-4 in early stages of tumor induction. MVD analyses, which were assessed in tumors of the same size, showed a significant increase in cells overexpressing flANGPTL-4 and cANGPTL-4 (FIGS. 2I and J). To investigate the relevance of the cleavage parts (cANGPTL-4 and nANGPTL-4) in another angiogenesis model, cornea pocket assays were performed. The cornea is an avascular tissue in the eye which is nourished by the tear fluid. Only the limbus is vascularized, from which in pathological conditions vessels can grow out. Inserting a small pellet containing the pro- or antiangiogenic agent into a pre-made pocket in the cornea shows outgrowing microvessels within one week of exposure. All vessels growing inside the cornea are newly formed vessels from the pre-existing limbus vessels. Corneas were harvested and stained for CD31 in order to analyze the microvessel density (FIGS. 2L and K). We observed a strong increase in MVD in this experiment when stimulated with fl- or cANGPTL-4. nANGPTL-4 alone did not show any angiogenic effect compared to control, but reduced VEGF-induced sprouting in this assay.
Example 3: Analyzing the Role of Different Cleavage Fragments of ANGPTL-4 in Metastasis Models
[0181] So far, most mouse metastasis models do not allow to decipher the effect of primary tumor cell secreted factors on metastasis as metastasis and primary tumors are not present in the mouse at the same timepoints. Therefore, we developed a new melanoma mouse model which allowed to study primary tumor and metastasis formation at the same time. First, we injected 1×10.sup.6 B16F10 tumor cells subcutaneously into the flank of C57Bl/6 mice. Some of these tumor cells were lentivirally modified to overexpressed flANGPTL-4, cANGPTL4 or nANGPTL-4. Five days later, when initial macroscopic tumor formation was visible, intravasation of tumor cells was mimicked via intravenous injection of 2.5×10.sup.5 wildtype B16F10 tumor cells. Wildtype cells were used for tail vein injection to exclude effects of ANGPTL-4 secreted by the circulating tumor cells. Mice were sacrificed 14 days after tail vein injection (total day 19) and the number of lung metastases was analyzed (FIG. 3A). When analyzing the lungs, we observed a significant reduction of lung metastasis when the primary tumors overexpressed flANGPTL-4 or nANGPTL-4. In contrast, overexpression of cANGPTL-4 by the primary tumor cells increased the number of lung metastasis compared to control (FIGS. 3B and C). When using this model, a substantial deviation between the mice occurred, e.g. in control group ranging from 1 to over 100 lung metastases. Considering the substantial deviation, we established a mass spectrometry analysis for murine ANGPTL-4 from mouse serum to check the levels of ANGPTL-4 in those mice. The ratio between the full-length protein and the N-Terminal part was determined in this analysis. Here we discovered that the nANGPTL-4/fl-ANGPTL-4 ratio inversely correlated with the number of metastasis (R=0.3972, P=0.0157). Mice from the control group (black symbols) and mice from the flANGPTL-4 group (red symbols) were included. The effect was detectable in both groups (FIG. 3D). Next, a lung carcinoma metastasis model was used in which lung metastasis form after primary tumor resection. To have comparable groups, we injected in all mice 1×10.sup.6 LLC tumor cells, either with a control construct or fl- or nANGPTL-4 overexpression and resected the tumors at the same size (FIG. 3E). Following resection, the mice underwent CT or PET-CT scans weekly for three consecutive weeks. Representative PET-CT scans are shown in FIG. 3F, indicating the location of the metastasis (red circles). All mice in the control group developed lung and/or liver metastasis. In flANGPTL-4, 2 out of 3 mice had metastasis and in nANGPTL-4 mice no metastases were detectable three weeks after resection. CT/PET-CT scans were done for three mice per group. All the mice were killed when predefined termination criteria were fulfilled (FIG. 3G). This curve shows a significant survival benefit for the mice overexpressing nANGPTL-4 in their tumors. To evaluate therapeutic potential of nANGPTL-4, we produced the murine N-Terminal fragment as recombinant protein. Mice were treated two days before intravenous tumor cell injection with 7 μg/mouse of protein. Thereafter, 2.5×10.sup.5 B16F10 tumor cells were injected into the tail vein. Following tumor cell injection, the mice were treated twice weekly with the above-mentioned amount of protein per injection (FIG. 3H). The mice were sacrificed two weeks after tail vein injection and lung metastases were evaluated. Here we saw a significant reduction of lung metastases upon treatment with recombinant murine nANGPTL-4 (FIG. 3I). Additionally, we tested the effect of nANGPTL4 on spontaneous metastasis using the LLC model. After having injected the tumor cells, we treated the mice with either PBS or recombinant mouse nANGPTL-4. The first injection was administered one day before resection of the primary tumor and three doses were given in an adjuvant manner after resection (FIG. 3J). Again, the readout of this experiment was the ‘survival’ of the mice when they had to be sacrificed due to predefined termination criteria. Treated mice had a median survival of 32 days in the PBS group and 37.5 days in the group treated with nANGPTL-4. Nonetheless, 38 days after tumor resection the curves of the two groups merged and were not significantly different anymore (FIG. 3K). Taken together, these data suggest that infusion of nANGPTL-4 may be a viable therapeutic option against aggressive metastatic cancers.
Example 4: Effect of nANGPTL-4 on Tumor and Endothelial Cells
[0182] Next, we set out experiments to address the question, how nANGPTL-4 mechanistically regulates metastasis formation. ANGPTL-4 is a key regulator of anoikis. We therefore checked if alteration of ANGPTL-4 expression (flANGPTL-4 or its fragments) affected anoikis in vitro. To do so, we plated B16F10 overexpressing cells under anoikis conditions for 48 h, stained the cells with Annexin V and implemented a quantitative FACS analysis to test the number of living cells. We observed in this experiment a significant reduction of living cells (Annexin V-FxCycle) in B16F10 overexpressing nANGPTL-4, (FIG. 4A), indicating that these cells have a lesser chance to survive in the circulation compared to control or flANGPTL-4 overexpressing cells. Extending experiments were performed to unravel if flANGPTL-4, nANGPTL-4 or c-ANGPTL-4 alter angiogenesis as an important step for macroscopic metastatic colony formation. A significant decrease of MVD was observed in a model with LLC tumors and two consecutive treatments with recombinant murine nANGPTL-4. Aiming to decipher the molecular mechanism for the observed antiangiogenic effect, microarrays of FACS-isolated EC, which had been treated with murine nANGPTL-4 or heat-inactivated murine n-ANGPTL-4 were performed (FIG. 4B). Analysis of the array revealed a strong downregulation of Pfkfb3, a potent regulator of glycolysis in endothelial cells (FIG. 4C). Endothelial cells rely on glycolysis rather than on oxidative phosphorylation for ATP production. The loss of the glycolytic activator PFKFB3 in endothelial cells impaired vessel formation and sprouting angiogenesis (Mandard et al., Cantelmo et al.). Next, we aimed to confirm the downregulation of Pfkfb3 in vitro upon stimulation with nANGPTL-4 in HUVEC cells in vitro. To further expose the cells to stress conditions as it may occur physiologically under metastatic conditions, the cells were stimulated after nANGPTL-4 treatment with Phorbol 12-myristate 13-acetate (PMA) for 1 h. Upon stimulation with PMA, we observed a substantial upregulation of Pfkfb3. When stimulating the cells with either recombinant human or mouse nANGPTL-4, we identified a reduction of Pfkfb3 expression (FIG. 4D). In order to investigate the effect of nANGPTL4 on the microanatomy of HUVEC, we took electron microscopic images of stimulated cells. Here we observed an increased number of microvesicular bodies, suggesting that the cells underwent a potent cell stress when stimulated with nANGPTL-4 (FIG. 4E).
Example 5: nANGPTL-4 Inhibits Metastasis Via SDC4-Dependent Sprouting Angiogenesis
[0183] Aiming to identify the molecular signaling targets of nANGPTL-4, mice were treated with nANGPTL-4 and compared with mice treated with heat-inactivated nANGPTL-4 (FIG. 5A). EC of both groups were isolated out of the lungs and compared with microarray technique (FIG. 5A). Among the top regulated genes, downregulation of PFKFB3 was observed. PFKFB3 is a key molecule for sprouting angiogenesis of EC (de Bock et al., Cell 2013). Therefore, we studied the effect of nANGPTL-4 and other variants of ANGPTL-4 in the cornea pocket assay in vivo (FIG. 5B). Here, treatment with nANGPTL-4 only, did not affect sprouting angiogenesis (compare PBS vs. NT alone). In contrast, the C-fragment of ANGPTL-4 enhanced sprouting angiogenesis significantly (compare PBS vs. CT alone). Strikingly, nANGPTL-4 inhibited VEGF-induced angiogenesis (compare VEGF low vs NT+VEGF). Similar results were obtained in the spheroid sprouting assay (FIG. 5C). While mono-stimulation with nANGPTL-4 had no effect on angiogenesis (compare unstim. vs. NT), nANGPTL-4 inhibited VEGF-induced sprouting angiogenesis (compare VEGF high vs VEGF high+NT). C- as well as FL-ANGTL-4 enhanced angiogenesis (FIG. 5C and data not included). This experiments show for the first time, that ANGPTL4 can have two opposing functions. It can be pro- or antiangiogenic. Interestingly, Cazes et al. observed an anti-angiogenic effect in their spheroid sprouting experiments (Cazes et al., 2006). Apparently, the full-length protein was tested. In contrast, this effect was not observed for the full-length protein in the studies underlying the present invention.
[0184] Recently, syndecans 2 and 4 (SDC2 and SDC4) were identified as the receptors of nANGPTL-4-induced signaling in a systematic screen using a pull-down and mass spectrometry approach (Kirsch et al., Dev. Cell 2017). We isolated endothelial cells (EC) of metastatic lung and compared it with non-metastatic lung. Here we observed that expression of SDC4 but not SDC2 was significantly upregulated in the EC of metastatic lungs (data not shown). Extending experiments with siRNA, proove that treatment of EC with nANGPTL-4 reduces expression PFKFB3, a master regulator of sprouting angiogenesis (de Bock et al., Cell 2013). This inhibitory effect is lost, when expression of SDC4 is downregulated with siRNA (FIG. 5D).
CONCLUSIONS
[0185] Angiopoietin-like 4 (ANGPTL-4) is a secreted glycoprotein for which conflicting pro-tumorigenic and antitumorigenic functions have been reported. We observed that ANGPTL-4 is proteolytically cleaved upon secretion into two fragments, a C-terminal and an N-terminal fragment. The uncleaved variant of ANGPTL-4 was detectable in every single tumor sample of various cancer entities (melanoma, breast cancer, colon cancer and hepatocellular carcinomas). In these tissues the fragments of ANGPTL-4 were only detectable in a minority of the samples. Using a broad array of tumor models, we discovered that nANGPTL-4 did not affect primary tumor growth, but profoundly inhibited distant site metastasis formation and enhanced overall survival in mice. Therefore, a therapeutic concept of surgical resection of the primary tumor combined with nANGPTL-4 treatment thereafter is promising. These findings are of major importance as i.) it has a direct therapeutic value using nANGPTL-4 as new anti-metastatic drug, ii.) it revitalize old concepts of primary tumors suppressing metastases through the systemic release of metastasis-inhibiting cytokines, and iii.) it is the first protein with a pro- and anti-(angiogenic) effect and therefore has more than the general on-/off-phenomena which can be found in different proteins.
[0186] In summary, the results show that nANGPTL-4 has a strong therapeutic potential to inhibit metastasis. By specifically suppressing metastatic outgrowth, the systemic disease cancer can be limited to a local tumor, for which various treatment options are already established.
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