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• W2589610792 abstract "•Tumor ECs frequently have activated Notch1 and this correlates with poor prognosis•Sustained EC Notch1 activity promotes TC migration across the vessel wall•Endothelial Notch1 hyperactivation promotes neutrophil infiltration and metastasis•Neutrophil infiltration and metastasis depend on Notch1-induced VCAM1 expression Endothelial cells (ECs) provide angiocrine factors orchestrating tumor progression. Here, we show that activated Notch1 receptors (N1ICD) are frequently observed in ECs of human carcinomas and melanoma, and in ECs of the pre-metastatic niche in mice. EC N1ICD expression in melanoma correlated with shorter progression-free survival. Sustained N1ICD activity induced EC senescence, expression of chemokines and the adhesion molecule VCAM1. This promoted neutrophil infiltration, tumor cell (TC) adhesion to the endothelium, intravasation, lung colonization, and postsurgical metastasis. Thus, sustained vascular Notch signaling facilitates metastasis by generating a senescent, pro-inflammatory endothelium. Consequently, treatment with Notch1 or VCAM1-blocking antibodies prevented Notch-driven metastasis, and genetic ablation of EC Notch signaling inhibited peritoneal neutrophil infiltration in an ovarian carcinoma mouse model. Endothelial cells (ECs) provide angiocrine factors orchestrating tumor progression. Here, we show that activated Notch1 receptors (N1ICD) are frequently observed in ECs of human carcinomas and melanoma, and in ECs of the pre-metastatic niche in mice. EC N1ICD expression in melanoma correlated with shorter progression-free survival. Sustained N1ICD activity induced EC senescence, expression of chemokines and the adhesion molecule VCAM1. This promoted neutrophil infiltration, tumor cell (TC) adhesion to the endothelium, intravasation, lung colonization, and postsurgical metastasis. Thus, sustained vascular Notch signaling facilitates metastasis by generating a senescent, pro-inflammatory endothelium. Consequently, treatment with Notch1 or VCAM1-blocking antibodies prevented Notch-driven metastasis, and genetic ablation of EC Notch signaling inhibited peritoneal neutrophil infiltration in an ovarian carcinoma mouse model. Endothelial cells (EC) are a major component of the tumor microenvironment. Beside their prominent role to form tumor blood vessels, they provide soluble factors and surface proteins that orchestrate tumor cell (TC) behavior. This study shows that TCs can stimulate EC Notch1 signaling activity. This happens frequently in various human tumor entities and correlates with worsened prognosis. Sustained EC Notch1 activation alters EC morphology and function in a manner that promotes TC transmigration, intravasation as well as homing at distant sites. An essential protein induced by Notch1 signaling in ECs is the vascular adhesion factor VCAM1. Blockade of this surface protein with monoclonal antibodies prevented the effect of Notch1on neutrophil infiltration and metastasis. Vascular endothelial cells (ECs) are a critical component of the tumor microenvironment and the metastatic niche. Beside their prominent role in blood vessel formation, ECs exert instructive roles for organ development (Lammert et al., 2001Lammert E. Cleaver O. Melton D. Induction of pancreatic differentiation by signals from blood vessels.Science. 2001; 294: 564-567Crossref PubMed Scopus (868) Google Scholar, Matsumoto et al., 2001Matsumoto K. Yoshitomi H. Rossant J. Zaret K.S. Liver organogenesis promoted by endothelial cells prior to vascular function.Science. 2001; 294: 559-563Crossref PubMed Scopus (697) Google Scholar, Nolan et al., 2013Nolan D.J. Ginsberg M. Israely E. Palikuqi B. Poulos M.G. James D. Ding B.S. Schachterle W. Liu Y. Rosenwaks Z. et al.Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.Dev. Cell. 2013; 26: 204-219Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar), regeneration (Hu et al., 2014Hu J. Srivastava K. Wieland M. Runge A. Mogler C. Besemfelder E. Terhardt D. Vogel M.J. Cao L. Korn C. et al.Endothelial cell-derived angiopoietin-2 controls liver regeneration as a spatiotemporal rheostat.Science. 2014; 343: 416-419Crossref PubMed Scopus (207) Google Scholar), and tumor progression (Butler et al., 2010Butler J.M. Kobayashi H. Rafii S. Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiocrine factors.Nat. Rev. Cancer. 2010; 10: 138-146Crossref PubMed Scopus (421) Google Scholar), referred to as angiocrine EC functions (Ramasamy et al., 2015Ramasamy S.K. Kusumbe A.P. Adams R.H. Regulation of tissue morphogenesis by endothelial cell-derived signals.Trends Cell Biol. 2015; 25: 148-157Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Tumor vasculature differs morphologically from normal vessels, expresses different signaling molecules, and provides cell surface molecules such as Notch ligands, which support tumor progression (Beck et al., 2011Beck B. Driessens G. Goossens S. Youssef K.K. Kuchnio A. Caauwe A. Sotiropoulou P.A. Loges S. Lapouge G. Candi A. et al.A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours.Nature. 2011; 478: 399-403Crossref PubMed Scopus (349) Google Scholar, Ghajar et al., 2013Ghajar C.M. Peinado H. Mori H. Matei I.R. Evason K.J. Brazier H. Almeida D. Koller A. Hajjar K.A. Stainier D.Y. et al.The perivascular niche regulates breast tumour dormancy.Nat. Cell Biol. 2013; 15: 807-817Crossref PubMed Scopus (747) Google Scholar). Notch signaling is an essential cell communication system during organ development (Roca and Adams, 2007Roca C. Adams R.H. Regulation of vascular morphogenesis by Notch signaling.Genes Dev. 2007; 21: 2511-2524Crossref PubMed Scopus (355) Google Scholar, Zhang et al., 2013Zhang R. Han P. Yang H. Ouyang K. Lee D. Lin Y.F. Ocorr K. Kang G. Chen J. Stainier D.Y. et al.In vivo cardiac reprogramming contributes to zebrafish heart regeneration.Nature. 2013; 498: 497-501Crossref PubMed Scopus (188) Google Scholar), and also plays a very prominent role in oncogenesis (Ntziachristos et al., 2014Ntziachristos P. Lim J.S. Sage J. Aifantis I. From fly wings to targeted cancer therapies: a centennial for notch signaling.Cancer Cell. 2014; 25: 318-334Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Activating Notch signaling mutations are frequently found in malignant tumors; however, the roles of Notch signaling in the tumor stroma are less understood. Notch ligands of the Delta-like (Dll) and Jagged (Jag) families are frequently present on tumor ECs (Kontomanolis et al., 2014Kontomanolis E. Panteliadou M. Giatromanolaki A. Pouliliou S. Efremidou E. Limberis V. Galazios G. Sivridis E. Koukourakis M.I. Delta-like ligand 4 (DLL4) in the plasma and neoplastic tissues from breast cancer patients: correlation with metastasis.Med. Oncol. 2014; 31: 945Crossref PubMed Scopus (29) Google Scholar, Mailhos et al., 2001Mailhos C. Modlich U. Lewis J. Harris A. Bicknell R. Ish-Horowicz D. Delta4, an endothelial specific notch ligand expressed at sites of physiological and tumor angiogenesis.Differentiation. 2001; 69: 135-144Crossref PubMed Scopus (259) Google Scholar, Patel et al., 2005Patel N.S. Li J.L. Generali D. Poulsom R. Cranston D.W. Harris A.L. Up-regulation of delta-like 4 ligand in human tumor vasculature and the role of basal expression in endothelial cell function.Cancer Res. 2005; 65: 8690-8697Crossref PubMed Scopus (314) Google Scholar, Reedijk et al., 2005Reedijk M. Odorcic S. Chang L. Zhang H. Miller N. McCready D.R. Lockwood G. Egan S.E. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival.Cancer Res. 2005; 65: 8530-8537Crossref PubMed Scopus (616) Google Scholar). Independent of their intrinsic roles for coordinating angiogenesis (Potente et al., 2011Potente M. Gerhardt H. Carmeliet P. Basic and therapeutic aspects of angiogenesis.Cell. 2011; 146: 873-887Abstract Full Text Full Text PDF PubMed Scopus (1903) Google Scholar), they can also promote Notch signaling in adjacent tumor cells (TCs). For example, Jag1 on ECs activates Notch2 on B cell lymphoma cells and this enhances TC aggressiveness (Cao et al., 2014Cao Z. Ding B.S. Guo P. Lee S.B. Butler J.M. Casey S.C. Simons M. Tam W. Felsher D.W. Shido K. et al.Angiocrine factors deployed by tumor vascular niche induce B cell lymphoma invasiveness and chemoresistance.Cancer Cell. 2014; 25: 350-365Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Jag1 secreted from tumor ECs promotes the cancer stem cell phenotype of colorectal carcinoma cells (Lu et al., 2013Lu J. Ye X. Fan F. Xia L. Bhattacharya R. Bellister S. Tozzi F. Sceusi E. Zhou Y. Tachibana I. et al.Endothelial cells promote the colorectal cancer stem cell phenotype through a soluble form of Jagged-1.Cancer Cell. 2013; 23: 171-185Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar); juxtacrine EC-to-TC signaling activates Notch1 in glioblastoma cells (Zhu et al., 2011Zhu T.S. Costello M.A. Talsma C.E. Flack C.G. Crowley J.G. Hamm L.L. He X. Hervey-Jumper S.L. Heth J.A. Muraszko K.M. et al.Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells.Cancer Res. 2011; 71: 6061-6072Crossref PubMed Scopus (282) Google Scholar); and ligands expressed on stromal cells activate Notch1 in colon TCs to facilitate metastasis (Sonoshita et al., 2011Sonoshita M. Aoki M. Fuwa H. Aoki K. Hosogi H. Sakai Y. Hashida H. Takabayashi A. Sasaki M. Robine S. et al.Suppression of colon cancer metastasis by Aes through inhibition of Notch signaling.Cancer Cell. 2011; 19: 125-137Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Notch ligands can also be secreted by TCs via exosomes (Sheldon et al., 2010Sheldon H. Heikamp E. Turley H. Dragovic R. Thomas P. Oon C.E. Leek R. Edelmann M. Kessler B. Sainson R.C. et al.New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes.Blood. 2010; 116: 2385-2394Crossref PubMed Scopus (300) Google Scholar) and be incorporated in EC membranes at distant sites to either activate or inhibit Notch signaling across long distances (Sharghi-Namini et al., 2014Sharghi-Namini S. Tan E. Ong L.L. Ge R. Asada H.H. Dll4-containing exosomes induce capillary sprout retraction in a 3D microenvironment.Scientific Rep. 2014; 4: 4031Crossref PubMed Scopus (77) Google Scholar, Sheldon et al., 2010Sheldon H. Heikamp E. Turley H. Dragovic R. Thomas P. Oon C.E. Leek R. Edelmann M. Kessler B. Sainson R.C. et al.New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes.Blood. 2010; 116: 2385-2394Crossref PubMed Scopus (300) Google Scholar). Ligand binding leads to Notch receptor cleavage. Following its cleavage, the Notch intracellular domain (NICD) translocates to the nucleus and forms an activating complex with the DNA binding protein Rbp-jk (Potente et al., 2011Potente M. Gerhardt H. Carmeliet P. Basic and therapeutic aspects of angiogenesis.Cell. 2011; 146: 873-887Abstract Full Text Full Text PDF PubMed Scopus (1903) Google Scholar). In mice, elevated Notch1 receptor activity was observed in ECs of atherosclerotic plaques (Liu et al., 2012Liu Z.J. Tan Y. Beecham G.W. Seo D.M. Tian R. Li Y. Vazquez-Padron R.I. Pericak-Vance M. Vance J.M. Goldschmidt-Clermont P.J. et al.Notch activation induces endothelial cell senescence and pro-inflammatory response: implication of Notch signaling in atherosclerosis.Atherosclerosis. 2012; 225: 296-303Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In cultured ECs, forced N1ICD expression impaired barrier functions (Venkatesh et al., 2011Venkatesh D. Fredette N. Rostama B. Tang Y. Vary C.P. Liaw L. Urs S. RhoA-mediated signaling in Notch-induced senescence-like growth arrest and endothelial barrier dysfunction.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 876-882Crossref PubMed Scopus (52) Google Scholar), induced EC quiescence (Rostama et al., 2015Rostama B. Turner J.E. Seavey G.T. Norton C.R. Gridley T. Vary C.P. Liaw L. DLL4/Notch1 and BMP9 interdependent signaling induces human endothelial cell quiescence via P27KIP1 and Thrombospondin-1.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 2626-2637Crossref PubMed Scopus (41) Google Scholar), and increased transcription of pro-inflammatory cytokines (Liu et al., 2012Liu Z.J. Tan Y. Beecham G.W. Seo D.M. Tian R. Li Y. Vazquez-Padron R.I. Pericak-Vance M. Vance J.M. Goldschmidt-Clermont P.J. et al.Notch activation induces endothelial cell senescence and pro-inflammatory response: implication of Notch signaling in atherosclerosis.Atherosclerosis. 2012; 225: 296-303Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The latter is particularly interesting, as studies have reported that an inflamed endothelium promotes adhesion with TCs (Giavazzi et al., 1993Giavazzi R. Foppolo M. Dossi R. Remuzzi A. Rolling and adhesion of human tumor cells on vascular endothelium under physiological flow conditions.J. Clin. Invest. 1993; 92: 3038-3044Crossref PubMed Scopus (193) Google Scholar, McDonald et al., 2009McDonald B. Spicer J. Giannais B. Fallavollita L. Brodt P. Ferri L.E. Systemic inflammation increases cancer cell adhesion to hepatic sinusoids by neutrophil mediated mechanisms.Int. J. Cancer. 2009; 125: 1298-1305Crossref PubMed Scopus (169) Google Scholar) and opening of EC junctions (Kumar et al., 2009Kumar P. Shen Q. Pivetti C.D. Lee E.S. Wu M.H. Yuan S.Y. Molecular mechanisms of endothelial hyperpermeability: implications in inflammation.Expert Rev. Mol. Med. 2009; 11: e19Crossref PubMed Scopus (320) Google Scholar). The passage of TCs across the capillary wall is (at least in part) related to leukocyte diapedesis (Reymond et al., 2013Reymond N. d'Agua B.B. Ridley A.J. Crossing the endothelial barrier during metastasis.Nat. Rev. Cancer. 2013; 13: 858-870Crossref PubMed Scopus (556) Google Scholar). As such, Notch activation in mature vessels could potentially generate a pro-metastatic tumor microenvironment. To test if EC Notch activation would be involved in tumor progression, we first determined the levels of active Notch1 protein (N1ICD) in blood vessels of non-tumorous human tissue samples. N1ICD expression was occasionally detected in microvascular ECs of lung, skin, ovary, breast, colon, bladder, stomach, uterus, prostate, lymph node, liver, pancreas, and testis (Figures S1A and 1A ). However, EC N1ICD expression was very robust in lung adenocarcinoma (13 of 20 samples, 65%), serous ovarian carcinoma (19 of 20 samples, 95%), colorectal carcinoma (241 of 395 samples, 61%, versus 92 of 390 control samples of the same patients, 23.5%), and breast carcinoma (38 of 52 samples, 73%) (Figures S1B, S1C, and 1A). Robust EC N1ICD expression was also detected in melanoma (17 of 19 samples, 89.5%) and melanoma metastases (17 of 34 samples, 50%) (Figure 1B). Correlation with clinical data revealed that, in breast cancer, the rate of N1ICD-positive versus negative tumor ECs (80% ± 10.3% versus 50% ± 12.5%) was higher in patients with positive sentinel lymph nodes (Figure S1D). In melanoma, EC N1ICD expression significantly correlated with higher rates of metastasis (stage IV tumors) and shorter progression-free survival compared with patients with low EC N1ICD expression (Figures 1C and 1D). It is not known what activates Notch signaling in tumor ECs. However, TCs frequently express Notch ligands (Guilmeau et al., 2010Guilmeau S. Flandez M. Mariadason J.M. Augenlicht L.H. Heterogeneity of Jagged1 expression in human and mouse intestinal tumors: implications for targeting Notch signaling.Oncogene. 2010; 29: 992-1002Crossref PubMed Scopus (38) Google Scholar, Leong et al., 2007Leong K.G. Niessen K. Kulic I. Raouf A. Eaves C. Pollet I. Karsan A. Jagged1-mediated Notch activation induces epithelial-to-mesenchymal transition through Slug-induced repression of E-cadherin.J. Exp. Med. 2007; 204: 2935-2948Crossref PubMed Scopus (393) Google Scholar, Mullendore et al., 2009Mullendore M.E. Koorstra J.B. Li Y.M. Offerhaus G.J. Fan X. Henderson C.M. Matsui W. Eberhart C.G. Maitra A. Feldmann G. Ligand-dependent Notch signaling is involved in tumor initiation and tumor maintenance in pancreatic cancer.Clin. Cancer Res. 2009; 15: 2291-2301Crossref PubMed Scopus (151) Google Scholar, Reedijk et al., 2005Reedijk M. Odorcic S. Chang L. Zhang H. Miller N. McCready D.R. Lockwood G. Egan S.E. High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival.Cancer Res. 2005; 65: 8530-8537Crossref PubMed Scopus (616) Google Scholar, Santagata et al., 2004Santagata S. Demichelis F. Riva A. Varambally S. Hofer M.D. Kutok J.L. Kim R. Tang J. Montie J.E. Chinnaiyan A.M. et al.JAGGED1 expression is associated with prostate cancer metastasis and recurrence.Cancer Res. 2004; 64: 6854-6857Crossref PubMed Scopus (291) Google Scholar), and this might trigger Notch receptor activation in adjacent stromal cells (Li et al., 2007Li J.L. Sainson R.C. Shi W. Leek R. Harrington L.S. Preusser M. Biswas S. Turley H. Heikamp E. Hainfellner J.A. Harris A.L. Delta-like 4 Notch ligand regulates tumor angiogenesis, improves tumor vascular function, and promotes tumor growth in vivo.Cancer Res. 2007; 67: 11244-11253Crossref PubMed Scopus (267) Google Scholar, Sethi et al., 2011Sethi N. Dai X. Winter C.G. Kang Y. Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.Cancer Cell. 2011; 19: 192-205Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). In cell culture, Lewis lung carcinoma (LLC) and B1F10 melanoma cells, which express the Notch ligands Dll4 and Jag1 (Figure 2A), induced higher Notch signaling activity in co-cultured ECs as indicated by increased luciferase activity of a Notch reporter. This was blocked by the gamma-secretase inhibitor DAPT, which prevents Notch receptor activation (Figure 2B). In addition, LLC and B16F10 cells also induced Notch receptor activation in co-cultured Notch-reporter CHO cells (Sprinzak et al., 2010Sprinzak D. Lakhanpal A. Lebon L. Santat L.A. Fontes M.E. Anderson G.A. Garcia-Ojalvo J. Elowitz M.B. Cis-interactions between Notch and Delta generate mutually exclusive signalling states.Nature. 2010; 465: 86-90Crossref PubMed Scopus (434) Google Scholar) at sites of direct cell-cell contacts (Figures S2A and S2B). Furthermore, tumors and metastatic niches are frequently infiltrated by immune cells, which could also interact with EC Notch receptors when crossing the vessel wall. To test Notch ligand expression on immune cells, wild-type C57BL/6 mice were intravenously injected with LLC or B16F10 TCs, which infiltrate the lungs. Sorted myeloid cells, but not lymphocytes, from lung cell suspensions strongly expressed the Notch ligand Jag1 on their surface (Figure S2C). Immunohistochemistry of lung sections showed that there was much stronger EC N1ICD expression in the peri-tumoral area compared with more distant healthy lung tissue (Figure 2C). This suggests that both TCs and infiltrated myeloid cells can activate endothelial Notch receptors via direct cell-cell contacts. Next, we analyzed if growth of a solid tumor could alter EC Notch1 activity at distant sites. Such regulation has been shown to occur, e.g., by exosomes carrying Notch ligands (Sharghi-Namini et al., 2014Sharghi-Namini S. Tan E. Ong L.L. Ge R. Asada H.H. Dll4-containing exosomes induce capillary sprout retraction in a 3D microenvironment.Scientific Rep. 2014; 4: 4031Crossref PubMed Scopus (77) Google Scholar, Sheldon et al., 2010Sheldon H. Heikamp E. Turley H. Dragovic R. Thomas P. Oon C.E. Leek R. Edelmann M. Kessler B. Sainson R.C. et al.New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes.Blood. 2010; 116: 2385-2394Crossref PubMed Scopus (300) Google Scholar). LLC or B16F10 cells were subcutaneously injected in the flanks of wild-type C57BL/6 mice. Lungs were isolated 2 weeks later. Importantly, at this time point no tumor foci were detectable by histological analysis. Immunostaining of lung sections revealed significantly higher rates of N1ICD positivity in lungs of tumor-bearing mice compared with non-tumorous controls (Figures 2D and 2E). To study the consequences of endothelial Notch1 receptor activation at the site of the primary tumor and in the potential metastatic site in an animal model, we used transgenic ecN1ICD mice (Ramasamy et al., 2014Ramasamy S.K. Kusumbe A.P. Wang L. Adams R.H. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone.Nature. 2014; 507: 376-380Crossref PubMed Scopus (567) Google Scholar). In these mice, the expression of Notch1-ICD inserted into the Rosa26 locus (Murtaugh et al., 2003Murtaugh L.C. Stanger B.Z. Kwan K.M. Melton D.A. Notch signaling controls multiple steps of pancreatic differentiation.Proc. Natl. Acad. Sci. USA. 2003; 100: 14920-14925Crossref PubMed Scopus (610) Google Scholar) is induced by CreERT2-recombinase upon tamoxifen injection. The recombinase is expressed under the control of the EC-specific Chd5 (VE-cadherin) promoter in all ECs (Benedito et al., 2012Benedito R. Rocha S.F. Woeste M. Zamykal M. Radtke F. Casanovas O. Duarte A. Pytowski B. Adams R.H. Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling.Nature. 2012; 484: 110-114Crossref PubMed Scopus (271) Google Scholar). To avoid interference with physiological angiogenesis in the postnatal and juvenile growth period, gene recombination was induced in 8- to 12-week-old mice. Cre-negative littermates and/or Cre-positive; Rosa26 wild-type mice injected with tamoxifen served as control. It is important to note that ecN1ICD mice showed only a very moderate overexpression of Notch target genes (Hes1 protein or Hey2 and Nrarp mRNAs up to 3-fold) in freshly isolated ECs (Figures S3A and S3B). Also, these mice did not develop macroscopically visible arteriovenous malformations within 8 weeks after gene recombination. This had been observed in a transgenic mouse model with strong N1ICD overexpression in the early perinatal period (Carlson et al., 2005Carlson T.R. Yan Y. Wu X. Lam M.T. Tang G.L. Beverly L.J. Messina L.M. Capobianco A.J. Werb Z. Wang R. Endothelial expression of constitutively active Notch4 elicits reversible arteriovenous malformations in adult mice.Proc. Natl. Acad. Sci. USA. 2005; 102: 9884-9889Crossref PubMed Scopus (155) Google Scholar), during which time point vascular remodeling occurs throughout the body. In a first approach, LLC cells were subcutaneously grafted 21 days after EC-specific N1ICD induction. Tumors were slightly smaller in ecN1ICD mice compared with controls and revealed decreased microvessel density with significantly enhanced mural cell coverage (Figures 3A–3C , day 21) as expected (Trindade et al., 2008Trindade A. Kumar S.R. Scehnet J.S. Lopes-da-Costa L. Becker J. Jiang W. Liu R. Gill P.S. Duarte A. Overexpression of delta-like 4 induces arterialization and attenuates vessel formation in developing mouse embryos.Blood. 2008; 112: 1720-1729Crossref PubMed Scopus (106) Google Scholar). In a second approach, TCs were injected 7 days after gene recombination. Growth kinetics of the primary tumors, microvessel density, vessel coverage with mural cells (Figures 3A–3C; day 7), Glut-1 expression as a marker for hypoxic tissue, and extravasation of fibrinogen as a marker of vessel leakiness were very similar in ecN1ICD mice compared with controls (Figures S3C and S3D). Similar data were observed with B16F10 tumors (Figures S4A–S4E). However, LLC tumors grown in ecN1ICD mice had a marked increase of infiltrated CD45+ immune cells compared with tumors from Cre-negative control animals (Figure S5A). In particular, we detected a stronger infiltration of the CD11b+/CD11c−/F4/80− population, but not macrophages (CD11b+/F4/80+), into the tumor mass (data not shown). These cells were CD11b+/Ly6G+, indicating that these infiltrated cells are neutrophils (Figure 3D). The increase of neutrophil numbers in primary tumors was associated with increased neutrophil numbers in blood (Figure 3E) and in lungs of ecN1ICD mice compared with controls 2 weeks after subcutaneous (s.c.) tumor implantation (Figure 3F). Notably, this difference was only detected in tumor-bearing mice, but not under basal conditions (Figures 3E and 3F). To test if EC N1ICD expression would also increase TC intravasation we quantified circulating TCs in the blood of ecN1ICD mice with primary LLC tumors (2 weeks after implantation). Significantly more circulating TCs were detected by flow cytometry in whole blood taken from in ecN1ICD mice compared with controls (Figure 3G). Since growth of the primary tumor is rate-limiting and precludes analyses of metastasis in this model, we established a postsurgical metastasis model. Two weeks after subcutaneous TC implantation, LLCs were completely resected and postsurgical lung metastasis was evaluated 3 weeks later. Although primary tumor size, microvessel density and vessel coverage was similar between groups (Figures S5B–S5D), the number of metastatic foci and the overall area of lung metastases were strongly increased in ecN1ICD mice (Figures 3H and 3I). This clearly indicates that sustained endothelial Notch signaling facilitates TC spreading to distant sites. Peritoneal injection of serous ovarian cancer ID8 cells is an established model for the study of cancer-associated inflammation. Recruitment of myeloid cells into the peritoneal fluid strongly contributes to tumor progression mainly through suppression of T lymphocytes. This model mimics stage III/IV ovarian carcinoma. In the first 8 weeks after implantation the cells grow as a liquid tumor without induction of tumor angiogenesis (Lee et al., 2011Lee H.J. Tantawy M.N. Nam K.T. Choi I. Peterson T.E. Price R.R. Evaluation of an intraperitoneal ovarian cancer syngeneic mouse model using 18F-FDG MicroPET imaging.Int. J. Gynecol. Cancer. 2011; 21: 22-27Crossref PubMed Scopus (5) Google Scholar). Therefore this model is ideally suited to study the consequences of EC Notch signaling, independently of its functions during angiogenesis (Potente et al., 2011Potente M. Gerhardt H. Carmeliet P. Basic and therapeutic aspects of angiogenesis.Cell. 2011; 146: 873-887Abstract Full Text Full Text PDF PubMed Scopus (1903) Google Scholar). Three weeks after tamoxifen-induced gene recombination, ID8 cells were injected orthotopically and peritoneal lavage was analyzed by flow cytometry 4 weeks later. There were significantly increased numbers of myeloid cells, in particular neutrophils in ecN1ICD mice compared with control, whereas the number of lymphocytes was not changed (Figures 3J and S5E). To further test the specificity of EC Notch signaling for neutrophil recruitment, we performed the same experiment in the well-established conditional EC-specific Rbp-jk-deficient mice (RbpjiΔEC mice). Inactivation of Rbp-jk blocks canonical signaling of all four Notch receptors (Ramasamy et al., 2014Ramasamy S.K. Kusumbe A.P. Wang L. Adams R.H. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone.Nature. 2014; 507: 376-380Crossref PubMed Scopus (567) Google Scholar). Growth of solid tumors is severely disturbed after EC Notch inhibition due to deregulated angiogenesis (Potente et al., 2011Potente M. Gerhardt H. Carmeliet P. Basic and therapeutic aspects of angiogenesis.Cell. 2011; 146: 873-887Abstract Full Text Full Text PDF PubMed Scopus (1903) Google Scholar). Therefore the angiogenesis-independent ID8 model is more reliable to study cancer-associated inflammation compared with solid tumors. Myeloid cell recruitment into peritoneal fluid was almost abolished in RbpjiΔEC mice 4 weeks after ID8 cell implantation, while again lymphocyte recruitment was unaffected (Figures 3J and S5E). This indicates a pivotal role for EC Notch signaling to recruit specifically myeloid cells into tumors. To study the cellular consequences of Notch1 activation, primary human endothelial cells from umbilical cords (HUVEC) were transduced with adenoviral particles expressing N1ICD or the constitutively active Notch nuclear transducer Rbp-jk (RBPJ-VP16). We observed not only EC growth arrest after forced Notch activation as reported previously (Eilken and Adams, 2010Eilken H.M. Adams R.H. Dynamics of endothelial cell behavior in sprouting angiogenesis.Curr. Opin. Cell Biol. 2010; 22: 617-625Crossref PubMed Scopus (411) Google Scholar, Rostama et al., 2015Rostama B. Turner J.E. Seavey G.T. Norton C.R. Gridley T. Vary C.P. Liaw L. DLL4/Notch1 and BMP9 interdependent signaling induces human endothelial cell quiescence via P27KIP1 and Thrombospondin-1.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 2626-2637Crossref PubMed Scopus (41) Google Scholar), but also a senescence-like cell morphology with enlarged and flattened cell shape in N1ICD expressing ECs (Figures 4A and 4B ). Consistently, these ECs contained elevated levels of p53 protein and expressed senescence-associated beta-galactosidase (SA-β-Gal) (Figures 4C and 4D). Likewise, Notch activation with immobilized DLL4 ligands also led to stronger induction of SA-β-Gal expression compared with control (Figure 4D, right column). Senescent cells typically secrete inflammatory cytokines as part of the senescence-associated secretory phenotype. This changes the cellular microenvironment and, e.g., facilitates the recruitment of immune cells (Sharpless and Sherr, 2015Sharpless N.E. Sherr C.J. Forging a signature of in vivo senescence.Nat. Rev. Cancer. 2015; 15: 397-408Crossref PubMed Scopus (571) Google Scholar). In accordance, we" @default.
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• W2589610792 title "Endothelial Notch1 Activity Facilitates Metastasis" @default.
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• W2589610792 doi "https://doi.org/10.1016/j.ccell.2017.01.007" @default.
• W2589610792 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28238683" @default.
• W2589610792 hasPublicationYear "2017" @default.
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