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Interleukin-6–mediated epigenetic control of the VEGFR2 gene induces disorganized angiogenesis in human breast tumors

2020; Elsevier BV; Volume: 295; Issue: 34 Linguagem: Inglês

10.1074/jbc.ra120.012590

1083-351X

Mangala Hegde, Kanive Parashiva Guruprasad, Lingadakai Ramachandra, Kapaettu Satyamoorthy, Manjunath B. Joshi,

Cytokine Signaling Pathways and Interactions

Disorganized vessels in the tumor vasculature lead to impaired perfusion, resulting in reduced accessibility to immune cells and chemotherapeutic drugs. In the breast tumor–stroma interplay, paracrine factors such as interleukin-6 (IL-6) often facilitate disordered angiogenesis. We show here that epigenetic mechanisms regulate the crosstalk between IL-6 and vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in myoepithelial (CD10+) and endothelial (CD31+, CD105+, CD146+, and CD133−) cells isolated from malignant and nonmalignant tissues of clinically characterized human breast tumors. Tumor endothelial (Endo-T) cells in 3D cultures exhibited higher VEGFR2 expression levels, accelerated migration, invasion, and disorganized sprout formation in response to elevated IL-6 levels secreted by tumor myoepithelial (Epi-T) cells. Constitutively, compared with normal endothelial (Endo-N) cells, Endo-T cells differentially expressed DNA methyltransferase isoforms and had increased levels of IL-6 signaling intermediates such as IL-6R and signal transducer and activator of transcription 3 (STAT3). Upon IL-6 treatment, Endo-N and Endo-T cells displayed altered expression of the DNA methyltransferase 1 (DNMT1) isoform. Mechanistic studies revealed that IL-6 induced proteasomal degradation of DNMT1, but not of DNMT3A and DNMT3B and subsequently led to promoter hypomethylation and expression/activation of VEGFR2. IL-6–induced VEGFR2 up-regulation was inhibited by overexpression of DNMT1. Transfection of a dominant-negative STAT3 mutant, but not of STAT1, abrogated VEGFR2 expression. Our results indicate that in the breast tumor microenvironment, IL-6 secreted from myoepithelial cells influences DNMT1 stability, induces the expression of VEGFR2 in endothelial cells via a promoter methylation–dependent mechanism, and leads to disordered angiogenesis. Disorganized vessels in the tumor vasculature lead to impaired perfusion, resulting in reduced accessibility to immune cells and chemotherapeutic drugs. In the breast tumor–stroma interplay, paracrine factors such as interleukin-6 (IL-6) often facilitate disordered angiogenesis. We show here that epigenetic mechanisms regulate the crosstalk between IL-6 and vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in myoepithelial (CD10+) and endothelial (CD31+, CD105+, CD146+, and CD133−) cells isolated from malignant and nonmalignant tissues of clinically characterized human breast tumors. Tumor endothelial (Endo-T) cells in 3D cultures exhibited higher VEGFR2 expression levels, accelerated migration, invasion, and disorganized sprout formation in response to elevated IL-6 levels secreted by tumor myoepithelial (Epi-T) cells. Constitutively, compared with normal endothelial (Endo-N) cells, Endo-T cells differentially expressed DNA methyltransferase isoforms and had increased levels of IL-6 signaling intermediates such as IL-6R and signal transducer and activator of transcription 3 (STAT3). Upon IL-6 treatment, Endo-N and Endo-T cells displayed altered expression of the DNA methyltransferase 1 (DNMT1) isoform. Mechanistic studies revealed that IL-6 induced proteasomal degradation of DNMT1, but not of DNMT3A and DNMT3B and subsequently led to promoter hypomethylation and expression/activation of VEGFR2. IL-6–induced VEGFR2 up-regulation was inhibited by overexpression of DNMT1. Transfection of a dominant-negative STAT3 mutant, but not of STAT1, abrogated VEGFR2 expression. Our results indicate that in the breast tumor microenvironment, IL-6 secreted from myoepithelial cells influences DNMT1 stability, induces the expression of VEGFR2 in endothelial cells via a promoter methylation–dependent mechanism, and leads to disordered angiogenesis. Pathologically activated tissue microenvironment has been shown to shape breast tumor growth, progression, nature, evolution and response to therapy. Persistent proliferation, immune cell recruitment, and angiogenesis are the key determinants of tumor growth (1Hanahan D. Weinberg R.A. Hallmarks of cancer: The next generation.Cell. 2011; 144 (21376230): 646-67410.1016/j.cell.2011.02.013Abstract Full Text Full Text PDF PubMed Scopus (42713) Google Scholar). 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Loss of MMP-8 in ductal carcinoma in situ (DCIS)-associated myoepithelial cells contributes to tumour promotion through altered adhesive and proteolytic function.Breast Cancer Res. 2017; 19 (28330493): 3310.1186/s13058-017-0822-9Crossref PubMed Scopus (18) Google Scholar, 4Katarina Sirka O. Shamir E.R. Ewald A.J. Myoepithelial cells are a dynamic barrier to epithelial dissemination.J. Cell Biol. 2018; 217 (30061105): 3368-338110.1083/jcb.201802144Crossref PubMed Google Scholar). Alterations in gene expression patterns in tumor-associated myoepithelial cells result in secretion of paracrine molecules including IL-6 and promote tumor growth (5Martinez E.F. Napimoga M.H. Montalli V.A.M. de Araújo N.S. de Araújo A.V. In vitro cytokine expression in in situ-like areas of malignant neoplasia.Arch. Oral Biol. 2013; 58 (22749497): 552-55710.1016/j.archoralbio.2012.06.001Crossref PubMed Scopus (11) Google Scholar). 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Hodivala-Dilke K. Leinster D.A. Zhong H. Hollingsworth R.E. Thompson R. Whiteford J.R. Balkwill F. Interleukin-6 stimulates defective angiogenesis.Cancer Res. 2015; 75 (26081809): 3098-310710.1158/0008-5472.CAN-15-1227Crossref PubMed Scopus (143) Google Scholar). Blocking of IL-6 results in restoration of pericyte coverage in vessels (12Gopinathan G. Milagre C. Pearce O.M. Reynolds L.E. Hodivala-Dilke K. Leinster D.A. Zhong H. Hollingsworth R.E. Thompson R. Whiteford J.R. Balkwill F. Interleukin-6 stimulates defective angiogenesis.Cancer Res. 2015; 75 (26081809): 3098-310710.1158/0008-5472.CAN-15-1227Crossref PubMed Scopus (143) Google Scholar). Epigenetic mechanisms including DNA methylation and histone acetylation are reported as key regulators of endothelial gene expression and functions including tumor induced neovascularization (13Yan M.S. Turgeon P.J. Man H.S.J. Dubinsky M.K. Ho J.J.D. El-Rass S. Wang Y.D. Wen X.Y. Marsden P.A. Histone acetyltransferase 7 (KAT7)-dependent intragenic histone acetylation regulates endothelial cell gene regulation.J. Biol. Chem. 2018; 293 (29414790): 4381-440210.1074/jbc.RA117.001383Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 14Pirola L. Ciesielski O. B A. The methylation status of the epigenome: Its emerging role in the regulation of tumor angiogenesis and tumor growth, and potential for drug targeting.Cancers (Basel). 2018; 10: 26810.3390/cancers10080268Crossref Scopus (25) Google Scholar). Earlier studies have shown that critical genes such as VEGF and its receptors, eNOS, MMPs, TMP, TSP, and RECK in maintaining angiogenic homeostasis are influenced by promoter DNA methylation. Inhibitors of DNA methyltransferases (DNMTs) exhibit significant angiostatic effects in various tumor models (14Pirola L. Ciesielski O. B A. The methylation status of the epigenome: Its emerging role in the regulation of tumor angiogenesis and tumor growth, and potential for drug targeting.Cancers (Basel). 2018; 10: 26810.3390/cancers10080268Crossref Scopus (25) Google Scholar, 15Hellebrekers D.M.E.I. Jair K.-W. Viré E. Eguchi S. Hoebers N.T.H. Fraga M.F. Esteller M. Fuks F. Baylin S.B. van Engeland M. Griffioen A.W. Angiostatic activity of DNA methyltransferase inhibitors.Mol. Cancer Ther. 2006; 5 (16505122): 467-47510.1158/1535-7163.MCT-05-0417Crossref PubMed Scopus (84) Google Scholar). Our earlier studies have revealed IL-6 as potential modulator of DNMT isoforms and significantly altered epigenome of endothelial cells (16Balakrishnan A. Guruprasad K.P. Satyamoorthy K. Joshi M. Interleukin-6 determines protein stabilization of DNA methyltransferases and alters DNA promoter methylation of genes associated with insulin signaling and angiogenesis.Lab. Invest. 2018; 98 (29955086): 1143-115810.1038/s41374-018-0079-7Crossref PubMed Scopus (27) Google Scholar). Microarray analysis indicated IL-6–induced significant hypomethylation of VEGFR2 promoter in HUVECs. These findings led us to investigate the paracrine effects of IL-6 derived from CD10+ myoepithelial cells on endothelial cells (CD31+, CD105+; CD133−) in human breast tissue microenvironment. From clinically characterized breast tumors, we isolated four cell types: (a) myoepithelial cells from malignant tissues (Epi-T), (b) myoepithelial cells from nonmalignant tissues (Epi-N), (c) endothelial cells from malignant tissues (Endo-T), and (d) endothelial cells from nonmalignant tissues (Endo-N). In these cell types, we examined the impact of IL-6 on epigenetic control of VEGFR2 expression and its role in modulating normal and tumor associated angiogenesis. In HUVECs, IL-6 elevated STAT3Tyr-705 phosphorylation to nearly 6-fold by 6 h, which was sustained until 24 h and subsequently increased to 8-fold at 36 h. The total VEGFR2 levels were increased to 2-fold after 6 h and further increased to 8-fold by 36 h of IL-6 treatment (Fig. 1A). We then isolated myoepithelial and endothelial cells from the malignant and nonmalignant part of human breast tissues to examine paracrine effects of IL-6 between these cell types (Fig. S1A). Endothelial and myoepithelial cells were extensively characterized from four individuals diagnosed with infiltrative ductal carcinoma graded and staged as ER+/PR+/Her2/neu+ based on immunohistochemistry analysis. Endo-N and Endo-T cells were characterized based on abundance of CD31 (>95%) and CD105 (>95%) expression Fig. S1B. Further characterization of Endo-T cells revealed increased expression of endothelial cell specifc markers such as total and phospho-eNOS, VE-cadherin, and CD146 compared with Endo-N cells. Endo-T, Endo-N, and HUVEC cells were negative for cytokeratins, E-cadherin, and smooth muscle α actin precluding the contamination of nonEC cell types (Fig. S2). These cells when cultured for seven consecutive passages showed persistent expression of CD31 and CD105 (Fig. S3). However, we observed phenotype change in seventh passage cells. Endo-T cells did not express CD133 (Fig. S1B) and karyotype (Fig. S4) matched with Endo-N cells, suggesting these cells were not tumor derived and of host origin. Further, Epi-N and Epi-T cells were characterized by presence of CD10 (>95%) and keratin by flow cytomtery and immunoblotting, respectively (Fig. S1, C and D). IL-6 quantification from culture supernatants showed a 4-fold increase in IL-6 levels in Epi-T cells (656 ± 20 pg/105 cells) compared with Epi-N cells (138 ± 9 pg/105 cells) (Fig. 1B). Breast tumor cell lines such as MCF-10A, MCF-7, MDA-MB-231, and MDA-MB-468 also secreted IL-6, albeit at lower levels than primary cells. Immunoblotting analysis indicated significanlty elevated levels of VEGFA-121 (6- to 8-fold) in Epi-T cell–conditioned medium compared with that of Epi-N cells (Fig. 1C and Fig. S5). We further examined influence of Epi-N and Epi-T cell–conditioned medium on HUVECs for the effect on STAT3/VEGFR2 expression. Treatment with Epi-T cell–conditioned medium showed nearly 4-fold higher STAT3 phosphorylation. Further, IL-6R neutralizing antibody significantly reduced both phospho-STAT3Tyr-705 (nearly 75%) and total VEGFR2 and phospho-VEGFR2Tyr-1175 (nearly 50%) levels (Fig. 1D and Fig. S6) suggesting that IL-6 in epithelial cell conditioned medium is the key to induce STAT3 phsophorylation and VEGFR2 expression. We next prepared 3D spheriods from HUVECs and cultured them in presence of Epi-T cell conditioned media and compared with Epi-N cell medium. The results showed an increase in sprout length and numbers (Fig. 1E) when cultured in presence of Epi-T cell medium than Epi-N cell medium (p < 0.001). IL-6R antibody (p < 0.001) and VEGFR2 inhibitor (tyrosine kinase) sorafenib (p < 0.001) significantly reduced sprout numbers and length (Fig. 1, F and G), suggesting that IL-6 and VEGFA secreted by Epi-T cells induced VEGFR2-dependent angiogenesis. We further investigated the underlying mechanisms for IL-6–dependent VEGFR2 expression in endothelial cells. As has been observed in our earlier studies (16Balakrishnan A. Guruprasad K.P. Satyamoorthy K. Joshi M. Interleukin-6 determines protein stabilization of DNA methyltransferases and alters DNA promoter methylation of genes associated with insulin signaling and angiogenesis.Lab. Invest. 2018; 98 (29955086): 1143-115810.1038/s41374-018-0079-7Crossref PubMed Scopus (27) Google Scholar), treatment of HUVECs with IL-6 increased DNMT1 levels at early time points to nearly 4- to 5-fold, which was sustained up to 12 h and subsequently decreased to less than the control levels by 24 h (Fig. 2A). HUVECs when cultured with conditioned medium of Epi-N and Epi-T cells in presence of IL-6R neutralizing antibody, we observed a significant increase in DNMT1 expression. The effects of IL-6R antibody was more robust in endothelial cells treated with conditioned medium from Epi-T cells (p < 0.01) than Epi-N cells (Fig. 2B and Fig. S6). Therefore, Epi-T cell secretome containing IL-6 may be responsible for the changes in DNMT1 levels in endothelial cells in tumor microenvironment. Treatment of HUVECs with actinomycin D, a transcription inhibitor, showed nearly 50% degradation of DNMT1 transcripts by 2 h. However, pretreatment of HUVECs with IL-6 for 3 h prior to blocking of transcription did not alter DNMT1 levels (Fig. 2C). Inhibition of protein translation by cycloheximide alone or in cells pretreated with IL-6 showed significant reduction in the levels of DNMT1 by 60 min (Fig. 2D). Taken together, these results indicate that regulation of DNMT1 expression by IL-6 is not at the level of transcription but probably a post-transcriptional or post-translational event. To test whether increased expression of VEGFR2 in response to IL-6 is as a consequence of DNMT1 degradation, we cultured HUVECs in presence of MG132 (carbobenzoxyleu-leu-leucinal), a potent inhibitor of 16S subunit of proteasomal complex. MG132 restored IL-6–induced degradation of DNMT1 to nearly 50% and, at the same time, enhanced both total and phospho-VEGFR2 expression to normalized basal levels. Thus, IL-6 facilitated proteasomal degradation of DNMT1, which subsequently led to elevation of VEGFR2 expression (Fig. 2E). Next, we examined the constitutive levels of IL-6 signaling intermediates, DNMT isoforms, and total/phospho-VEGFR2 levels in Endo-N and Endo-T cells (Fig. 3). Endo-T cells showed consitutively elevated expression of IL-6 signaling intermediates such as IL-6R, total and phospho-STAT3Tyr-705. Interestingly, phosphorylation of STAT1Tyr-701 were significantly reduced in Endo-T cells compared with Endo-N cells. However, levels of total and phospho-STAT1Ser-727 did not alter between Endo-N and Endo-T cells. Levels of DNMT protein isoforms such as DNMT1 and DNMT3A were significantly elevated in Endo-T cells as opposed to DNMT3B which was down-regulated in Endo-T cells compared with its normal counterpart. Therefore, aberrant endothelial cell function in tumor microenvironment might be associated with dynamic reprogramming in methylome. Basal levels of phospho- and total VEGFR2 were also significantly increased in Endo-T cells. Next, we examined the differential response of Endo-N and Endo-T cells to IL-6 signaling pathway proteins (Fig. S7, A and B). IL-6 induced 2-fold increase in IL-6R levels in Endo-N cells but not in Endo-T cells. However, Endo-T cells showed constitutively higher levels of IL-6R. Kinetic analysis revealed both endothelial cell types responded to IL-6 treatment which led to induction of STAT3Tyr-705 phosphorylation at similar magnitude (2- to 3-fold increase). IL-6 also facilitated an increase in total STAT3 expression. Similar to HUVECs, Endo-N cells showed significant down-regulation of DNMT1 in response to chronic IL-6 treatment although the reduction in levels of DNMT1 varied among the three individual isolates. Endo-T cells expressed constitutively higher levels of DNMT1 and treatment with IL-6 induced its degradation. However, IL-6 effects on DNMT1 down-regulation was more prominent in Endo-N cells than in Endo-T cells. Both the cell types showed 2- to 3-fold increase in VEGFR2 expression upon IL-6 treatment. In response to IL-6, we observed nearly 50% reduction in DNMT1 protein levels and an increase in nearly 2- to 3-fold of VEGFR2 levels by 36 h in HUVECs. These effects were abrogated upon treatment with IL-6R neutralizing antibody, where DNMT1 levels were restored and VEGFR2 levels remained similar to that of control (Fig. S8). Further, transient overexpression of WT STAT3 in HUVECs enhanced the effects of IL-6 on VEGFR2 expression and activation (Fig. 4A). We observed 50% reduction in DNMT1 levels in IL-6–treated cells in control group transfected with empty vector (p < 0.001) and, interestingly, cells overexpressing STAT3 showed further 10% reduction in DNMT1 levels in response to IL-6 (p < 0.001). IL-6–induced VEGFR2 levels in empty vector and STAT3 overexpressing cells remained unaltered. However, IL-6 increased the phosphorylation of VEGFR2Tyr-1175 to nearly 3-fold in cells overexpressing STAT3, suggesting IL-6 dependent STAT3 phosphorylation is the key determinant for increased activation of VEGFR2 (Fig. 4A). Role of STAT3 in regulating VEGFR2 expression was further confirmed by transfection of plasmid with dominant negative mutatnt of STAT3(Y705F). Overexpression of dominant negative STAT3 revealed significant abrogation of total and phospho-VEGFR2Tyr-1175 levels in presence or absence of IL-6 (Fig. 4B). As IL-6 has been demonstrated to activate STAT1 phosphorylation, we examined effects of dominant negative STAT1(Y701F) on VEGFR2 expression. We observed overexpression of STAT1(Y701F) did not influence expression of VEGFR2 in both presence and absence of IL-6, suggesting STAT3 but not STAT1 as a key regulator of VEGFR2 (Fig. 4C). Interestingly, overexpression of WT, dominant negative STAT3, and dominant negative STAT1 did not show any influence on DNMT1 levels. These results suggest that IL-6–induced reduction in DNMT1 levels were mediated by proteasomal degradation. As we observed differential expression of DNMT isoforms in tumor and normal endothelial cells, we tested influence of DNMT isoforms on VEGFR2 gene regulation and protein expression in HUVECs. DNMT1 overexpression completely abrogated the VEGFR2 expression independent of IL-6. However, VEGFR2 levels remained unaltered in DNMT3A and DNMT3B transfected cells under normal culture condition and in response to IL-6 treatment (Fig. 4D). This suggested DNMT1 but not DNMT3A or DNMT3B are essential for regulating VEGFR2 expression. Further, inhibition of DNMT isoforms by 5′-aza-deoxycytidine resulted in 3-fold increase in VEGFR2 expression (Fig. 4E). Therefore, blocking of DNMTs has similar effects to that of IL-6 on VEGFR2 expression. Further, we determined whether IL-6 modulates DNA methylation levels of VEGFR2 promoter in HUVECs. The bisulfite DNA sequencing of the promoter region spanning –408 to +6 corresponding to 55083071–55083531 coordinates on chromosome 4 showed significant hypomethylation of VEGFR2 promoter region in response to IL-6 treatment for 36 h (Fig. 4F). Compared with basal levels of CpG methylation, a significant demethylation was observed at −54 position (80 versus 10–15%) and a complete demethylation at −75 (40 versus 0%) position relative to the transcription start site in the promoter region. Subsequently, we examined status of DNA promoter methylation of VEGFR2 gene in Endo-N and Endo-T cells. Endo-T cells from all three subjects showed significant constitutive hypomethylation when compared with controls in VEGFR2 promoter at −54 (45% versus 0; 42 versus 2%; and 30% versus 0) and −75 (15% versus 0; 12% versus 0; 10% versus 0) positions (Fig. 4F). We observed Endo-T cells displayed increased expression of CD105/endoglin (Fig. S1B) and proliferated more robustly than Endo-N cells (Fig. S9). Cell cycle analysis showed that 60.23 ± 9.6% of Endo-T cells were in G0/G1 phase and 29.04 ± 6.63% in G2/M phase or proliferative phase constitutively, whereas 84.13 ± 3% of Endo-N cells were in G0/G1 phase and 4.28 ± 2.6% were in G2/M phase (Fig. S10, A–C). The percentages of cell cycle phase distribution for each subject are given in Table S1. Further, we tested the response of IL-6 in serum-depleted Endo-N and Endo-T cells. The 24 h serum starvation arrested 3–4% more cells in G0/G1 phase and 2–3% fewer cells in G2/M phase in both cell types (Fig. S10, A–C). Addition of IL-6 did not significantly effect the redistribution of cells into various phases of cell cycle both in Endo-N and Endo-T cells (Fig. S10, A–C). This indicated that the Endo-T cells possessed increased inherent ability to proliferate and IL-6 signaling does not induce abnormal proliferation. In conventional scratch assays, Endo-T cells displayed inherent pro-migratory properties by closing the wound in 12 h and in presence of IL-6, the migration was complete in 6 h. The Endo-N cells required 18 h to close the wound and in response to IL-6, for these cells, complete migration was observed in 12 h (Fig. 5, A–C, and Fig. S11). Similar results were observed when HUVECs were treated with supernatants of myoepithelial cells. The HUVEC treated with Epi-N cell culture supernatant for 12 h showed 80% of gap closure which was significantly inhibited upon inclusion of IL-6R neutralizing antibody. When treated with Epi-T cell supernatant, complete wound closure was observed within 12 h and these effects were inhibited in presence of IL-6R neutralizing antibody (Fig. S12). This indicated IL-6 secreted from the Epi-T cell–facilitated endothelial cell migration. To examine the invasive properties, endothelial cell types were co-cultured with epithelial cells with or without IL-6R neutralizing antibody. In transwell invasion assays, the cells were co-cultured in four different combinations as indicated in Fig. 5, D and E. The Endo-N cells when co-cultured with Epi-N derived from the same patient did not invade the membrane and IL-6R neutralizing antibody did not show any effect. The Endo-N cells when co-cultured with Epi-T cells showed a 75% increase in invasion and the percentage of migration was decreased to 26% in presence of IL-6R neutralizing antibody (Fig. 5, D and F). The Endo-T cells showed increased invasion (140%) when cultured with Epi-N cells and in presence of IL-6R neutralizing antibody, 53% inhibition of invasion was observed. Interestingly, in the Endo-T cells cultured with Epi-T, the invasive ability was significantly higher (600%) and IL-6R neutralizing antibody decreased it to 290% (Fig. 5, E and F). This indicated that the IL-6 secreted by Epi-T cells induced invasiveness of endothelial cells in tumor environment. Further, we examined cytoskeletal changes in IL-6–treated endothelial cell types. Actin phalloidin staining showed that Endo-T cells possessed constitutively higher number of cells with lamellipodia than Endo-N cells which correlated with the invasion assay. In response to IL-6, Endo-N cells formed lamellipodia, which is a characteristic migratory phenotype. On the other hand, IL-6 induced stellate phenotype in the Endo-T cells in 6 h, facilitating an aggressive migratory behavior (Fig. 5G). We then tested the angiogenic behavior of endothelial cell types in 3D culture/sprout assays and Matrigel tube-forming assays. Endo-T cells showed inherently formed higher number of sprouts (2-fold) and with increased length (1.5-fold) than Endo-N cells in the absence of any stimulant. Upon IL-6 treatment, Endo-N cells formed 3-fold increase in number of sprouts compared with untreated spheroids and was completely inhibited in presence of IL-6R neutralizing antibody. In response to IL-6, the tumor endothelial cells failed to form intact sprouts which was associated with the deflection of cells from core indicating their invasive behavior (Fig. 6, A and B). The addition of IL-6R neutralizing antibody reversed the effect of IL-6 on tumor endothelial cells and showed formation of rudimentary sprouts. The spheriods made from Endo-T cells when treated with bFGF resulted in significantly enhanced and intact sprouts, unlike IL-6, which showed pro-invasive phenotype (Fig. S13). In Matrigel tube-forming assays, we observed Endo-T cells started rearranging themselves without any stimulation to form tube like structures by 2 h, indicating inherent reprogramming of Endo-T cells leading to angiogenic switch. Both Endo-N cells and HUVECs showed 2.5-fold increased number of tube formation in response to IL-6 treatment which was abrogated by the addition of IL-6R neutralizing antibody. In response to IL-6, Endo-T cells lost their ability to form intact hexagonal tubes and IL-6R neutralizing antibody resensitized tube formation in these cells (Fig. 6, C and D, and Fig. S14). This clearly suggested that IL-6 might be one of the potential molecules to induce disorganized vessels in the tumor microenvironment. Because IL-6 induces VEGFR2 up-regulation and aberrant angiogenesis (Fig. 6, A and B), we tested the influence of VEGFR2 antibody in spheriod models. IL-6 effects on angiogenesis in HUVECs were inhibited by addition of VEGFR2 blocking antibody in dose-dependent manner (Fig. 7, A and D). Endo-N cells showed 3-fold increase in sprout numbers and length in response to IL-6 (Fig. 7, B and D), and VEGFR2 antibody at 1.5 μg/ml was sufficient to block the IL-6 effects. In contrast, the Endo-T cells formed higher number of sprouts constitutively (3-fold increase versus normal) with increased length (4-fold increase). In response to IL-6, the tumor-derived endothelial cells failed to form the additional sprouts and deflected away from the spheroid core (Fig. 7, C and D). The VEGFR2 antibody concentrations at 1.0 μg/ml and 1.5 μg/ml were able to restore the sprout formation in Endo-T cells in presence of IL-6, and further increase in the concentration of VEGFR2 antibody (2.0 μg/ml) deteriorated the spheroid architecture. Taken together, this indicated excess VEGFR2 facilitates disorganized angiogenesis as a functional consequence of IL-6 effects. Our study deciphered key molecules and mechanisms that cause disordered angiogenesis in breast tumors. Paracrine signaling and interrelationship between IL-6 and VEGFR2 in myoepithelial and endothelial cells in human breast t