Tie2-expressing monocytes and tumor angiogenesis: regulation by hypoxia and angiopoietin-2

Recent findings indicate that tumor-associated macrophages are important drivers of tumor angiogenesis. Here, we review the essential role played by Tie2-expressing monocytes (TEM) in this phenomenon. TEMs are present in human blood and tumors and their elimination in various tumor models suppresses tumor angiogenesis. A ligand for Tie2, angiopoietin-2 (Ang-2), is produced by angiogenic tumor vessels and is a chemoattractant for TEMs. Hypoxia up-regulates Tie2 expression on TEMs and, together with Ang-2, down-regulates their antitumor functions. Learning more about the regulation of TEMs by the tumor microenvironment may yield new strategies to ablate the tumor vasculature.     

Direct binding of the human homologue of the Drosophila disc large tumor suppressor gene to seven-pass transmembrane proteins, tumor endothelial marker 5 (TEM5), and a novel TEM5-like protein

The human homologue of the Drosophila discs large tumor suppressor gene (hDlg) is a member of the membrane-associated guanylate kinase family with three PSD-95/Dlg/ZO-1 (PDZ) domains. hDlg has been shown to bind tumor suppressor proteins, adenomatous polyposis coli (APC) and protein tyrosine phosphatase and tensin homologue (PTEN), and several viral oncoproteins, and has been implicated in the negative regulation of cell proliferation. hDlg has furthermore been shown to localize at the plasma membrane of synapses and to scaffold cell surface receptors and channels. In epithelial cells, hDlg localizes at the basolateral plasma membrane, but its localization mechanism is unknown. We searched here for a transmembrane protein that directly bound to hDlg. hDlg bound tumor endothelial marker 5 (TEM5), a seven-pass transmembrane protein that is homologous to the family B of G-protein-coupled receptors (GPCRs). TEM5 has previously been reported to display elevated expression during tumor angiogenesis and neoangiogenesis. The PDZ domains of hDlg bound the C-terminal PDZ-binding motif of TEM5. The expression of TEM5 was detected in endothelial cells of embryonic liver, where hDlg colocalized with TEM5. hDlg furthermore bound a novel seven-pass transmembrane protein, which was homologous to TEM5, and was named here a TEM5-like protein (TEM5-like). These results suggest that hDlg localizes at the plasma membrane through TEM5 and TEM5-like and furthermore scaffolds these GPCRs in endothelial cells during tumor angiogenesis and neoangiogenesis.     

Tumor endothelial markers: new targets for cancer therapy

PURPOSE OF REVIEW: Targeting the endothelial cells that line tumor infiltrating blood vessels is a new anticancer strategy that has gained widespread support from biologists and clinicians. Here we highlight different approaches currently being used to target tumor endothelium and discuss new avenues for intervention that have been opened through the recent identification of tumor endothelial markers (TEMs). RECENT FINDINGS: The ability of Avastin to prolong survival in a Phase III clinical trial of human colorectal cancer has established the validity of the anti-angiogenic approach. However, realization of the full potential of a vascular targeting strategy may require the exploitation of molecules which are highly restricted in expression to tumor endothelium. Here we explore the potential of TEMs as new targets for cancer therapy. Current knowledge of these markers and their relation to other family members in the context of tumor angiogenesis is discussed. In particular, we highlight those molecules which, by virtue of their structure, cell-surface location and expression pattern, appear to hold promise as targets for future drug development. The identification of TEM8 as the anthrax toxin receptor and the successful targeting of this receptor in preclinical tumor models make this molecule a particularly attractive candidate for future vascular targeting studies. SUMMARY: Technological advances in cellular fractionation and genomics enabled the identification of several markers preferentially expressed on human tumor endothelium. Studies of these TEMs are expected to aid in our understanding of angiogenesis and could lead to the development of new imaging and diagnostic agents for cancer.     

人类肿瘤中Tie2阳性单核巨噬细胞研究进展

Tie2阳性单核巨噬细胞（TEMs）是一类表达酪氨酸激酶受体Tie2的单核细胞或巨噬细胞,在人类和鼠类中均有发现,可存在于外周血和组织中,参与肿瘤血管生成、淋巴管生成和免疫抑制等肿瘤微环境的形成。现有研究发现TEMs对于多种肿瘤具有潜在的临床诊断及预后指导意义,有望为肿瘤治疗提供新的靶向策略,本文就TEMs在肿瘤中的研究进展作一综述     

Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors

Bone marrow-derived cells contribute to tumor angiogenesis. Here, we demonstrate that monocytes expressing the Tie2 receptor (Tie2-expressing monocytes [TEMs]) (1) are a distinct hematopoietic lineage of proangiogenic cells, (2) are selectively recruited to spontaneous and orthotopic tumors, (3) promote angiogenesis in a paracrine manner, and (4) account for most of the proangiogenic activity of myeloid cells in tumors. Remarkably, TEM knockout completely prevented human glioma neovascularization in the mouse brain and induced substantial tumor regression. Besides TEMs and endothelial cells (ECs), Tie2 expression distinguished a rare population of tumor stroma-derived mesenchymal progenitors representing a primary source of tumor pericytes. Therefore, Tie2 expression characterizes three distinct cell types required for tumor neovascularization: ECs, proangiogenic cells of hematopoietic origin, and pericyte precursors of mesenchymal origin.     

Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis by impairing angiogenesis and disabling rebounds of proangiogenic myeloid cells

Tumor-infiltrating myeloid cells convey proangiogenic programs that counteract the efficacy of antiangiogenic therapy. Here, we show that blocking angiopoietin-2 (ANG2), a TIE2 ligand and angiogenic factor expressed by activated endothelial cells (ECs), regresses the tumor vasculature and inhibits progression of late-stage, metastatic MMTV-PyMT mammary carcinomas and RIP1-Tag2 pancreatic insulinomas. ANG2 blockade did not inhibit recruitment of MRC1(+) TIE2-expressing macrophages (TEMs) but impeded their upregulation of Tie2, association with blood vessels, and ability to restore angiogenesis in tumors. Conditional Tie2 gene knockdown in TEMs was sufficient to decrease tumor angiogenesis. Our findings support a model wherein the ANG2-TIE2 axis mediates cell-to-cell interactions between TEMs and ECs that are important for tumor angiogenesis and can be targeted to induce effective antitumor responses.     

表达Tie2的单核细胞在肿瘤研究中的进展

表达Tie2的单核细胞(TEM)仅存在于外周血及肿瘤组织中,在肿瘤血管生成中发挥重要作用.TEM能够被血管生成素2(Ang2)及缺氧化学信号募集进入肿瘤组织,分化成表达Tie2的巨噬细胞,通过旁分泌作用参与肿瘤血管生成.肝癌、结肠癌、乳腺癌、恶性胶质瘤等肿瘤中检测到TEM水平升高,这对于肿瘤的诊断及预后判断有一定的提示作用.近年研究发现,TEM可用于抗肿瘤药物的靶向递送,能有效抑制肿瘤的生长和转移,同时TEM也是抗肿瘤治疗的潜在靶点.然而TEM在肿瘤微血管密度、临床分级及预后判断中的作用并不明确,目前针对TEM具体功能的探究引起了学者的广泛关注.     

Apigenin inhibits tumor angiogenesis through decreasing HIF-1alpha and VEGF expression

Apigenin is a non-toxic dietary flavonoid with anti-tumor properties. We recently showed that apigenin-inhibited hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) expression in human ovarian cancer cells under normoxic condition. However, the effect of apigenin in angiogenesis remains to be elucidated. Angiogenesis is the formation of new blood vessels and is required for tumor growth and metastasis. In this study, we showed that apigenin-inhibited expression of HIF-1 and VEGF in different cancer cells under both normoxic and hypoxic conditions. We demonstrated that apigenin significantly inhibited tumor angiogenesis in vivo, by using both the chicken chorioallantoic membrane and Matrigel plug assays. The inhibition of tumor angiogenesis was associated with the decrease of HIF-1 and VEGF in tumor tissues. Taken together, our results show that apigenin suppresses tumor angiogenesis through HIF-1 and VEGF expression.     

CXCL12–CXCR7 axis is important for tumor endothelial cell angiogenic property

We reported that tumor endothelial cells (TECs) differ from normal endothelial cells (NECs) in many aspects, such as gene expression profiles. Although CXCR7 is reportedly highly expressed in blood vessels of several tumors, its function in TECs is still unknown. To investigate this role, we isolated TECs from mouse tumor A375SM xenografts, and compared them with NECs from normal mouse dermis. After confirming CXCR7 upregulation in TECs, we analyzed its function using CXCR7 siRNA and CXCR7 inhibitor; CCX771. CXCR7 siRNA and CCX771 inhibited migration, tube formation and resistance to serum starvation in TECs but not in NECs. ERK1/2 phosphorylation was inhibited by CXCR7 knockdown in TECs. These results suggest that CXCR7 promotes angiogenesis in TECs via ERK1/2 phosphorylation. Using ELISA, we also detected CXCL12, a ligand of CXCR7, in conditioned medium from TECs, but not from NECs. CXCL12 neutralizing antibody significantly inhibited TEC random motility. VEGF stimulation upregulated CXCR7 expression in NECs, implying that VEGF mediates CXCR7 expression in endothelial cells. A CXCR7 inhibitor, CCX771 also inhibited tumor growth, lung metastasis and tumor angiogenesis in vivo. Taken together, the CXCL12–CXCR7 autocrine loop affects TEC proangiogenic properties, and could be the basis for an antiangiogenic therapy that specifically targets tumor blood vessels rather than normal vessels.     

Novel anti-denatured collagen humanized antibody D93 inhibits angiogenesis and tumor growth: An extracellular matrix-based therapeutic approach

Matrix metalloproteases (MMPs) secreted by both tumor and endothelial cells proteolytically degrade collagen during tumor growth and neo-vascularization. This exposes cryptic binding sites on collagen with functional relevance for angiogenesis. In this report, we characterized a novel humanized monoclonal IgG1 antibody, D93. After humanization, the antibody retained the binding specificity of the parental murine IgM antibody for denatured (dn) collagen. D93 recognized dn-collagen but not native (nat) collagen of different species, including mouse, chicken, and human, indicating that its cryptic binding site(s) is conserved across species. In immunohistochemistry (IHC) studies, D93 stained the basement membrane of blood vessels in several xenograft human tumors or in surgically removed tumor tissues from patients with different types of malignancies. D93 staining was rarely or not present in normal blood vessels of healthy tissues. In in vivo experiments, D93 significantly inhibited basic fibroblast growth factor (bFGF)-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) and the tumor growth of pre-established orthotopic human breast (MDA-MB-435) tumors in mice. D93 i.v. administered in mice was subsequently detected in the subendothelial basement membrane of tumor blood vessels but not blood vessels of normal tissues. Inhibition of growth of pre-established orthotopic human breast MDA-MB-435 tumors was more effective when D93 was combined with Taxol, than either treatment alone. In addition, tumors from animals treated with D93 and/or Taxol showed significantly reduced levels of the endothelial cell-marker CD31. Our data suggest that blockade of cryptic epitopes exposed on collagen IV during angiogenesis and tumor growth by a monoclonal antibody may provide a novel therapeutic modality for treatment of cancer and pathogenic neo-vascularization.     

Antiangiogenic cancer therapy

Like most embryonic tissues, tumors have the ability to build up their own blood vessel networks. However, the architecture of tumor vessels is fundamentally different from that found in healthy tissues. Tumor vessels are usually irregular, heterogeneous, leaky, and poorly associated with mural cells. Endothelial cells in tumor vessels are also disorganized and express imbalanced surface molecules. These unusual features may provide some molecular and structural basis for selective inhibition or even destruction of tumor vessels by angiogenesis inhibitors. In animal tumor models, several angiogenesis inhibitors seem to inhibit tumor angiogenesis specifically without obvious effects on the normal vasculature. As a result, these inhibitors produced potent antitumor effects in mice. Excited by these preclinical studies, more than 60 angiogenesis inhibitors are being evaluated for their anticancer effects in human patients. Although the ultimate outcome of antiangiogenic clinical trials remains to be seen, several early observations have reported some disappointing results. These early clinical data have raised several important questions. Can we cure human cancers with angiogenesis inhibitors? Have we found the ideal angiogenesis inhibitors for therapy? What is the difference between angiogenesis in an implanted mouse tumor and in a spontaneous human tumor? What are the molecular mechanisms of these angiogenesis inhibitors? Should angiogenesis inhibitors be used alone or in combinations with other existing anticancer drugs? In this review, we will discuss these important issues in relation to ongoing antiangiogenic clinical trials.     

Dysregulated expression of HOX and ParaHOX genes in human esophageal squamous cell carcinoma

Homeobox genes function as master regulators in embryonic morphogenesis. We hypothesized that homeobox genes are essential to maintain tissue- or organ-specificity even in adult body and that the dysregulated expression of homeobox genes results in tumor development and progression. To better understand the roles of homeobox genes in development and progression of esophageal cancer, we analyzed the expression patterns of 39 HOX genes and 4 ParaHOX (CDX1, CDX2, CDX4 and PDX1) genes in esophageal squamous cell carcinoma (ESCC) and normal esophageal mucosa tissues. A total of 48 primary ESCC tissues and 7 normal esophageal mucosa tissues were resected from patients who underwent radical surgery without any preoperative chemotherapy or radiotherapy. The expression of HOX and ParaHOX genes were analyzed by a quantitative real-time RT-PCR method and immunohistochemistry. The expression levels of 24 HOX genes, CDX1, CDX2 and PDX1 were significantly higher in ESCC compared to normal mucosa (p<0.01, Mann-Whitney U test). The Immunohistochemical study revealed that HOXA5 and D9 proteins were more cytoplasmic in ESCC than normal mucosa cells. Our data indicate that the disordered expression of HOX and ParaHOX genes are involved in the development of ESCC or its malignancy.     

Expression of matrix metalloproteinase-7 on cancer cells and tissue endothelial cells in renal cell carcinoma: prognostic implications and clinical significance for invasion and metastasis.

PURPOSE: The expression of matrix metalloproteinase-7 (MMP-7) correlates with the malignant potential of various tumors and patient survival. We investigated the clinical and prognostic significance of MMP-7 expression in cancer cells and endothelial cells in human renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: We reviewed tissue samples of 156 patients with RCC who had undergone radical operation. MMP-7 expression was examined by immunohistochemistry. Sections containing MMP-7-positive vessels were also stained for CD34. The density of MMP-7-positive vessels was determined by a computer-aided image analysis system. Multivariate analysis was done to assess relevant variables for invasion, metastasis, and cause-specific survival. RESULTS: The proportion of MMP-7-expressing tumor cells were significantly higher (P < 0.001) than that of normal cells. MMP-7-positive vessels were considered blood vessels based on staining for CD34, and their density was increased in tumor areas. The proportion of MMP-7-expressing cancer cells and density of MMP-7-positive vessels correlated with grade, pathologic tumor stage, and metastasis. Multivariate analysis showed that MMP-7 expression on cancer cells correlated with pathologic tumor stage only, whereas MMP-7-positive vessel density correlated with metastasis only. The elevated status of MMP-7 in cancer tissues was an independent predictor for cause-specific survival (odds ratio, 8.61; P = 0.040) by multivariate analysis. CONCLUSIONS: Our results showed that MMP-7 influences tumor progression by regulating invasion and angiogenesis. Multivariate analysis showed that MMP-7 status of cancer tissues was strong predictor of poor prognosis. Our results suggest that MMP-7 targeting treatment may be a potential target against RCC.     

Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis

Phage that display a surface peptide with the NGR sequence motif home selectively to tumor vasculature in vivo. A drug coupled to an NGR peptide has more potent antitumor effects than the free drug [W. Arap et al., Science (Washington DC), 279: 377-380, 1998]. We show here that the receptor for the NGR peptides in tumor vasculature is aminopeptidase N (APN; also called CD13). NGR phage specifically bound to immunocaptured APN and to cells engineered to express APN on their surface. Antibodies against APN inhibited in vivo tumor homing by the NGR phage. Immunohistochemical staining showed that APN expression is up-regulated in endothelial cells within mouse and human tumors. In another tissue that undergoes angiogenesis, corpus luteum, blood vessels also expressed APN, but APN was not detected in blood vessels of various other normal tissues stained under the same conditions. APN antagonists specifically inhibited angiogenesis in chorioallantoic membranes and in the retina and suppressed tumor growth. Thus, APN is involved in angiogenesis and can serve as a target for delivering drugs into tumors and for inhibiting angiogenesis.     

Downregulation of a newly identified laminin, laminin-3B11, in vascular basement membranes of invasive human breast cancers

Laminins present in the basement membranes (BM) of blood vessels are involved in angiogenesis and other vascular functions that are critical for tumor growth and metastasis. Two major vascular laminins, the α4 (laminin-411/421) and α5 (laminin-511/521) types, have been well characterized. We recently found a third type of vascular laminin, laminin-3B11, consisting of the α3B, β1 and γ1 chains, and revealed its biological activity. Laminin-3B11 potently stimulates vascular endothelial cells to extend lamellipodial protrusions. To understand the roles of laminin-3B11 in blood vessel functions and tumor growth, we examined localization of the laminin α3B chain in normal mammary glands and breast cancers, in comparison with the α4 and α5 laminins. In the immunohistochemical analysis, the α3B laminin was co-localized with the α4 and α5 laminins in the BM of venules and capillaries of normal breast tissues, but α3B was scarcely detected in vessels near invasive breast carcinoma cells. In contrast, the α4 laminin was overexpressed in capillaries of invasive carcinomas, where a large number of macrophages were found. The α5 laminin appeared to be weakly downregulated in cancer tissues, especially in capillary vessels. Furthermore, our in vitro analysis indicated that TNF-α significantly suppressed the laminin α3B expression in vascular endothelial cells, while it, as well as IL-1β and TGF-α, upregulated the α4 expression. These results suggest that Lm3B11/3B21 may be required for normal mature vessels and interfere with tumor angiogenesis.     

Macrophage-derived SHP-2 inhibits the metastasis of colorectal cancer via Tie2-PI3K signals

This research aimed to explore the influence of Src homology-2 containing protein tyrosine phosphatase (SHP- 2) on the functions of tyrosine kinase receptors with immunoglobulin and EGF homology domains 2 (Tie2)-expressing monocyte/macrophages (TEMs) and the influence of the angiopoietin(Ang)/Tie2-phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) (Ang/Tie2-PI3K/Akt/mTOR) signaling pathway on the tumor microvascular remodeling in an immunosuppressive microenvironment. In vivo, SHP-2- deficient mice were used to construct colorectal cancer (CRC) liver metastasis models. SHP-2-deficient mice had significantly more metastatic cancer and inhibited nodules on the liver surface than wild-type mice, and the high-level expression of p-Tie2 was found in the liver tissue of the macrophages’ specific SHP-2-deficient mice (SHP-2MACKO) + planted tumor mice. Compared with the SHP-2 wild type mice (SHP-2WT) + planted tumor group, the SHP-2MAC-KO + planted tumor group experienced increased expression of p-Tie2, p-PI3K, p-Akt, p-mTOR, vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), matrix metalloproteinase 2 (MMP2), and MMP9 in the liver tissue. TEMs selected by in vitro experiments were co-cultured with remodeling endothelial cells and tumor cells as carriers. It was found that when Angpt1/2 was used for stimulation, the SHP-2MAC-KO + Angpt1/2 group displayed evident increases in the expression of the Ang/Tie2-PI3K/Akt/mTOR pathway. The number of cells passing through the lower chamber and the basement membrane and the number of blood vessels formed by cells compared with the SHP-2WT + Angpt1/2 group, while these indexes were subjected to no changes under the simultaneous stimulation of Angpt1/2 + Neamine. To sum up, the conditional knockout of SHP-2 can activate the Ang/Tie2-PI3K/Akt/mTOR pathway in TEMs, thereby strengthening tumor micro angiogenesis in the microenvironment and facilitating CRC liver metastasis.