The Tie2 receptor tyrosine kinase is expressed in vascular endothelial cells, tumor-associated macrophages, and tumor cells and has been a major focus of research in therapies targeting the tumor microenvironment. The most extensively studied Tie2 ligands are Angiopoietin 1 and 2 (Ang1, Ang2). Ang1 plays a critical role in vessel maturation, endothelial cell migration, and survival. Ang2, depending on the context, may function to disrupt connections between the endothelial cells and perivascular cells, promoting vascular regression. However, in the presence of VEGF-A, Ang2 instead promotes angiogenesis. Tie2-expressing macrophages play a critical role in both tumor angiogenesis and the dissemination of tumor cells from the primary tumor to secondary sites. Therefore, Ang-Tie2 signaling functions as an angiogenic switch during tumor progression and metastasis.
2. Role of Ang-Tie2 in Tumor Angiogenesis
The Ang-Tie2 signaling pathway wields a critical and rate-limiting control over the early stages of tumor vascularization. Although Ang2 is only weakly expressed in quiescent ECs under physiological conditions, its expression—along with Tie2—is dramatically upregulated during hypoxia, which is a hallmark of the tumor microenvironment, making Ang2 an attractive candidate for anti-angiogenic therapies. To sustain tumor growth, the tumor must activate the angiogenic switch and co-opt the pre-existing host vasculature 
, leading to increased Ang2 production and secretion 
. Acting as a Tie2 antagonist, this causes increased EC permeability and vessel regression leading to an increase in hypoxia, which in turn increases VEGF-A expression and causes a robust angiogenic response. Working in concert with VEGF signaling, the Ang-Tie2 system subsequently signals in a manner analogous to that occurring during physiological angiogenesis.
In the tumor vasculature, Ang2 is upregulated and its expression is correlated with malignancy in several cancers 
. Ang2 can be detected at significant concentrations in the blood of tumor patients with esophageal squamous cell cancer 
, lung cancer 
, and hepatocellular carcinoma 
and the expression of Ang2 correlates with tumor progression in melanoma 
. The vasculature of glioblastoma tumors displays increased levels of Ang2 
, particularly in the necrotic and hypoxic regions 
and in vessels deficient in smooth muscle cell coverage 
. Although Ang2 expression is increased in the tumor vasculature and with tumor progression, the results of Ang2 expression modulation have been shown to be context-dependent and complicated. This is not surprising, given that Ang2 has been demonstrated to be a dose-dependent agonist or antagonist of Tie2 and to exhibit differential signaling based on the presence or absence of VEGF-A.
The overexpression of Ang2 in a rat glioma model led to aberrant vessel formation and decreased smooth muscle cell coverage 
whereas Ang2-neutralizing antibodies decreased tumor growth 
and also prevented VEGF-induced EC migration during angiogenesis 
, underscoring the role of Ang2 during VEGF-induced angiogenesis. The induction of Ang2 in glioma, breast cancer, pancreatic neuroendocrine tumors, and lung carcinoma inhibited tumor growth and metastasis 
. Ang2-deficient mice with Lewis lung carcinoma or melanoma exhibited a decreased initial tumor growth and a more mature vasculature with pericyte coverage compared with wildtype mice. However, the tumor growth rates were similar during the later stages 
. In spontaneous mouse models of breast cancer, Ang2 inhibition led to decreased tumor angiogenesis and metastasis 
. This could be due to the effects of Ang2 on Tie2-expressing macrophages (discussed below) as Ang2 inhibition decreases the upregulation of Tie2 in tumor-associated macrophages and their association with the tumor vasculature 
. These studies highlight that the modulation of Ang2 expression (either up- or downregulation) causes altered mural cell coverage and vessel maturity resulting in delayed tumor growth.
The mechanisms by which the modulation of Ang2 expression affects tumor growth and metastasis are not yet fully understood. The studies described above suggest that the overall balance between Ang1 and Ang2 expression likely plays a crucial role in determining the effect on tumor angiogenesis and metastasis. Indeed, in many cancer types, tumor angiogenesis and a poor prognosis are associated with an increased ratio of Ang2 expression levels relative to Ang1 
. Recently, combination therapies of Ang2 inhibition and cytotoxic drugs with agents targeting tyrosine kinases or VEGF-A have shown enhanced anti-tumor effects compared with monotherapy 
. DAAP (double anti-angiogenic protein), a chimeric decoy receptor that simultaneously blocks all angiopoietins and VEGF-A, inhibited tumor angiogenesis and metastasis 
. These studies suggest that utilizing therapies to simultaneously inhibit both VEGF and Ang-Tie2 signaling is a promising strategy to block vascular permeability, tumor angiogenesis, and metastasis.
3. Role of Tie2+ Macrophages in Tumor Progression and Metastasis Formation
Hematopoietic cells of diverse lineages, including macrophages, contribute to tumor progression 
. Monocytes are recruited from the bloodstream into tumors where they differentiate into tumor-associated macrophages. There, they contribute to tumor progression by blocking anti-tumor immunity and promoting angiogenesis, cell migration, invasion, and metastasis 
. High numbers of macrophages in human primary tumor specimens correlate with increased tumor vascularization 
. Specifically, a subset of tumor-associated macrophages that express Tie2 plays a crucial role in both tumor angiogenesis 
and the dissemination of tumor cells from the primary tumor to secondary sites 
. In this section, we will describe our current understanding of the function of Tie2-expressing macrophages in both tumor angiogenesis and dissemination.
3.1. Tie2 Macrophages Regulate Tumor Angiogenesis
As described in Section 2
, during angiogenesis, tumor cells and associated immune cells recruit endothelial cells from the surrounding vasculature to form new blood vessels 
. Interactions between these cell types trigger signaling pathways that result in the formation of new blood vessels and finally in tumor vascularization 
. In particular, tumor-associated macrophages that express Tie2 receptors (previously thought to be restricted to endothelial and hematopoietic stem cells) migrate toward, and adhere to, sprouting blood vessels. These macrophages are essential for vascular anastomosis and the formation of a functional vessel system 
. The elimination of Tie2 macrophages by a suicide gene impairs angiogenesis and reduces tumor growth 
. Additionally, recent studies suggest that Tie2 macrophages contribute to metastatic relapse because, following chemotherapy treatments, they are recruited into tumors to promote vascular reconstruction 
. The deletion of Tie2 macrophages impairs the metastatic relapse after chemotherapy and the mechanism by which Tie2 macrophages contribute to this process is becoming clearer.
3.2. Tie2 Macrophages Regulate Vascular Permeably and Intravasation
In addition to tumor angiogenesis, Tie2-expressing macrophages play an important role in tumor cell intravasation and metastasis 
. Using multiphoton intravital imaging to study mammary carcinomas, it was found that tumor cell intravasation does not occur throughout the cancer-associated vascular endothelium. Rather, entry is localized to specific microanatomical doorways called TMEM (tumor microenvironment of metastasis) 
. Following intravasation via TMEM doorways, the tumor cells enter the circulation and disseminate to secondary sites. Each TMEM doorway is composed of a three cell complex involving an endothelial cell, a perivascular Tie2high
macrophage, and a tumor cell expressing high levels of Mena (an actin-regulatory protein that influences cell cohesion and motility), all in direct and stable contact with each other 
. Although many macrophage subtypes may be present in perivascular regions, only macrophages expressing high levels of Tie2 are capable of assembling functional TMEM structures 
. The TMEM macrophage reduces the cohesion of the endothelial junctions in the immediate vicinity of TMEM through the transient secretion of VEGF-A. This causes localized and transient vascular opening, during which MenaINVhigh
migratory tumor cells intravasate and disseminate to secondary sites 
The critical role of Tie2high
macrophages in TMEM doorway function was further demonstrated by the conditional ablation of macrophages in PyMT transgenic mice and by VEGF deletion in the monocyte/macrophage lineage in FVB mice; in both cases, there was a dramatic inhibition of TMEM function and, consequently, of tumor cell intravasation and metastasis formation 
. Additionally, these proangiogenic perivascular Tie2 macrophages can be inhibited by the Tie2 inhibitor rebastinib 
. This offers new treatment options by targeting TMEM-associated vascular opening and tumor cell dissemination. The pharmacological inhibition of Tie2 macrophages is discussed in the following section.
TMEM doorways have been detected in human mammary carcinomas and their density is a clinically validated prognostic marker for distant recurrence in breast cancer patients 
. Recently, it was found that TMEM doorways are also present at secondary sites in the mouse and human lung metastases of breast cancer patients 
as well as in lymph node tumor cell nests 
. This finding suggests that TMEM doorway-mediated tumor cell dissemination occurs not only from the primary tumor but also from the secondary sites, leading to an increased metastatic burden and hastening the death of stage IV breast cancer patients. Indeed, using photo-convertible fluorescent protein-based fate mapping, it has been possible to mark tumor cells within established lung metastases and track redisseminated tumor cells to tertiary sites such as the bone marrow 
3.3. Targeting Tie2 to Prevent Tumor Progression and Metastasis Formation
The identification of Tie2 macrophages as a key player in tumor angiogenesis, intravasation, and immunosuppression offers the possibility of combination therapies that, in addition to targeting tumor cells, target the cells of the tumor microenvironment. Although anti-angiogenic treatments such as bevacizumab and other VEGF-A pathway inhibitors have shown an initial efficacy in decreasing tumor angiogenesis and disease burden, patients rapidly develop a resistance to these agents 
. This is due to tumor infiltration by immune cells, especially Tie2-expressing macrophages, in response to hypoxia and cell death after vascular regression 
. Adding agents that target Ang-Tie2 signaling between ECs and macrophages such as Tie2-neutralizing antibodies, siRNA-targeting Tie2, and inducible Tie2 knockdown in the hematopoietic stem cells in addition to anti-angiogenic agents has yielded promising pre-clinical results, blocking tumor angiogenesis, growth, and metastasis 
As tumor cells disseminate via the TMEM doorways 
, the pharmacological inhibition of the TMEM doorways to seal them to tumor cell intravasation is a very promising novel therapeutic approach to block intravasation, the dissemination of tumor cells, and, ultimately, metastasis formation. As previously described, the TMEM doorway is composed of a perivascular Tie2high
macrophage, a Mena-expressing tumor cell, and an endothelial cell, all in direct stable contact with each other 
. To date, there is not a specific pharmacological approach to target Mena-expressing tumor cells nor a specific approach to block the association of ECs with TMEM doorways. Therefore, blocking the Tie2 macrophage is the most promising strategy to seal the TMEM doorways to intravasation.
Recent pre-clinical studies of metastatic mammary carcinoma and pancreatic neuroendocrine tumors in orthotopic mouse models showed that rebastinib reduces tumor growth, angiogenesis, and metastasis and significantly increases the overall survival of paclitaxel-treated mice even after the resection of the primary tumor 
. By inhibiting Tie2 in tumor-associated macrophages, this drug prevented the VEGF-dependent vascular opening associated with the TMEM doorways. Consequently, rebastinib reduced the circulating tumor cells and their dissemination to the lungs 
. Rebastinib is now being tested in several clinical trials; three in solid tumors and one in leukemias. NCT02824575 is a phase 1b trial for breast cancer in women with HER2-negative adenocarcinoma of the breast, metastatic breast cancer, or women with prior chemotherapy and/or endocrine therapy where rebastinib is being used in combination with an anti-tubulin therapy, paclitaxel, or eribulin. NCT03717415 is phase 1b/2 trial for patients with locally advanced or metastatic solid tumors testing rebastinib in combination with carboplatin. The inclusion criterion for the dose escalation phase (Part 1) is patients who have been diagnosed with locally advanced or metastatic solid tumors for which carboplatin, an alkylating agent, is considered to be an appropriate treatment. The dose expansion phase (Part 2) includes patients with previously treated triple negative breast cancer, recurrent platinum-sensitive ovarian cancer, or pleural or peritoneal malignant mesothelioma. NCT03601897 is a phase 1b/2 trial for patients with locally advanced or metastatic solid tumors for which paclitaxel is considered to be an appropriate treatment. Part 2 of the trial will be completed next year and includes patients with triple negative and stage IV inflammatory breast cancer, recurrent ovarian cancer, recurrent or metastatic endometrial cancer, or advanced gynecological carcinosarcoma.
Several other small-molecule tyrosine kinase inhibitors that target Tie2 along with VEGFR2 and other RTKs have been tested in recent clinical trials. Ripretinib is a c-Kit, VEGFR2, PDGFRα, and Tie2 inhibitor approved for use as a salvage therapy for patients with advanced gastrointestinal stroma tumors who have received prior treatments with three or more kinase inhibitors (INVICTUS/NCT03353753) 
. Regorafenib is a multikinase inhibitor of Tie2, VEGFR1/2/3, and FGFRs and has achieved approval as a salvage therapy for metastatic colorectal cancer, advanced gastrointestinal stromal tumors, and advanced hepatocellular carcinoma (NCT01103323) 
The identification of functional TMEM doorways not only in primary tumors but also in secondary metastatic sites demonstrates that TMEM doorways are not restricted solely to the primary tumor microenvironment but are additionally implicated in tumor cell redissemination from metastatic sites. These studies suggest that TMEM doorways are a common mechanism of tumor cell dissemination during all stages of breast cancer progression, indicating that it is never too late to block dissemination and to treat metastatic patients