Lung endothelium exploits susceptible tumor cell states to instruct metastatic latency.

In metastasis, cancer cells travel around the circulation to colonize distant sites. Due to the rarity of these events, the immediate fates of metastasizing tumor cells (mTCs) are poorly understood while the role of the endothelium as a dissemination interface remains elusive. Using a newly developed combinatorial mTC enrichment approach, we provide a transcriptional blueprint of the early colonization process. Following their arrest at the metastatic site, mTCs were found to either proliferate intravascularly or extravasate, thereby establishing metastatic latency. Endothelial-derived angiocrine Wnt factors drive this bifurcation, instructing mTCs to follow the extravasation-latency route. Surprisingly, mTC responsiveness towards niche-derived Wnt was established at the epigenetic level, which predetermined tumor cell behavior. Whereas hypomethylation enabled high Wnt activity leading to metastatic latency, methylated mTCs exhibited low activity and proliferated intravascularly. Collectively the data identify the predetermined methylation status of disseminated tumor cells as a key regulator of mTC behavior in the metastatic niche.

The angiopoietin receptor Tie2 is atheroprotective in arterial endothelium.

Leukocytes and resident cells in the arterial wall contribute to atherosclerosis, especially at sites of disturbed blood flow. Expression of endothelial Tie1 receptor tyrosine kinase is enhanced at these sites, and attenuation of its expression reduces atherosclerotic burden and decreases inflammation. However, Tie2 tyrosine kinase function in atherosclerosis is unknown. Here we provide genetic evidence from humans and from an atherosclerotic mouse model to show that TIE2 is associated with protection from coronary artery disease. We show that deletion of Tie2, or both Tie2 and Tie1, in the arterial endothelium promotes atherosclerosis by increasing Foxo1 nuclear localization, endothelial adhesion molecule expression and accumulation of immune cells. We also show that Tie2 is expressed in a subset of aortic fibroblasts, and its silencing in these cells increases expression of inflammation-related genes. Our findings indicate that unlike Tie1, the Tie2 receptor functions as the dominant endothelial angiopoietin receptor that protects from atherosclerosis.

Systemic Reprogramming of Endothelial Cell Signaling in Metastasis and Cachexia.

Proliferating cancer cells secrete a multitude of factors impacting metabolism, interorgan communication, and tumor progression. The distribution of tumor-derived factors to distant organs occurs via the circulation, which provides an extensive reactive surface lined by endothelial cells. Primary tumor-derived proteins impact cancer progression by modulating endothelial cell activation at the (pre-)metastatic niche, which affects tumor cell dissemination as well as the outgrowth of seeded metastatic cells into overt tumors. In addition, new insight indicates that endothelial cell signaling contributes to metabolic symptoms of cancer, including cancer-associated cachexia, opening a new field of vascular metabolism research. This review addresses how tumor-derived factors systemically affect endothelial cell signaling and activation and impact distant organs as well as tumor progression.

Active Remodeling of Capillary Endothelium via Cancer Cell-Derived MMP9 Promotes Metastatic Brain Colonization.

Crossing the blood-brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and endpoint in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (EC) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by cross-talk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it. SIGNIFICANCE: Tracking single extravasating cancer cells using multimodal correlative microscopy uncovers a brain seeding mechanism involving endothelial remodeling driven by cancer cell-derived MMP9, which might enable the development of approaches to prevent brain metastasis. See related commentary by McCarty, p. 1167.

Sphingosine-1-Phosphate Recruits Macrophages and Microglia and Induces a Pro-Tumorigenic Phenotype That Favors Glioma Progression.

Glioblastoma is the most aggressive brain tumor in adults. Treatment failure is predominantly caused by its high invasiveness and its ability to induce a supportive microenvironment. As part of this, a major role for tumor-associated macrophages/microglia (TAMs) in glioblastoma development was recognized. Phospholipids are important players in various fundamental biological processes, including tumor-stroma crosstalk, and the bioactive lipid sphingosine-1-phosphate (S1P) has been linked to glioblastoma cell proliferation, invasion, and survival. Despite the urgent need for better therapeutic approaches, novel strategies targeting sphingolipids in glioblastoma are still poorly explored. Here, we showed that higher amounts of S1P secreted by glioma cells are responsible for an active recruitment of TAMs, mediated by S1P receptor (S1PR) signaling through the modulation of Rac1/RhoA. This resulted in increased infiltration of TAMs in the tumor, which, in turn, triggered their pro-tumorigenic phenotype through the inhibition of NFkB-mediated inflammation. Gene set enrichment analyses showed that such an anti-inflammatory microenvironment correlated with shorter survival of glioblastoma patients. Inhibition of S1P restored a pro-inflammatory phenotype in TAMs and resulted in increased survival of tumor-bearing mice. Taken together, our results establish a crucial role for S1P in fine-tuning the crosstalk between glioma and infiltrating TAMs, thus pointing to the S1P-S1PR axis as an attractive target for glioma treatment.

Activating NO-sGC crosstalk in the mouse vascular niche promotes vascular integrity and mitigates acute lung injury.

Disruption of endothelial cell (ECs) and pericytes interactions results in vascular leakage in acute lung injury (ALI). However, molecular signals mediating EC-pericyte crosstalk have not been systemically investigated, and whether targeting such crosstalk could be adopted to combat ALI remains elusive. Using comparative genome-wide EC-pericyte crosstalk analysis of healthy and LPS-challenged lungs, we discovered that crosstalk between endothelial nitric oxide and pericyte soluble guanylate cyclase (NO-sGC) is impaired in ALI. Indeed, stimulating the NO-sGC pathway promotes vascular integrity and reduces lung edema and inflammation-induced lung injury, while pericyte-specific sGC knockout abolishes this protective effect. Mechanistically, sGC activation suppresses cytoskeleton rearrangement in pericytes through inhibiting VASP-dependent F-actin formation and MRTFA/SRF-dependent de novo synthesis of genes associated with cytoskeleton rearrangement, thereby leading to the stabilization of EC-pericyte interactions. Collectively, our data demonstrate that impaired NO-sGC crosstalk in the vascular niche results in elevated vascular permeability, and pharmacological activation of this crosstalk represents a promising translational therapy for ALI.

Primary tumor-derived systemic nANGPTL4 inhibits metastasis.

Primary tumors and distant site metastases form a bidirectionally communicating system. Yet, the molecular mechanisms of this crosstalk are poorly understood. Here, we identified the proteolytically cleaved fragments of angiopoietin-like 4 (ANGPTL4) as contextually active protumorigenic and antitumorigenic contributors in this communication ecosystem. Preclinical studies in multiple tumor models revealed that the C-terminal fragment (cANGPTL4) promoted tumor growth and metastasis. In contrast, the N-terminal fragment of ANGPTL4 (nANGPTL4) inhibited metastasis and enhanced overall survival in a postsurgical metastasis model by inhibiting WNT signaling and reducing vascularity at the metastatic site. Tracing ANGPTL4 and its fragments in tumor patients detected full-length ANGPTL4 primarily in tumor tissues, whereas nANGPTL4 predominated in systemic circulation and correlated inversely with disease progression. The study highlights the spatial context of the proteolytic cleavage-dependent pro- and antitumorigenic functions of ANGPTL4 and identifies and validates nANGPTL4 as a novel biomarker of tumor progression and antimetastatic therapeutic agent.

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression.

Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C-induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C-induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.

Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice.

The gut has a specific vascular barrier that controls trafficking of antigens and microbiota into the bloodstream. However, the molecular mechanisms regulating the maintenance of this vascular barrier remain elusive. Here, we identified Caspase-8 as a pro-survival factor in mature intestinal endothelial cells that is required to actively maintain vascular homeostasis in the small intestine in an organ-specific manner. In particular, we find that deletion of Caspase-8 in endothelial cells results in small intestinal hemorrhages and bowel inflammation, while all other organs remained unaffected. We also show that Caspase-8 seems to be particularly needed in lymphatic endothelial cells to maintain gut homeostasis. Our work demonstrates that endothelial cell dysfunction, leading to the breakdown of the gut-vascular barrier, is an active driver of chronic small intestinal inflammation, highlighting the role of the intestinal vasculature as a safeguard of organ function.

Viewpoints: Dual-blocking antibody against VEGF-A and angiopoietin-2 for treating vascular diseases of the eye.

Faricimab, a bispecific antibody that targets the endothelial cell growth factors vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Angpt2), was recently approved for treating neovascular age-related macular degeneration and diabetic macular edema. Here, Koh and Augustin review how mechanistic studies have translated into therapies, while Campochiaro evaluates their impact and value for clinical practice.

Tie2 Receptor in Tumor-Infiltrating Macrophages Is Dispensable for Tumor Angiogenesis and Tumor Relapse after Chemotherapy.

Tumor relapse after chemotherapy relies on the reconstruction of damaged tumor vasculature. In this context, proangiogenic Tie2-expressing macrophages have been suggested to serve as crucial instructors of tumor revascularization by secreting angiogenic factors while being closely associated with the vessel wall. Although the proangiogenic nature of Tie2+ macrophages is well described, the functional contribution of macrophage Tie2 expression remains elusive. Here, we employed a Cre-loxP system to specifically delete Tie2 in macrophages. In multiple syngeneic solid tumor models and two distinct chemotherapeutic treatment regimens, macrophage-expressed Tie2 did not contribute to primary tumor growth, tumor revascularization after chemotherapy, tumor recurrence, or metastasis. Exposing cultured murine macrophage cell lines and bone marrow-derived macrophages to hypoxia or stimulating them with Ang2 did not induce expression of Tie2 at the RNA or protein level. Furthermore, a comprehensive meta-analysis of publicly available single cell RNA sequencing datasets of human and murine tumor-infiltrating CD11b+ myeloid cells did not reveal a transcriptionally distinct macrophage population marked by the expression of Tie2. Collectively, these data question the previously reported critical role of Tie2-expressing macrophages for tumor angiogenesis and tumor relapse after chemotherapy. Moreover, lack of Tie2 inducibility and absence of Tie2-positive macrophages in multiple recently published tumor studies refute a possible prognostic value of macrophage-expressed Tie2. SIGNIFICANCE: Multiple preclinical tumor models, cell stimulation experiments, and meta-analysis of published tumor single cell RNA sequencing data challenge the reported role of Tie2-positive macrophages for tumor angiogenesis, metastasis, and relapse after chemotherapy. See related commentary by Zhang and Brekken, p. 1172.

Antiangiogenesis: Vessel Regression, Vessel Normalization, or Both?

The concepts of antiangiogenic tumor therapy were pioneered on the assumption that the inhibition of tumor angiogenesis should lead to the complete regression of the tumor-associated vasculature and thereby hold the tumor in an avascular dormant state. Yet, clinical trials revealed limited efficacy of angiogenesis inhibitors when used as monotherapy. Instead, antiangiogenic drugs proved effective to extend overall survival when used in combination with chemotherapy. This counterintuitive observation-inhibition of tumor vascularization should lead to less and not more delivery of chemotherapy to the tumor-led to the concepts of "vessel normalization." This refers to the notion that antiangiogenic drugs prune the most immature tumor vessels and spare mature vessels, thereby resulting in a more normal-appearing vasculature that leads to better access of chemotherapy to the tumor. The concepts of vessel normalization were first laid out in a landmark publication in Cancer Research in 2004. More than 600 studies on different aspects of vessel normalization have been published since then. Nevertheless, it is to this day less clear than ever to what extent vessel regression (leading to tumor starvation) and vessel normalization (facilitating chemotherapy) contribute to the clinical efficacy of antiangiogenic tumor therapy. This "Landmark Commentary" puts the concepts of tumor vessel normalization in historical context and develops thereupon some of the most burning questions in the field of translational angiogenesis research that need to be answered to further advance the application of tumor vascular stroma reprogramming therapies.See related article by Tong and colleagues, Cancer Res 2004;64:3731-6.

STAT3-YAP/TAZ signaling in endothelial cells promotes tumor angiogenesis.

The nuclear translocation and activity of the cotranscriptional activators YAP and TAZ (YAP/TAZ) in endothelial cells (ECs) are crucial during developmental angiogenesis. Here, we studied the role of YAP/TAZ signaling in ECs in tumor angiogenesis and found that the expression of YAP/TAZ and downstream target genes in ECs correlated with tumor vascularization in human colorectal carcinomas and skin melanoma. Treatment with the YAP/TAZ inhibitor verteporfin reduced vessel density and tumor progression in a mouse colorectal cancer (CRC) model. Conditional deletion of YAP/TAZ in ECs reduced tumor angiogenesis and growth in a mouse B16-F10 melanoma model. Using cultured ECs and mice with EC-specific ablation, we showed that signal transducer and activator of transcription 3 (STAT3) was required for the activation of YAP/TAZ in tumor-associated ECs. Moreover, we showed that STAT3-mediated signaling promoted YAP/TAZ activity and that the nuclear shuttling machinery for STAT3 was also required for YAP/TAZ nuclear translocation. Together, our data highlight the role of YAP/TAZ as critical players in ECs during tumor angiogenesis and provide insight into the signaling pathways leading to their activation.

Temporal multi-omics identifies LRG1 as a vascular niche instructor of metastasis.

Metastasis is the primary cause of cancer-related mortality. Tumor cell interactions with cells of the vessel wall are decisive and potentially rate-limiting for metastasis. The molecular nature of this cross-talk is, beyond candidate gene approaches, hitherto poorly understood. Using endothelial cell (EC) bulk and single-cell transcriptomics in combination with serum proteomics, we traced the evolution of the metastatic vascular niche in surgical models of lung metastasis. Temporal multiomics revealed that primary tumors systemically reprogram the body's vascular endothelium to perturb homeostasis and to precondition the vascular niche for metastatic growth. The vasculature with its enormous surface thereby serves as amplifier of tumor-induced instructive signals. Comparative analysis of lung EC gene expression and secretome identified the transforming growth factor­ß (TGFß) pathway specifier LRG1, leucine-rich alpha-2-glycoprotein 1, as an early instructor of metastasis. In the presence of a primary tumor, ECs systemically up-regulated LRG1 in a signal transducer and activator of transcription 3 (STAT3)­dependent manner. A meta-analysis of retrospective clinical studies revealed a corresponding up-regulation of LRG1 concentrations in the serum of patients with cancer. Functionally, systemic up-regulation of LRG1 promoted metastasis in mice by increasing the number of prometastatic neural/glial antigen 2 (NG2)+ perivascular cells. In turn, genetic deletion of Lrg1 hampered growth of lung metastasis. Postsurgical adjuvant administration of an LRG1-neutralizing antibody delayed metastatic growth and increased overall survival. This study has established a systems map of early primary tumor-induced vascular changes and identified LRG1 as a therapeutic target for metastasis.

The angiopoietin-Tie2 pathway regulates Purkinje cell dendritic morphogenesis in a cell-autonomous manner.

Neuro-vascular communication is essential to synchronize central nervous system development. Here, we identify angiopoietin/Tie2 as a neuro-vascular signaling axis involved in regulating dendritic morphogenesis of Purkinje cells (PCs). We show that in the developing cerebellum Tie2 expression is not restricted to blood vessels, but it is also present in PCs. Its ligands angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are expressed in neural cells and endothelial cells (ECs), respectively. PC-specific deletion of Tie2 results in reduced dendritic arborization, which is recapitulated in neural-specific Ang1-knockout and Ang2 full-knockout mice. Mechanistically, RNA sequencing reveals that Tie2-deficient PCs present alterations in gene expression of multiple genes involved in cytoskeleton organization, dendritic formation, growth, and branching. Functionally, mice with deletion of Tie2 in PCs present alterations in PC network functionality. Altogether, our data propose Ang/Tie2 signaling as a mediator of intercellular communication between neural cells, ECs, and PCs, required for proper PC dendritic morphogenesis and function.

Visceral obesity and insulin resistance associate with CD36 deletion in lymphatic endothelial cells.

Disruption of lymphatic lipid transport is linked to obesity and type 2 diabetes (T2D), but regulation of lymphatic vessel function and its link to disease remain unclear. Here we show that intestinal lymphatic endothelial cells (LECs) have an increasing CD36 expression from lymphatic capillaries (lacteals) to collecting vessels, and that LEC CD36 regulates lymphatic integrity and optimizes lipid transport. Inducible deletion of CD36 in LECs in adult mice (Cd36ΔLEC) increases discontinuity of LEC VE-cadherin junctions in lacteals and collecting vessels. Cd36ΔLEC mice display slower transport of absorbed lipid, more permeable mesenteric lymphatics, accumulation of inflamed visceral fat and impaired glucose disposal. CD36 silencing in cultured LECs suppresses cell respiration, reduces VEGF-C-mediated VEGFR2/AKT phosphorylation and destabilizes VE-cadherin junctions. Thus, LEC CD36 optimizes lymphatic junctions and integrity of lymphatic lipid transport, and its loss in mice causes lymph leakage, visceral adiposity and glucose intolerance, phenotypes that increase risk of T2D.

A spatial vascular transcriptomic, proteomic, and phosphoproteomic atlas unveils an angiocrine Tie-Wnt signaling axis in the liver.

Single-cell transcriptomics (scRNA-seq) has revolutionized the understanding of the spatial architecture of tissue structure and function. Advancing the "transcript-centric" view of scRNA-seq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyze post-translational modifications. Here, by combining spatial cell sorting with transcriptomics and quantitative proteomics/phosphoproteomics, we established the spatially resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signaling mechanisms. Phosphorylation of receptor tyrosine kinases was detected preferentially in the central vein area, resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation identified Tie receptor signaling as a selective and specific regulator of vascular Wnt activity orchestrating angiocrine signaling, thereby controlling hepatocyte function during liver regeneration. Taken together, the study has yielded fundamental insight into the spatial organization of liver endothelial cell signaling. Spatial sorting may be employed as a universally adaptable strategy for multiomic analyses of scRNA-seq-defined cellular (sub)-populations.