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.

Timed Ang2-Targeted Therapy Identifies the Angiopoietin-Tie Pathway as Key Regulator of Fatal Lymphogenous Metastasis.

Recent clinical and preclinical advances have highlighted the existence of a previously hypothesized lymphogenous route of metastasis. However, due to a lack of suitable preclinical modeling tools, its contribution to long-term disease outcome and relevance for therapy remain controversial. Here, we established a genetically engineered mouse model (GEMM) fragment-based tumor model uniquely sustaining a functional network of intratumoral lymphatics that facilitates seeding of fatal peripheral metastases. Multiregimen survival studies and correlative patient data identified primary tumor-derived Angiopoietin-2 (Ang2) as a potent therapeutic target to restrict lymphogenous tumor cell dissemination. Mechanistically, tumor-associated lymphatic endothelial cells (EC), in contrast to blood vascular EC, were found to be critically addicted to the Angiopoietin-Tie pathway. Genetic manipulation experiments in combination with single-cell mapping revealed agonistically acting Ang2-Tie2 signaling as key regulator of lymphatic maintenance. Correspondingly, acute presurgical Ang2 neutralization was sufficient to prolong survival by regressing established intratumoral lymphatics, hence identifying a therapeutic regimen that warrants further clinical evaluation. SIGNIFICANCE: Exploiting multiple mouse tumor models including a unique GEMM-derived allograft system in combination with preclinical therapy designs closely matching the human situation, this study provides fundamental insight into the biology of tumor-associated lymphatic EC and defines an innovative presurgical therapeutic window of migrastatic Ang2 neutralization to restrict lymphogenous metastasis.This article is highlighted in the In This Issue feature, p. 211.

Preclinical validation of a novel metastasis-inhibiting Tie1 function-blocking antibody.

The angiopoietin (Ang)-Tie pathway has been intensely pursued as candidate second-generation anti-angiogenic target. While much of the translational work has focused on the ligand Ang2, the clinical efficacy of Ang2-targeting drugs is limited and failed to improve patient survival. In turn, the orphan receptor Tie1 remains therapeutically unexplored, although its endothelial-specific genetic deletion has previously been shown to result in a strong reduction in metastatic growth. Here, we report a novel Tie1 function-blocking antibody (AB-Tie1-39), which suppressed postnatal retinal angiogenesis. During primary tumor growth, neoadjuvant administration of AB-Tie1-39 strongly impeded systemic metastasis. Furthermore, the administration of AB-Tie1-39 in a perioperative therapeutic window led to a significant survival advantage as compared to control-IgG-treated mice. Additional in vivo experimental metastasis and in vitro transmigration assays concurrently revealed that AB-Tie1-39 treatment suppressed tumor cell extravasation at secondary sites. Taken together, the data phenocopy previous genetic work in endothelial Tie1 KO mice and thereby validate AB-Tie1-39 as a Tie1 function-blocking antibody. The study establishes Tie1 as a therapeutic target for metastasis in a perioperative or neoadjuvant setting.

Cytokine-Like 1 Is a Novel Proangiogenic Factor Secreted by and Mediating Functions of Endothelial Progenitor Cells.

RATIONALE: Endothelial colony forming cells (ECFCs) or late blood outgrowth endothelial cells can be isolated from human cord or peripheral blood, display properties of endothelial progenitors, home into ischemic tissues and support neovascularization in ischemic disease models. OBJECTIVE: To assess the functions of CYTL1 (cytokine-like 1), a factor we found preferentially produced by ECFCs, in regard of vessel formation. METHODS AND RESULTS: We show by transcriptomic analysis that ECFCs are distinguished from endothelial cells of the vessel wall by production of high amounts of CYTL1. Modulation of expression demonstrates that the factor confers increased angiogenic sprouting capabilities to ECFCs and can also trigger sprouting of mature endothelial cells. The data further display that CYTL1 can be induced by hypoxia and that it functions largely independent of VEGF-A (vascular endothelial growth factor-A). By recombinant production of CYTL1 we confirm that the peptide is indeed a strong proangiogenic factor and induces sprouting in cellular assays and functional vessel formation in animal models comparable to VEGF-A. Mass spectroscopy corroborates that CYTL1 is specifically O-glycosylated on 2 neighboring threonines in the C-terminal part and this modification is important for its proangiogenic bioactivity. Further analyses show that the factor does not upregulate proinflammatory genes and strongly induces several metallothionein genes encoding anti-inflammatory and antiapoptotic proteins. CONCLUSIONS: We conclude that CYTL1 can mediate proangiogenic functions ascribed to endothelial progenitors such as ECFCs in vivo and may be a candidate to support vessel formation and tissue regeneration in ischemic pathologies.

Endothelial cell fitness dictates the source of regenerating liver vasculature.

Neoangiogenesis plays a key role in diverse pathophysiological conditions, including liver regeneration. Yet, the source of new endothelial cells (ECs) remains elusive. By analyzing the regeneration of the liver vasculature in irradiation-based myeloablative and nonmyeloablative bone marrow transplantation mouse models, we discovered that neoangiogenesis in livers with intact endothelium was solely mediated by proliferation of resident ECs. However, following irradiation-induced EC damage, bone marrow-derived mononuclear cells were recruited and incorporated into the vasculature. Further experiments with direct bone marrow infusion or granulocyte colony-stimulating factor (G-CSF)-mediated progenitor cell mobilization, which resembles clinically relevant stem cell therapy, demonstrated that bone marrow-derived cells did not contribute to the regeneration of liver vasculature after two-thirds partial hepatectomy (PHx). Taken together, the data reconcile many of the discrepancies in the literature and highlight that the cellular source of regenerating endothelium depends on the fitness of the residual vasculature.

Hepatic stellate cells limit hepatocellular carcinoma progression through the orphan receptor endosialin.

Hepatocellular carcinoma (HCC) is among the most common and deadliest cancers worldwide. A major contributor to HCC progression is the cross talk between tumor cells and the surrounding stroma including activated hepatic stellate cells (HSC). Activation of HSC during liver damage leads to upregulation of the orphan receptor endosialin (CD248), which contributes to regulating the balance of liver regeneration and fibrosis. Based on the established role of endosialin in regulating HSC/hepatocyte cross talk, we hypothesized that HSC-expressed endosialin might similarly affect cell proliferation during hepatocarcinogenesis. Indeed, the histological analysis of human HCC samples revealed an inverse correlation between tumor cell proliferation and stromal endosialin expression. Correspondingly, global genetic inactivation of endosialin resulted in accelerated tumor growth in an inducible mouse HCC model. A candidate-based screen of tumor lysates and differential protein arrays of cultured HSC identified several established hepatotropic cytokines, including IGF2, RBP4, DKK1, and CCL5 as being negatively regulated by endosialin. Taken together, the experiments identify endosialin-expressing HSC as a negative regulator of HCC progression.

Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage.

Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (EN(KO)) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in EN(KO) mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings.

Postsurgical adjuvant tumor therapy by combining anti-angiopoietin-2 and metronomic chemotherapy limits metastatic growth.

Antiangiogenic tumor therapy has failed in the adjuvant setting. Here we show that inhibition of the Tie2 ligand angiopoietin-2 (Ang2) effectively blocks metastatic growth in preclinical mouse models of postsurgical adjuvant therapy. Ang2 antibody treatment combines well with low-dose metronomic chemotherapy (LDMC) in settings in which maximum-dose chemotherapy does not prove effective. Mechanistically, Ang2 blockade could be linked to quenching the inflammatory and angiogenic response of endothelial cells (ECs) in the metastatic niche. Reduced EC adhesion molecule and chemokine expression inhibits the recruitment of tumor-promoting CCR2(+)Tie2(-) metastasis-associated macrophages. Moreover, LDMC contributes to therapeutic efficacy by inhibiting the recruitment of protumorigenic bone marrow-derived myeloid cells. Collectively, these data provide a rationale for mechanism-guided adjuvant tumor therapies.

Endothelial cell-derived angiopoietin-2 controls liver regeneration as a spatiotemporal rheostat.

Liver regeneration requires spatially and temporally precisely coordinated proliferation of the two major hepatic cell populations, hepatocytes and liver sinusoidal endothelial cells (LSECs), to reconstitute liver structure and function. The underlying mechanisms of this complex molecular cross-talk remain elusive. Here, we show that the expression of Angiopoietin-2 (Ang2) in LSECs is dynamically regulated after partial hepatectomy. During the early inductive phase of liver regeneration, Ang2 down-regulation leads to reduced LSEC transforming growth factor-ß1 production, enabling hepatocyte proliferation by releasing an angiocrine proliferative brake. During the later angiogenic phase of liver regeneration, recovery of endothelial Ang2 expression enables regenerative angiogenesis by controlling LSEC vascular endothelial growth factor receptor 2 expression. The data establish LSECs as a dynamic rheostat of liver regeneration, spatiotemporally orchestrating hepatocyte and LSEC proliferation through angiocrine- and autocrine-acting Ang2, respectively.

Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling.

Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.

Endosialin (Tem1) is a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells.

Endosialin (Tem1) has been identified by two independent experimental approaches as an antigen of tumor-associated endothelial cells, and it has been claimed to be the most abundantly expressed tumor endothelial antigen, making it a prime candidate for vascular targeting purposes. Recent experiments have challenged the endothelial expression of endosialin and suggested an expression by activated fibroblasts and pericytes. Thus, clarification of the controversial cellular expression of endosialin is critically important for an understanding of its role during tumor progression and its validation as a potential therapeutic target. We have therefore performed extensive expression profiling analyses of endosialin. The experiments unambiguously demonstrate that endosialin is expressed by tumor-associated myofibroblasts and mural cells and not by endothelial cells. Endosialin expression is barely detectable in normal human tissues with moderate expression only detectable in the stroma of the colon and the prostate. Corresponding cellular experiments confirmed endosialin expression by mesenchymal cells and indicated that it may in fact be a marker of mesenchymal stem cells. Silencing endosialin expression in fibroblasts strongly inhibited migration and proliferation. Collectively, the experiments validate endosialin as a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells. The data warrant further functional analysis of endosialin during tumor progression and its exploitation as marker of tumor vessel-associated mural cells, expression of which may reflect the non-normalized phenotype of the tumor vasculature.