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Table S1

Supplementary Figures S1 - S28

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

The angiopoietin (Angpt)–TIE signaling pathway controls vascular maturation and maintains the quiescent phenotype of resting vasculature. The contextual agonistic and antagonistic Tie2 ligand ANGPT2 is believed to be exclusively produced by endothelial cells, disrupting constitutive ANGPT1–TIE2 signaling to destabilize the microvasculature during pathologic disorders like inflammation and cancer. However, scattered reports have also portrayed tumor cells as a source of ANGPT2. Employing ISH-based detection of ANGPT2, we found strong tumor cell expression of ANGPT2 in a subset of patients with melanoma. Comparative analysis of biopsies revealed a higher fraction of ANGPT2-expressing tumor cells in metastatic versus primary sites. Tumor cell–expressed Angpt2 was dispensable for primary tumor growth, yet in-depth analysis of primary tumors revealed enhanced intratumoral necrosis upon silencing of tumor cell Angpt2 expression in the absence of significant immune and vascular alterations. Global transcriptional profiling of Angpt2-deficient tumor cells identified perturbations in redox homeostasis and an increased response to cellular oxidative stress. Ultrastructural analyses illustrated a significant increase of dysfunctional mitochondria in Angpt2-silenced tumor cells, thereby resulting in enhanced reactive oxygen species (ROS) production and downstream MAPK stress signaling. Functionally, enhanced ROS in Angpt2-silenced tumor cells reduced colonization potential in vitro and in vivo. Taken together, these findings uncover the hitherto unappreciated role of tumor cell–expressed ANGPT2 as an autocrine-positive regulator of metastatic colonization and validate ANGPT2 as a therapeutic target for a well-defined subset of patients with melanoma.Significance:This study reveals that tumor cells can be a source of ANGPT2 in the tumor microenvironment and that tumor cell-derived ANGPT2 augments metastatic colonization by protecting tumor cells from oxidative stress.

This file consists of supplementary figures (SF) 1-11 consisting of: SF1: Melanoma cells express ANGPT2 (A-F). SF2: Melanoma cell-expressed Angpt2 does not modulate tumor cell proliferation (A-B). SF3: Tumor cell knockdown of Angpt2 does not impact growth of primary tumors (A-J). SF4: Angpt2-depletion in melanoma cells does not affect primary tumor microenvironment (A-F). SF5: Angpt2-depletion alters pathways governing metastasis and oxidative stress (A-B). SF6: Angpt2 knockdown in tumor cells affects the cellular redox balance (A-G). SF7: Tumor cell-expressed Angpt2 alters mitochondrial dynamics (A-E). SF8: Tumor cell-expressed Angpt2 promotes metastasis (A-G). SF9: Tumor cell-expressed Angpt2 does not affect early stages of metastasis in vitro (A-H). SF10: Silencing of Angpt2 impairs invasion through the basement membrane and transmigration across the endothelial barrier (A-H). SF11: Silencing of Angpt2 reduces in vitro colony formation (A-F).

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.

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

The angiopoietin (Angpt)–TIE signaling pathway controls vascular maturation and maintains the quiescent phenotype of resting vasculature. The contextual agonistic and antagonistic Tie2 ligand ANGPT2 is believed to be exclusively produced by endothelial cells, disrupting constitutive ANGPT1–TIE2 signaling to destabilize the microvasculature during pathologic disorders like inflammation and cancer. However, scattered reports have also portrayed tumor cells as a source of ANGPT2. Employing ISH-based detection of ANGPT2, we found strong tumor cell expression of ANGPT2 in a subset of patients with melanoma. Comparative analysis of biopsies revealed a higher fraction of ANGPT2-expressing tumor cells in metastatic versus primary sites. Tumor cell–expressed Angpt2 was dispensable for primary tumor growth, yet in-depth analysis of primary tumors revealed enhanced intratumoral necrosis upon silencing of tumor cell Angpt2 expression in the absence of significant immune and vascular alterations. Global transcriptional profiling of Angpt2-deficient tumor cells identified perturbations in redox homeostasis and an increased response to cellular oxidative stress. Ultrastructural analyses illustrated a significant increase of dysfunctional mitochondria in Angpt2-silenced tumor cells, thereby resulting in enhanced reactive oxygen species (ROS) production and downstream MAPK stress signaling. Functionally, enhanced ROS in Angpt2-silenced tumor cells reduced colonization potential in vitro and in vivo. Taken together, these findings uncover the hitherto unappreciated role of tumor cell–expressed ANGPT2 as an autocrine-positive regulator of metastatic colonization and validate ANGPT2 as a therapeutic target for a well-defined subset of patients with melanoma. Significance: This study reveals that tumor cells can be a source of ANGPT2 in the tumor microenvironment and that tumor cell-derived ANGPT2 augments metastatic colonization by protecting tumor cells from oxidative stress.

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 factor–β (TGFβ) 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)

The Tie receptors with their Angiopoietin ligands act as regulators of angiogenesis and vessel maturation. Tie2 exerts its functions through its supposed endothelial-specific expression. Yet, Tie2 is also expressed at lower levels by pericytes and it has not been unravelled through which mechanisms pericyte Angiopoietin/Tie signalling affects angiogenesis. Here we show that human and murine pericytes express functional Tie2 receptor. Silencing of Tie2 in pericytes results in a pro-migratory phenotype. Pericyte Tie2 controls sprouting angiogenesis in in vitro sprouting and in vivo spheroid assays. Tie2 downstream signalling in pericytes involves Calpain, Akt and FOXO3A. Ng2-Cre-driven deletion of pericyte-expressed Tie2 in mice transiently delays postnatal retinal angiogenesis. Yet, Tie2 deletion in pericytes results in a pronounced pro-angiogenic effect leading to enhanced tumour growth. Together, the data expand and revise the current concepts on vascular Angiopoietin/Tie signalling and propose a bidirectional, reciprocal EC-pericyte model of Tie2 signalling., The angiopoietins regulate vascular maturation, angiogenesis and lymphangiogenesis via their Tie receptors that were long believed to be endothelium-specific. Here the authors show that angiopoietins activate and control pericyte function through pericyte-expressed Tie2 triggering of Calpain, Akt and FOXO3A signalling cascades.

Simple Summary Myeloid-derived suppressor cells (MDSC) represent a heterogeneous myeloid cell population that is expanded in tumor bearing hosts and substantially contributes to immunosuppression, representing thereby a valuable therapeutic target. Our study analyzes polymorphonuclear (PMN) and monocytic (M) MDSC subsets regarding their immunosuppressive capacity and recruitment mechanisms in murine melanoma. The immunosuppressive activity of both subsets was comparable. We identified the C-X-C Motif Chemokine Receptor (CXCR) 2/chemokine C-X-C motif ligand (CXCL) 1 axis as an important mediator of PMN-MDSC recruitment. Inhibition of CXCR2 resulted in a decreased infiltration of tumors with PMN-MDSC and increased survival of melanoma bearing mice. Furthermore, adjuvant treatment of mice with resected tumors reduced the infiltration of pre-metastatic sites with PMN-MDSC and the occurrence of distant metastasis. The decrease in PMN-MDSC infiltration was accompanied by an increase in natural killer (NK) cell frequency, suggesting an important role of PMN-MDSC in suppressing the NK cell-mediated anti-tumor response. Abstract Background: Despite recent improvement in the treatment of malignant melanoma by immune-checkpoint inhibitors, the disease can progress due to an immunosuppressive tumor microenvironment (TME) mainly represented by myeloid-derived suppressor cells (MDSC). However, the relative contribution of the polymorphonuclear (PMN) and monocytic (M) MDSC subsets to melanoma progression is not clear. Here, we compared both subsets regarding their immunosuppressive capacity and recruitment mechanisms. Furthermore, we inhibited PMN-MDSC migration in vivo to determine its effect on tumor progression. Methods: Using the RET transgenic melanoma mouse model, we investigated the immunosuppressive function of MDSC subsets and chemokine receptor expression on these cells. The effect of CXCR2 inhibition on PMN-MDSC migration and tumor progression was studied in RET transgenic mice and in C57BL/6 mice after surgical resection of primary melanomas. Results: Immunosuppressive capacity of intratumoral M- and PMN-MDSC was comparable in melanoma bearing mice. Anti-CXCR2 therapy prolonged survival of these mice and decreased the occurrence of distant metastasis. Furthermore, this therapy reduced the infiltration of melanoma lesions and pre-metastatic sites with PMN-MDSC that was associated with the accumulation of natural killer (NK) cells. Conclusions: We provide evidence for the tumor−promoting properties of PMN-MDSC as well as for the anti-tumor effects upon their targeting in melanoma bearing mice.

In this issue of JEM, Singhal et al. explore the cellular mechanisms involved in endothelial cell regeneration in the liver. Using a combination of myeloablative and nonmyeloablative approaches, the authors found that repair of the endothelium is mediated by endothelial cells themselves, but when injured, endothelial cells enlist myeloid counterparts that aid in vascular repair., In this issue of JEM, Singhal et al. (https://doi.org/10.1084/jem.20180008) explore the cellular mechanisms involved in endothelial cell regeneration in the liver. Using a combination of myeloablative and nonmyeloablative approaches, the authors found that repair of the endothelium is mediated by endothelial cells themselves, but when injured, endothelial cells enlist myeloid counterparts that aid in vascular repair.

Epithelial to mesenchymal transition (EMT) is a developmental process which is aberrantly activated during cancer invasion and metastasis. Elevated expression of EMT-inducers like ZEB1 enables tumor cells to detach from the primary tumor and invade into the surrounding tissue. The main antagonist of ZEB1 in controlling EMT is the microRNA-200 family that is reciprocally linked to ZEB1 in a double negative feedback loop. Here, we further elucidate how the ZEB1/miR-200 feedback loop controls invasion of tumor cells. The process of EMT is attended by major changes in the actin cytoskeleton. Via in silico screening of genes encoding for actin interacting proteins, we identified two novel targets of miR-200c - TKS5 and MYLK (MLCK). Co-expression of both genes with ZEB1 was observed in several cancer cell lines as well as in breast cancer patients and correlated with low miR-200c levels. Depletion of TKS5 or MYLK in breast cancer cells reduced their invasive potential and their ability to form invadopodia. Whereas TKS5 is known to be a major component, we could identify MYLK as a novel player in invadopodia formation. In summary, TKS5 and MYLK represent two mediators of invasive behavior of cancer cells that are regulated by the ZEB1/miR-200 feedback loop.

Oncology research in humans is limited to analytical and observational studies for obvious ethical reasons, with therapy-focused clinical trials being the one exception to this rule. Preclinical mouse tumour models therefore serve as an indispensable intermediate experimental model system bridging more reductionist in vitro research with human studies. Based on a systematic survey of preclinical mouse tumour studies published in eight scientific journals in 2016, this Analysis provides an overview of how contemporary preclinical mouse tumour biology research is pursued. It thereby identifies some of the most important challenges in this field and discusses potential ways in which preclinical mouse tumour models could be improved for better relevance, reproducibility and translatability.