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Organ-specific metastasis to secondary organs is dependent on the formation of a supportive pre-metastatic niche. This tissue-specific microenvironmental response is thought to be mediated by mutational and epigenetic changes to primary tumour cells resulting in altered cross-talk between cell types. This response is augmented through the release of tumour and stromal signalling mediators including cytokines, chemokines, exosomes and growth factors. Although researchers have elucidated some of the cancer-promoting features that are bespoke to organotropic metastasis to the lungs, it remains unclear if these are organ-specific or generic between organs. Understanding the mechanisms that mediate the metastasis-promoting synergy between the host microenvironment, immunity, and pulmonary structures may elucidate predictive, prognostic and therapeutic markers that could be targeted to reduce the metastatic burden of disease. Herein, we give an updated summary of the known cellular and molecular mechanisms that contribute to the formation of the lung pre-metastatic niche and tissue-specific metastasis.

Metastasis via the lymphatic vasculature is an important step in cancer progression. The formation of new lymphatic vessels (lymphangiogenesis), or remodeling of existing lymphatics, is thought to facilitate the entry and transport of tumor cells into lymphatic vessels and on to distant organs. The migration of lymphatic endothelial cells (LEC) toward guidance cues is critical for lymphangiogenesis. While chemokines are known to provide directional navigation for migrating immune cells, their role in mediating LEC migration during tumor-associated lymphangiogenesis is not well defined. Here, we undertook gene profiling studies to identify chemokine–chemokine receptor pairs that are involved in tumor lymphangiogenesis associated with lymph node metastasis. CCL27 and CCL28 were expressed in tumor cells with metastatic potential, while their cognate receptor, CCR10, was expressed by LECs and upregulated by the lymphangiogenic growth factor VEGFD and the proinflammatory cytokine TNFα. Migration assays demonstrated that LECs are attracted to both CCL27 and CCL28 in a CCR10-dependent manner, while abnormal lymphatic vessel patterning in CCR10-deficient mice confirmed the significant role of CCR10 in lymphatic patterning. In vivo analyses showed that LECs are recruited to a CCL27 or CCL28 source, while VEGFD was required in combination with these chemokines to enable formation of coherent lymphatic vessels. Moreover, tumor xenograft experiments demonstrated that even though CCL27 expression by tumors enhanced LEC recruitment, the ability to metastasize was dependent on the expression of VEGFD. These studies demonstrate that CCL27 and CCL28 signaling through CCR10 may cooperate with inflammatory mediators and VEGFD during tumor lymphangiogenesis.Significance:The study shows that the remodeling of lymphatic vessels in cancer is influenced by CCL27 and CCL28 chemokines, which may provide a future target to modulate metastatic spread.

Supplementary Figures S1-S8 S1 Heat map of chemokines and receptors S2 Immunohistochemical staining of lymphatic vessels S3 Expression of VEGF-D and chemokines in cancer cell lines S4 Expression of genes in melanoma patient samples S5 Chemokine secretion by tumour cells S6 Chemokine induced migration of LEC S7 Chemokines attract LEC in vivo S8 CCL-293EBNA tumour growth and CCL27 secretion

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Lymph node metastasis, an early and prognostically important event in the progression of many human cancers, is associated with expression of VEGF-D. Changes to lymph node vasculature that occur during malignant progression may create a metastatic niche capable of attracting and supporting tumor cells. In this study, we sought to characterize molecules expressed in lymph node endothelium that could represent therapeutic or prognostic targets. Differential mRNA expression profiling of endothelial cells from lymph nodes that drained metastatic or nonmetastatic primary tumors revealed genes associated with tumor progression, in particular bone morphogenetic protein-4 (BMP-4). Metastasis driven by VEGF-D was associated with reduced BMP-4 expression in high endothelial venules, where BMP-4 loss could remodel the typical high-walled phenotype to thin-walled vessels. VEGF-D expression was sufficient to suppress proliferation of the more typical BMP-4–expressing high endothelial venules in favor of remodeled vessels, and mechanistic studies indicated that VEGF receptor-2 contributed to high endothelial venule proliferation and remodeling. BMP-4 could regulate high endothelial venule phenotype and cellular function, thereby determining morphology and proliferation responses. Notably, therapeutic administration of BMP-4 suppressed primary tumor growth, acting both at the level of tumor cells and tumor stromal cells. Together, our results show that VEGF-D–driven metastasis induces vascular remodeling in lymph nodes. Furthermore, they implicate BMP-4 as a negative regulator of this process, suggesting its potential utility as a prognostic marker or antitumor agent. Cancer Res; 71(20); 6547–57. ©2011 AACR.

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Lymphangiogenesis (growth of new lymphatic vessels), and lymphatic remodelling more broadly, are important for disease progression in cancer, lymphedema and the pulmonary disease lymphangioleiomyomatosis. Multiple molecular pathways which signal for aspects of lymphangiogenesis are known but little is understood about their co-ordinate regulation in lymphatic endothelial cells (LECs). Small RNA molecules co-ordinately regulate complex biological processes, but knowledge about their involvement in lymphangiogenesis is limited. Here we used high-throughput small RNA sequencing of LECs to identify microRNAs (miRs) regulating lymphatic remodelling driven by the lymphangiogenic growth factors VEGF-C and VEGF-D. We identified miR-132 as up-regulated by both growth factors, and demonstrated that inhibiting miR-132 in LECs in vitro blocked cell proliferation and tube formation, key steps in lymphangiogenesis. We showed that miR-132 is expressed in human LECs in vivo in the lymphatics of human breast tumours expressing VEGF-D. Importantly, we demonstrated that inhibiting miR-132 in vivo blocked many aspects of lymphangiogenesis in mice. Finally, we identified mRNAs regulated by miR-132 in LECs, by sequencing after RNA-protein cross-linking and Argonaute immunoprecipitation, which demonstrated how miR-132 co-ordinately regulates signalling pathways in lymphangiogenesis. This study shows miR-132 is a critical regulator of lymphangiogenesis and a potential target for therapeutically manipulating lymphatic remodelling in disease.

Supplemental Digital Content is available in the text., Objective: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. Conclusions: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Angiogenesis underlies development, physiology and pathogenesis of cancer, eye and cardiovascular diseases. Inhibiting aberrant angiogenesis using anti-angiogenic therapy (AAT) has been successful in the clinical treatment of cancer and eye diseases. However, resistance to AAT inevitably occurs and its molecular basis remains poorly understood. Here, we uncover molecular modifiers of the blood endothelial cell (EC) response to a widely used AAT bevacizumab by performing a pooled genetic screen using three-dimensional microcarrier-based cell culture and CRISPR–Cas9. Functional inhibition of the epigenetic reader BET family of proteins BRD2/3/4 shows unexpected mitigating effects on EC survival and/or proliferation upon VEGFA blockade. Moreover, transcriptomic and pathway analyses reveal an interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs via the cell cycle regulator CDC25B phosphatase. Collectively, our findings provide insight into epigenetic regulation of the EC response to VEGFA blockade and may facilitate development of quality biomarkers and strategies for overcoming resistance to AAT., Through three-dimensional CRISPR screening, He et al. report that functional inhibition of BET family of proteins BRD2/3/4 shows mitigating effects on blood endothelial cell (EC) survival and/or proliferation upon VEGFA blockade. An interaction between epigenetic regulation and anti-angiogenesis, which may affect chromosomal structure and activity in ECs through CDC25B phosphatase, is potentially involved with EC resistance to anti-angiogenic therapy.

Background: Lymphatic vascular development is regulated by well-characterised signalling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity and morphogenesis. Canonical and non-canonical WNT signalling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Results: Here we find that loss of non-canonical WNT signalling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1;Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1-/- mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a-/- mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signalling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Conclusion: Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signalling during the formation of lymphatic vascular networks. Refereed/Peer-reviewed

Lymphatic vascular development is regulated by well-characterised signalling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity and and morphogenesis. Canonical and non-canonical WNT signalling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. Here we find that loss of non-canonical WNT signalling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1/Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1−/− mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a−/− mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild-type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signalling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signalling during the formation of lymphatic vascular networks.

Tumour angiogenesis and lymphangiogenesis are hallmarks of cancer and have been associated with tumour progression, tumour metastasis and poor patient prognosis. Many factors regulate angiogenesis and lymphangiogenesis in cancer including non-coding RNAs which are a category of RNAs that do not encode proteins and have important regulatory functions at transcriptional and post-transcriptional levels. Non-coding RNAs can be encapsulated in extracellular vesicles called exosomes which are secreted by tumour cells or other cells in the tumour microenvironment and can then be taken up by the endothelial cells of blood vessels and lymphatic vessels. The “delivery” of these non-coding RNAs to endothelial cells in tumours can facilitate tumour angiogenesis and lymphangiogenesis. Here we review recent findings about exosomal non-coding RNAs, specifically microRNAs and long non-coding RNAs, which regulate tumour angiogenesis and lymphangiogenesis in cancer. We then focus on the potential use of these molecules as cancer biomarkers and opportunities for exploiting ncRNAs for the treatment of cancer.

Between embryonic days 10.5 and 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, which is secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we have investigated two known WNT5A receptors: ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a(−/−) phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair WNT5A-RYK acts as a navigation system to instruct filopodial pathfinding, a process that is crucial for continuous cell cycling to fuel rapid midgut elongation.

Primary tumours, particularly from major solid organs, are able to disseminate into the blood and lymphatic system and spread to distant sites. These secondary metastases to other major organs are the most lethal aspect of cancer, accounting for the majority of cancer deaths. The brain is a frequent site of metastasis, and brain metastases are often fatal due to the critical role of the nervous system and the limited options for treatment, including surgery. This creates a need to further understand the complex cell and molecular biology associated with the establishment of brain metastasis, including the changes to the environment of the brain to enable the arrival and growth of tumour cells. Local changes in the vascular network, immune system and stromal components all have the potential to recruit and foster metastatic tumour cells. This review summarises our current understanding of brain vascular microenvironments, fluid circulation and drainage in the context of brain metastases, as well as commenting on current cutting-edge experimental approaches used to investigate changes in vascular environments and alterations in specialised subsets of blood and lymphatic vessel cells during cancer spread to the brain.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference. Fil: Nowak Sliwinska, Patrycja. Université of Lausanne; Suiza. Univeristé of Geneve; Suiza Fil: Alitalo, Kari. Katholikie Universiteit Leuven; Bélgica Fil: Allen, Elizabeth. Katholikie Universiteit Leuven; Bélgica Fil: Anisimov, Andrey. Katholikie Universiteit Leuven; Bélgica Fil: Aplin, Alfred C.. University of Washington; Estados Unidos Fil: Auerbach, Robert. University of Wisconsin; Estados Unidos Fil: Augustin, Hellmut G.. Heidelberg University; Alemania. German Cancer Consortium; Alemania Fil: Bates, David O.. University of Nottingham; Reino Unido Fil: Beijnum, Judy R. van. Cancer Center Amsterdam; Países Bajos Fil: Bender, R. Hugh F.. University of California; Estados Unidos Fil: Bergers, Gabriele. Katholikie Universiteit Leuven; Bélgica Fil: Bikfalvi, Andreas. Universite de Bordeaux; Francia Fil: Bischoff, Joyce. Harvard Medical School; Estados Unidos Fil: Böck, Barbara C.. Heidelberg University; Alemania. German Cancer Consortium; Alemania Fil: Brooks, Peter C.. Maine Medical Center Research Institute; Estados Unidos Fil: Bussolino, Federico. Università di Torino; Italia. Candiolo Cancer Institute; Italia Fil: Cakir, Bertan. Harvard Medical School; Estados Unidos Fil: Carmeliet, Peter. Katholikie Universiteit Leuven; Bélgica Fil: Castranova, Daniel. Harvard Medical School; Estados Unidos Fil: Cimpean, Anca M.. Victor Babes University of Medicine and Pharmacy; Rumania Fil: Cleaver, Ondine. University Of Texas At Brownsville; Estados Unidos Fil: Coukos, George. Universida de Lausanne; Suiza Fil: Davis, George E.. University of Missouri; Estados Unidos Fil: De Palma, Michele. Swiss Federal Institute of Technology; Suiza Fil: Dimberg, Anna. Uppsala University; Suiza Fil: Dings, Ruud P. M.. University of Arkansas for Medical Sciences; Estados Unidos Fil: Djonov, Valentin. University of Bern; Suiza Fil: Dudley, Andrew C.. University of Virginia; Estados Unidos Fil: Dufton, Neil P.. Imperial College London; Reino Unido Fil: Fendt, Sarah-Maria. VIB Center for Cancer Biology; Bélgica Fil: Ferrara, Napoleone. University of California at San Diego; Estados Unidos Fil: Fruttiger, Marcus. University College London; Estados Unidos Fil: Fukumura, Dai. Harvard Medical School; Estados Unidos Fil: Ghesquière, Bart. Harvard Medical School; Estados Unidos Fil: Gong, Yan. Harvard Medical School; Estados Unidos Fil: Griffin, Robert J.. VIB Center for Cancer Biology; Bélgica Fil: Harris, Adrian L.. University of Oxford; Reino Unido Fil: Hughes, Christopher C. W.. University of California at Irvine; Estados Unidos Fil: Hultgren, Nan W.. University of California at Irvine; Estados Unidos Fil: Iruela-Arispe, M. Luisa. University of California at Los Angeles; Estados Unidos Fil: Irving, Melita. Universida de Lausanne; Suiza Fil: Maidana, Agostina Jainen. Harvard Medical School; Estados Unidos Fil: Kalluri, Raghu. Texas A&M University; Estados Unidos Fil: Kalucka, Joanna. Katholikie Universiteit Leuven; Bélgica Fil: Kerbel, Robert S.. University of Toronto; Canadá Fil: Kitajewski, Jan. University of Illinois; Estados Unidos Fil: Klaassen, Ingeborg. University of Amsterdam; Países Bajos Fil: Kleinmann, Hynda K.. The George Washington University; Estados Unidos Fil: Koolwijk, Pieter. Fondation Asile des Aveugles; Suiza. Universida de Lausanne; Suiza Fil: Kuczynski, Elisabeth. University of Toronto; Canadá Fil: Kwak, Brenda R.. University of Geneva; Suiza Fil: Koen, Marien. HistoGeneX; Bélgica Fil: Melero Martin, Juan M.. University of Liège; Bélgica Fil: Munn, Lance L.. Harvard Medical School; Estados Unidos Fil: Nicosia, Roberto F.. VA Puget Sound Health Care System; Estados Unidos Fil: Noel, Agnes. University of Liège; Bélgica Fil: Nurro, Jussi. University of Eastern Finland; Finlandia Fil: Olsson, Anna-Karin. Uppsala University; Suiza Fil: Petrova, Tatiana V.. Ludwig Institute for Cancer Research Lausanne; Suiza Fil: Pietras, Kristian. Division of Translational Cancer Research; Suecia Fil: Pili, Roberto. Indiana University Simon Cancer Center; Estados Unidos Fil: Pollard, Jeffrey W.. University of Edinburgh; Reino Unido Fil: Post, Mark J.. Maastricht University; Países Bajos Fil: Quax, Paul H. A.. Einthoven Laboratory for Experimental Vascular Medicine; Países Bajos Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Raica, Marius. Victor Babes University of Medicine and Pharmacy; Rumania Fil: Randi, Anna M.. Imperial College London; Reino Unido Fil: Ribatti, Domenico. Università degli Studi di Bari; Italia Fil: Ruegg, Curzio. University of Fribourg; Suiza Fil: Schlingemann, Reinier O.. University of Amsterdam; Países Bajos Fil: Schulte Merker, Stefan. Institute of Cardiovascular Organogenesis and Regeneration; Alemania Fil: Smith, Lois E. H.. Harvard Medical School; Estados Unidos Fil: Song, Jonathan W.. Ohio State University; Estados Unidos Fil: Stacker, Steven A.. University of Melbourne; Australia Fil: Stalin, Jimmy. Institute of Cardiovascular Organogenesis and Regeneration; Alemania Fil: Stratman, Amber N.. National Institutes of Health; Estados Unidos Fil: Van de Velde, Maureen. University of Liège; Bélgica Fil: van Hinsbergh, Victor W. M.. Universida de Lausanne; Suiza Fil: Vermeulen, Peter B.. HistoGeneX; Bélgica. University of Antwerp; Bélgica Fil: Waltenberger, Johannes. University of Münster; Alemania Fil: Weinstein, Brant M.. National Institutes of Health; Estados Unidos Fil: Xin, Hong. University of California at San Diego; Estados Unidos Fil: Yetkin Arik, Bahar. University of Amsterdam; Países Bajos Fil: Yla Herttuala, Seppo. University of Eastern Finland; Finlandia Fil: Yoder, Mervin C.. Indiana University; Estados Unidos Fil: Griffioen, Arjan W.. VU University Medical Center; Países Bajos

Even though we have known for over 250 years that cancers spread to regional lymph nodes (LNs) and distant organs, the fundamental question of which anatomical routes are taken by tumor cells has remained a mystery. Two recently published papers in Science, by Pereira et al. and Brown et al., directly address this important issue in tumor biology by assessing the capacity of tumor cells in LNs to spread to distant sites.

While the link between the lymphatic system and the metastatic spread of cancer is centuries old, understanding of the underlying mechanisms is still evolving. Lymphatic vessels provide a route for tumour cells to reach regional lymph nodes (LNs), which is prognostic of distant organ metastasis and poor survival. However, genomic analyses of metastatic cancer now reveal complex patterns of dissemination. The lymphatic endothelial cells lining lymphatics respond to molecular cues from the tumour microenvironment, mediating growth and remodelling of lymphatic vessels at the primary tumour, draining LNs and distant premetastatic niches. Recent studies emphasise that this not only supports metastasis but also influences antitumour immunity. Understanding the complex interactions between tumour cells, the immune system and lymphatics will be essential to inform developing therapeutic and prognostic approaches to cancer.

Ryk is a member of the receptor tyrosine kinase (RTK) family of proteins that control and regulate cellular processes. It is distinguished by binding Wnt ligands and having no detectable intrinsic protein tyrosine kinase activity suggesting Ryk is a pseudokinase. Here, we show an essential role for Ryk in directing morphogenetic events required for normal cardiac development through the examination of Ryk-deficient mice. We employed vascular corrosion casting, vascular perfusion with contrast dye, and immunohistochemistry to characterize cardiovascular and pharyngeal defects in Ryk-/- embryos. Ryk-/- mice exhibit a variety of malformations of the heart and outflow tract that resemble human congenital heart defects. This included stenosis and interruption of the aortic arch, ventriculoarterial malalignment, ventricular septal defects and abnormal pharyngeal arch artery remodelling. This study therefore defines a key intersection between a subset of growth factor receptors involved in planar cell polarity signalling, the Wnt family and mammalian cardiovascular development.

Remodelling of lymphatic vessels in tumours facilitates metastasis to lymph nodes. The growth factors VEGF-C and VEGF-D are well known inducers of lymphatic remodelling and metastasis in cancer. They are initially produced as full-length proteins requiring proteolytic processing in order to bind VEGF receptors with high affinity and thereby promote lymphatic remodelling. The fibrinolytic protease plasmin promotes processing of VEGF-C and VEGF-D in vitro, but its role in processing them in cancer was unknown. Here we explore plasmin’s role in proteolytically activating VEGF-D in vivo, and promoting lymphatic remodelling and metastasis in cancer, by co-expressing the plasmin inhibitor α2-antiplasmin with VEGF-D in a mouse tumour model. We show that α2-antiplasmin restricts activation of VEGF-D, enlargement of intra-tumoural lymphatics and occurrence of lymph node metastasis. Our findings indicate that the fibrinolytic system influences lymphatic remodelling in tumours which is consistent with previo...

Chemokines are a family of small protein cytokines that act as chemoattractants to migrating cells, in particular those of the immune system. They are categorized functionally as either homeostatic, constitutively produced by tissues for basal levels of cell migration, or inflammatory, where they are generated in association with a pathological inflammatory response. While the extravasation of leukocytes via blood vessels is a key step in cells entering the tissues, the lymphatic vessels also serve as a conduit for cells that are recruited and localized through chemoattractant gradients. Furthermore, the growth and remodeling of lymphatic vessels in pathologies is influenced by chemokines and their receptors expressed by lymphatic endothelial cells (LECs) in and around the pathological tissue. In this review we summarize the diverse role played by specific chemokines and their receptors in shaping the interaction of lymphatic vessels, immune cells, and other pathological cell types in physiology and disease.

The lymphatic system is a series of vessels that transport cells and excess fluid from tissues to the blood vascular system. Normally quiescent, the lymphatics can grow or remodel in response to developmental, immunological, or cells pathological stimuli. Lymphatic vessels comprise lymphatic endothelial cells (LECs) that can respond to external growth factors by undergoing proliferation, migration, adhesion, and tube and lumen formation into new vessel structures, a process known as lymphangiogenesis. To understand the key gene and signaling pathways necessary for lymphangiogenesis and lymphatic vessel remodeling, we have developed a three-dimensional LEC tube formation assay to explore the role of kinase signaling in these processes. The collagen-overlay-based assay was used with primary human adult dermal LECs to investigate a library of 60 tyrosine kinase (TK) and TK-like genes by siRNA knockdown. Nine candidate genes were identified and characterized for their ability to modify key parameters of lymphatic tube formation, including tube length, area, thickness, branching, and number of blind-ended sacs. Four genes-ZAP70, IRAK4, RIPK1, and RIPK2-were identified as high-confidence hits after tertiary deconvolution screens and demonstrate the utility of the assay to define LEC genes critical for the formation of tube structures. This assay facilitates the identification of potential molecular targets for novel drugs designed to modulate the remodeling of lymphatics that is important for the metastatic spread of cancer and other pathologies.

VEGF-C and VEGF-D are secreted glycoproteins that induce angiogenesis and lymphangiogenesis in cancer, thereby promoting tumor growth and spread. They exhibit structural homology and activate VEGFR-2 and VEGFR-3, receptors on endothelial cells that signal for growth of blood vessels and lymphatics. VEGF-C and VEGF-D were thought to exhibit similar bioactivities, yet recent studies indicated distinct signaling mechanisms (e.g. tumor-derived VEGF-C promoted expression of the prostaglandin biosynthetic enzyme COX-2 in lymphatics, a response thought to facilitate metastasis via the lymphatic vasculature, whereas VEGF-D did not). Here we explore the basis of the distinct bioactivities of VEGF-D using a neutralizing antibody, peptide mapping, and mutagenesis to demonstrate that the N-terminal α-helix of mature VEGF-D (Phe93–Arg108) is critical for binding VEGFR-2 and VEGFR-3. Importantly, the N-terminal part of this α-helix, from Phe93 to Thr98, is required for binding VEGFR-3 but not VEGFR-2. Surprisingly, the corresponding part of the α-helix in mature VEGF-C did not influence binding to either VEGFR-2 or VEGFR-3, indicating distinct determinants of receptor binding by these growth factors. A variant of mature VEGF-D harboring a mutation in the N-terminal α-helix, D103A, exhibited enhanced potency for activating VEGFR-3, was able to promote increased COX-2 mRNA levels in lymphatic endothelial cells, and had enhanced capacity to induce lymphatic sprouting in vivo. This mutant may be useful for developing protein-based therapeutics to drive lymphangiogenesis in clinical settings, such as lymphedema. Our studies shed light on the VEGF-D structure/function relationship and provide a basis for understanding functional differences compared with VEGF-C.

Leakage of fluid from blood vessels, leading to oedema, is a key feature of many diseases including hyperoxic acute lung injury (HALI), which can occur when patients are ventilated with high concentrations of oxygen (hyperoxia). The molecular mechanisms driving vascular leak and oedema in HALI are poorly understood. VEGF-D is a protein that promotes blood vessel leak and oedema when overexpressed in tissues, but the role of endogenous VEGF-D in pathological oedema was unknown. To address these issues, we exposed Vegfd-deficient mice to hyperoxia. The resulting pulmonary oedema in Vegfd-deficient mice was substantially reduced compared to wild-type, as was the protein content of bronchoalveolar lavage fluid, consistent with reduced vascular leak. Vegf-d and its receptor Vegfr-3 were more highly expressed in lungs of hyperoxic, versus normoxic, wild-type mice, indicating that components of the Vegf-d signalling pathway are up-regulated in hyperoxia. Importantly, VEGF-D and its receptors were co-localized on blood vessels in clinical samples of human lungs exposed to hyperoxia; hence, VEGF-D may act directly on blood vessels to promote fluid leak. Our studies show that Vegf-d promotes oedema in response to hyperoxia in mice and support the hypothesis that VEGF-D signalling promotes vascular leak in human HALI. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Counting nuclei released from intact cells is a convenient and reliable approach to assess cell density during microcarrier-based culture of adherent cells. However, commonly used methods for counting nuclei, such as crystal violet staining and quantification with a hemocytometer/ automated imaging system or a Coulter counter, are imprecise, laborious and, limited in throughput. Here, we describe the use of high-affinity pro-fluorescent nucleic acid stains and volumetric flow cytometry for automated counting of nuclei released from cells attached to microcarriers with improved precision and high sample throughput. This simple procedure facilitates rapid and precise assessment of cell attachment, survival, and proliferation on microcarriers, and can provide information about the cell cycle, all without the need for cell detachment. Consequently, various microcarrier-based applications, from small-scale multi-factor experiments to large-scale functional genetic screens and clinical/industrial cultures, could be enhanced by this approach.

Metastasis via the lymphatic vasculature is an important step in cancer progression. The formation of new lymphatic vessels (lymphangiogenesis), or remodeling of existing lymphatics, is thought to facilitate the entry and transport of tumor cells into lymphatic vessels and on to distant organs. The migration of lymphatic endothelial cells (LEC) toward guidance cues is critical for lymphangiogenesis. While chemokines are known to provide directional navigation for migrating immune cells, their role in mediating LEC migration during tumor-associated lymphangiogenesis is not well defined. Here, we undertook gene profiling studies to identify chemokine–chemokine receptor pairs that are involved in tumor lymphangiogenesis associated with lymph node metastasis. CCL27 and CCL28 were expressed in tumor cells with metastatic potential, while their cognate receptor, CCR10, was expressed by LECs and upregulated by the lymphangiogenic growth factor VEGFD and the proinflammatory cytokine TNFα. Migration assays demonstrated that LECs are attracted to both CCL27 and CCL28 in a CCR10-dependent manner, while abnormal lymphatic vessel patterning in CCR10-deficient mice confirmed the significant role of CCR10 in lymphatic patterning. In vivo analyses showed that LECs are recruited to a CCL27 or CCL28 source, while VEGFD was required in combination with these chemokines to enable formation of coherent lymphatic vessels. Moreover, tumor xenograft experiments demonstrated that even though CCL27 expression by tumors enhanced LEC recruitment, the ability to metastasize was dependent on the expression of VEGFD. These studies demonstrate that CCL27 and CCL28 signaling through CCR10 may cooperate with inflammatory mediators and VEGFD during tumor lymphangiogenesis. Significance: The study shows that the remodeling of lymphatic vessels in cancer is influenced by CCL27 and CCL28 chemokines, which may provide a future target to modulate metastatic spread.

The receptor tyrosine kinases (RTKs) are a well-characterized family of growth factor receptors that have central roles in human disease and are frequently therapeutically targeted. The RYK, ROR, PTK7 and MuSK subfamilies make up an understudied subset of WNT-binding RTKs. Numerous developmental, stem cell and pathological roles of WNTs, in particular WNT5A, involve signalling via these WNT receptors. The WNT-binding RTKs have highly context-dependent signalling outputs and stimulate the β-catenin-dependent, planar cell polarity and/or WNT/Ca2+ pathways. RYK, ROR and PTK7 members have a pseudokinase domain in their intracellular regions. Alternative signalling mechanisms, including proteolytic cleavage and protein scaffolding functions, have been identified for these receptors. This review explores the structure, signalling, physiological and pathological roles of RYK, with particular attention paid to cancer and the possibility of therapeutically targeting RYK. The other WNT-binding RTKs are compared with RYK throughout to highlight the similarities and differences within this subset of WNT receptors.

GABAergic interneurons have many important functions in cortical circuitry, a reflection of their diversity as a cell population. However, the developmental origins of this diversity are poorly understood. Here, we identify a rostral-caudal gradient of Wnt-responsiveness that delineates the specification of the two main interneuron subclasses. Caudallysituated medial ganglionic eminence (MGE) progenitors receive high levels of Wnt signaling and give rise to somatostatin (SST)-expressing cortical interneurons. Parvalbumin (PV)-expressing basket cells, by contrast, originate mostly from the rostral MGE where Wnt signaling is attenuated. Interestingly, rather than canonical signaling through beta-catenin, Wnt signaling transmitted via the non-canonical receptor, Ryk regulates interneuron cell-fate specification in vivo and in vitro. Indeed, gain-of-function of Ryk intracellular domain signaling regulates SST and PV fate in a dose-dependent manner, suggesting Ryk signaling acts in a graded fashion. These data reveal a complex and important role for non-canonical Wnt-Ryk signaling in establishing the correct ratios of mature cortical interneuron subtypes.

Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (Apc(Min/+)) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.

Blood vessels and lymphatic vessels are located in many tissues and organs throughout the body, and play important roles in a wide variety of prevalent diseases in humans. Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that can promote the remodeling of blood vessels and lymphatics in development and disease. Recent fundamental and translational studies have provided insight into the molecular mechanisms by which VEGF-D exerts its effects in human disease. Hence this protein is now of interest as a therapeutic and/or diagnostic target, or as a potential therapeutic agent, in a diversity of indications in cardiovascular medicine, cancer and the devastating pulmonary condition lymphangioleiomyomatosis. This has led to clinical trial programs to assess the effect of targeting VEGF-D signaling pathways, or delivering VEGF-D, in angina, cancer and ocular indications. This review summarizes our understanding of VEGF-D signaling in human disease, which is largely based on animal disease models and clinicopathological studies, and provides information about the outcomes of recent clinical trials testing agonists or antagonists of VEGF-D signaling.

Lymphatic vessels constitute a specialized vasculature that is involved in development, cancer, obesity, and immune regulation. The migration of lymphatic endothelial cells (LECs) is critical for vessel growth (lymphangiogenesis) and vessel remodeling, processes that modify the lymphatic network in response to developmental or pathological demands. Using the publicly accessible results of our genome-wide siRNA screen, we characterized the migratome of primary human LECs and identified individual genes and signaling pathways that regulate LEC migration. We compared our data set with mRNA differential expression data from endothelial and stromal cells derived from two in vivo models of lymphatic vessel remodeling, viral infection and contact hypersensitivity-induced inflammation, which identified genes selectively involved in regulating LEC migration and remodeling. We also characterized the top candidates in the LEC migratome in primary blood vascular endothelial cells to identify genes with functions common to lymphatic and blood vascular endothelium. On the basis of these analyses, we showed that LGALS1, which encodes the glycan-binding protein Galectin-1, promoted lymphatic vascular growth in vitro and in vivo and contributed to maintenance of the lymphatic endothelial phenotype. Our results provide insight into the signaling networks that control lymphangiogenesis and lymphatic remodeling and potentially identify therapeutic targets and biomarkers in disease specific to lymphatic or blood vessels.

Remodelling of lymphatic vessels in tumours facilitates metastasis to lymph nodes. The growth factors VEGF-C and VEGF-D are well known inducers of lymphatic remodelling and metastasis in cancer. They are initially produced as full-length proteins requiring proteolytic processing in order to bind VEGF receptors with high affinity and thereby promote lymphatic remodelling. The fibrinolytic protease plasmin promotes processing of VEGF-C and VEGF-D in vitro, but its role in processing them in cancer was unknown. Here we explore plasmin's role in proteolytically activating VEGF-D in vivo, and promoting lymphatic remodelling and metastasis in cancer, by co-expressing the plasmin inhibitor α

Many cell types undergo migration during embryogenesis and disease. Endothelial cells line blood vessels and lymphatics, which migrate during development as part of angiogenesis, lymphangiogenesis and other types of vessel remodelling. These processes are also important in wound healing, cancer metastasis and cardiovascular conditions. However, the molecular control of endothelial cell migration is poorly understood. Here, we present a dataset containing siRNA screens that identify known and novel components of signalling pathways regulating migration of lymphatic endothelial cells. These components are compared to signalling in blood vascular endothelial cells. Further, using high-content microscopy, we captured a dataset of images of migrating cells following transfection with a genome-wide siRNA library. These datasets are suitable for the identification and analysis of genes involved in endothelial cell migration and morphology, and for computational approaches to identify signalling networks controlling the migratory response and integration of cell morphology, gene function and cell signaling. This may facilitate identification of protein targets for therapeutically modulating angiogenesis and lymphangiogenesis in the context of human disease.

Background: Preoperative radiotherapy (RT) is commonly used to treat localised soft-tissue sarcomas (STS). Hypoxia is an important determinant of radioresistance. Whether antiangiogenic therapy can ‘normalise' tumour vasculature, thereby improving oxygenation, remains unknown. Methods: Two cohorts were prospectively enrolled. Cohort A evaluated the implications of hypoxia in STS, using the hypoxic tracer 18F-azomycin arabinoside (FAZA-PET). In cohort B, sunitinib was added to preoperative RT in a dose-finding phase 1b/2 design. Results: In cohort A, 13 out of 23 tumours were hypoxic (FAZA-PET), correlating with metabolic activity (r2=0.85; P

Summary: Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in an organ-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to be fully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis. : Lymphatic vessels display organotypic function and develop in an organ-specific manner. Vogrin et al. find that the zebrafish Kdr receptor is indispensible for craniofacial, but not trunk, lymphangiogenesis whereas Flt4 is essential for the latter. Thus, vascular endothelial growth factor (VEGF) receptor signaling pathways are differentially employed in different tissues to drive developmental lymphangiogenesis. Keywords: developmental biology, Kdr, lymphangiogenesis, lymphatic, organotypic, vascular biology, VEGF, VEGF receptor, Vegfd, zebrafish

The progression of cancer is supported by a wide variety of non-neoplastic cell types which make up the tumour stroma, including immune cells, endothelial cells, cancer-associated fibroblasts and nerve fibres. These host cells contribute molecular signals that enhance primary tumour growth and provide physical avenues for metastatic dissemination. This article provides an overview of the role of blood vessels, lymphatic vessels and nerve fibres in the tumour microenvironment and highlights the interconnected molecular signalling pathways that control their development and activation in cancer. Further, this article highlights the known pharmacological agents which target these pathways and discusses the potential therapeutic uses of drugs that target angiogenesis, lymphangiogenesis and stress-response pathways in the different stages of cancer care.

Mutations in SOX18, VEGFC and Vascular Endothelial Growth Factor 3 underlie the hereditary lymphatic disorders hypotrichosis-lymphedema-telangiectasia (HLT), Milroy-like lymphedema and Milroy disease, respectively. Genes responsible for hereditary lymphedema are key regulators of lymphatic vascular development in the embryo. To identify novel modulators of lymphangiogenesis, we used a mouse model of HLT (Ragged Opossum) and performed gene expression profiling of aberrant dermal lymphatic vessels. Expression studies and functional analysis in zebrafish and mice revealed one candidate, ArfGAP with RhoGAP domain, Ankyrin repeat and PH domain 3 (ARAP3), which is down-regulated in HLT mouse lymphatic vessels and necessary for lymphatic vascular development in mice and zebrafish. We position this known regulator of cell behaviour during migration as a mediator of the cellular response to Vegfc signalling in lymphatic endothelial cells in vitro and in vivo. Our data refine common mechanisms that are likely to contribute during both development and the pathogenesis of lymphatic vascular disorders. Refereed/Peer-reviewed

VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

Vascular endothelial growth factors (VEGFs) control angiogenesis and lymphangiogenesis during development and in pathological conditions. In the zebrafish trunk, Vegfa controls the formation of intersegmental arteries by primary angiogenesis and Vegfc is essential for secondary angiogenesis, the formation of veins and lymphatics. VEGFD has been largely thought of as dispensable for vascular development in vertebrates. Here, we generated a zebrafish vegfd mutant by genome editing. vegfd mutants display significant defects in facial lymphangiogenesis independent of vegfc function. Strikingly, we find that vegfc and vegfd cooperatively control lymphangiogenesis throughout the embryo, including during the formation of the trunk lymphatic vasculature. Interestingly, we find that vegfd and vegfc also redundantly drive artery hyperbranching phenotypes observed upon depletion of Flt1 or Dll4. Epistasis and biochemical binding assays suggest that during primary angiogenesis Vegfd influences these phenotypes through Kdr (Vegfr2) rather than Flt4 (Vegfr3). These data demonstrate that, rather than being dispensable during development, Vegfd plays context specific indispensible and also compensatory roles during both blood vessel angiogenesis and lymphangiogenesis.

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.