Key Points• Tumor-derived VEGF-A mediates endothelial cell activation, VWF release, and platelet aggregation provoking coagulation in tumor patients.• Local ADAMTS13 inhibition promotes VWF fiber formation in tumor microvessels.Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrinlike and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation. (Blood. 2015;125(20):3153-3163) IntroductionTo form new metastatic lesions, circulating melanoma cells have to interact with endothelial cells (ECs) and migrate through the vessel wall.1,2 In this context, our own in vitro studies show that melanoma cells activate ECs by an indirect, tissue factor (TF)-mediated thrombin generation.3 Next to this indirect melanoma-induced EC activation, recent findings identified melanoma-derived vascular endothelial growth factor-A (VEGF-A) as main mediator of direct EC activation. 4,5 Both the thrombin-and the VEGF-A-dependent pathways induce EC activation followed by Weibel-Palade body (WPB) exocytosis and the release of inflammatory cytokines and the highly procoagulatory glycoprotein von Willebrand factor (VWF), linking inflammation and coagulation. 6 On the one hand, luminally released VWF fibers are involved in hemostasis and vessel repair as mediators of platelet adhesion to the endothelium. 7,8 On the other hand, we showed that tumor cell-induced ultra-large VWF (ULVWF) fibers have the highest potential for platelet binding and aggregation.9,10 This effect may contribute not only to pathophysiologic vessel occlusion, 11 but also to the establishment of metastasis as platelets facilitate tumor cell extravasation. 12-14Indeed, it is well-known that cancer pati...

Low antigen expression and an absence of coimmunostimulatory signals may be partly responsible for the low immunogenicity of many tumors. It may be possible to overcome this situation by defining a combination of adjuvants and antigens that can activate a high-avidity antitumor response. Using the poorly immunogenic B16-OVA melanoma cells as tumor model, we tested different combinations of adjuvants and antigens to treat established tumors. In the absence of exogenous antigens, repeated administration of the TLR7 ligand Imiquimod together with anti-CD40 agonistic antibodies activated only innate immunity, which was insufficient to reject intradermal tumors. Administering this adjuvant combination together with OVA as a tumor antigen induced T-cell responses that delayed tumor growth. However, administering a combination of anti-CD40 plus TLR3 and TLR7 ligands, together with antigen targeting to dendritic cells through TLR4, was sufficient to induce tumor rejection in 50% of mice. This response was associated with a greater activation of innate immunity and induction of high-avidity polyfunctional CD8 þ T-cell responses, which each contributed to tumor rejection. This therapy activated T-cell responses not only against OVA, which conferred protection against a rechallenge with B16-OVA cells, but also activated T-cell responses against other melanoma-associated antigens. Our findings support the concept that multiple adjuvant combination and antigen targeting may be a useful immunotherapeutic strategy against poorly immunogenic tumors. Cancer Res; 71(9); 3214-24. Ó2011 AACR.