The coagulation and complement systems are evolutionarily related enzymatic cascades and it is known that both systems interact with each other. Although growing body of evidence suggest that tumor mediated hypercoagulation supports neutrophil recruitment and metastasis, the impact of complement effectors on melanoma metastasis formation are poorly understood. Increased activation of the complement system has been measured in various malignancies. Previous studies indicated that the complement activates endothelial cells (ECs) and neutrophils. However, in the context of tumor progression, knowledge on the crosstalk between the vascular endothelium, the complement system and the neutrophil associated innate immunity is still scarce. Here, we report the systemic complement activation in patients suffering from malignant melanoma. Using mouse and human tumor tissue samples, we showed that complement effectors such as C3b and C5a fragments deposit around tumor blood vessel walls. However the complement cascade terminated by the formation of the membrane attack complex (MAC) not on the endothelium but on perivascular neutrophils. In vitro experiments with human ECs and neutrophils could confirm this complement mediated crosstalk. Further in vitro experiments demonstrated that MAC positive neutrophils release reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). In close proximity to the endothelium, complement activated neutrophils were able to increase the vascular permeability allowing the transmigration of melanoma cells. Interference with the deposition of complement factors on the EC surface through the low-molecular weight heparin tinzaparin prevented MACs formation and thus ROS and NETs release from neutrophils. Moreover, tinzaparin treatment stabilized the vascular permeability and might contribute to a reduced metastasis as previously published. In summary, we discovered a triangular communication between the complement, neutrophils and the vascular endothelium mediating NETosis, endothelial dysfunction and subsequently melanoma cells extravasation. Therefore, targeting complement activation envisions a new therapeutic strategy for malignant melanoma.
