Membrane attack complex activated neutrophils increase the permeability of tumor blood vessels

Abstract

Increased activation of the complement system has been measured in various malignancies. Previous studies indicated that the complement system 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 tumor-specific complement activation in patients suffering from malignant melanoma. Using mouse and human tumor tissue samples, we showed that accumulation of complement effectors such as C3 fragments and C5a around tumor blood vessel walls were increased. Moreover, we detected high levels of the mannose binding lectin (MBL) at the endothelium suggesting the involvement of the lectin pathway as main trigger of the melanoma mediated complement activation. 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 confirmed this complement mediated crosstalk. Further in vitro experiments demonstrated that MAC positive neutrophils released 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. MAC deposition on tumor-associated neutrophils was also found in human melanomas but not in rarely metastasizing basal cell carcinomas, keratoacanthoma, or non-metastatic nevocytic nevi. Interference with the deposition of complement factors on the EC surface through the low-molecular weight heparin tinzaparin prevented MAC 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 system, neutrophils and the vascular endothelium mediating NETosis, endothelial dysfunction and subsequently melanoma cells extravasation. Therefore, targeting complement activation envisions a new therapeutic strategy for the treatment of malignant melanoma.