Neutrophil extracellular traps in the pathology of cancer and other inflammatory diseases.

Neutrophil extracellular trap (NET) formation, first described in 2004 as a previously unknown strategy of neutrophils to fight microbes, has attracted an increasing interest in the research community. NETs are formed when neutrophils externalize their decondensed chromatin together with content from their azurophilic granules. In addition to their role in defense against microbes, NETs have been implicated as mediators of pathology in sterile inflammation, such as cancer and autoimmunity, and their potential as therapeutic targets is actively explored. However, targeting of NETs is challenging since the beneficial effects of their removal need to be balanced against the potential harmful loss of their function in microbial defense. Moreover, depending on the stimuli or species, NETs can be formed via distinct mechanisms and are not always made up of the same components, making direct comparisons between various studies challenging. This review focuses on the role of NETs in cancer-associated pathology, such as thrombosis, organ dysfunction, and metastasis. Different strategies to target NETs, by either preventing their formation or degrading existing ones, are also discussed.

Platelet-Derived PDGFB Promotes Recruitment of Cancer-Associated Fibroblasts, Deposition of Extracellular Matrix and Tgfß Signaling in the Tumor Microenvironment.

Platelets constitute a major reservoir of platelet-derived growth factor B (PDGFB) and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. To address the specific role of platelet-derived PDGFB in the tumor microenvironment, we have created a mouse model with conditional knockout of PDGFB in platelets (pl-PDGFB KO). Lack of PDGFB in platelets resulted in 10-fold lower PDGFB concentration in the tumor microenvironment, fewer cancer-associated fibroblasts and reduced deposition of the extracellular matrix (ECM) molecules fibronectin and collagen I in the orthotopic RIP1-Tag2 model for pancreatic neuroendocrine cancer. Myosin light chain phosphorylation, promoting cell contraction and, consequently, the mechano-induced release of active transforming growth factor (TGF) ß from extracellular compartments, was reduced in tumors from pl-PDGFB KO mice. In agreement, TGFß signaling, measured as phosphorylated Smad2, was significantly hampered in tumors from mice lacking PDGFB in their platelets, providing a plausible explanation for the reduced deposition of extracellular matrix. These findings indicate a major contribution of platelet-derived PDGFB to a malignant transformation of the tumor microenvironment and address for the first time the role of PDGFB released specifically from platelets in the remodeling of the ECM in tumors.

Inflammation and neutrophil extracellular traps in cerebral cavernous malformation.

Cerebral Cavernous Malformation (CCM) is a brain vascular disease with various neurological symptoms. In this study, we describe the inflammatory profile in CCM and show for the first time the formation of neutrophil extracellular traps (NETs) in rodents and humans with CCM. Through RNA-seq analysis of cerebellum endothelial cells from wild-type mice and mice with an endothelial cell-specific ablation of the Ccm3 gene (Ccm3iECKO), we show that endothelial cells from Ccm3iECKO mice have an increased expression of inflammation-related genes. These genes encode proinflammatory cytokines and chemokines, as well as adhesion molecules, which promote recruitment of inflammatory and immune cells. Similarly, immunoassays showed elevated levels of these cytokines and chemokines in the cerebellum of the Ccm3iECKO mice. Consistently, both flow cytometry and immunofluorescence analysis showed infiltration of different subsets of leukocytes into the CCM lesions. Neutrophils, which are known to fight against infection through different strategies, including the formation of NETs, represented the leukocyte subset within the most pronounced increase in CCM. Here, we detected elevated levels of NETs in the blood and the deposition of NETs in the cerebral cavernomas of Ccm3iECKO mice. Degradation of NETs by DNase I treatment improved the vascular barrier. The deposition of NETs in the cavernomas  of patients with CCM confirms the clinical relevance of NETs in CCM.

Vaccination against galectin-1 promotes cytotoxic T-cell infiltration in melanoma and reduces tumor burden.

Galectin-1 (Gal1) is a glycan-binding protein that promotes tumor progression by several distinct mechanisms. Through direct binding to vascular endothelial growth factor (VEGF)-receptor 2, Gal1 is able to induce VEGF-like signaling, which contributes to tumor angiogenesis. Furthermore, several studies have demonstrated an immunosuppressive function of Gal1 through effects on both effector and regulatory T cells. Elevated Gal1 expression and secretion have been shown in many tumor types, and high Gal1 serum levels have been connected to poor prognosis in cancer patients. These findings suggest that therapeutic strategies directed against Gal1 would enable simultaneous targeting of angiogenesis, immune evasion and metastasis. In the current study, we have analyzed the potential of Gal1 as a cancer vaccine target. We show that it is possible to generate high anti-Gal1 antibody levels in mice immunized with a recombinant vaccine protein consisting of bacterial sequences fused to Gal1. Growth of Gal1 expressing melanomas was significantly impaired in the immunized mice compared to the control group. This was associated with improved perfusion of the tumor vasculature, as well as increased infiltration of macrophages and cytotoxic T cells (CTLs). The level of granzyme B, mainly originating from CTLs in our model, was significantly elevated in Gal1 vaccinated mice and correlated with a decrease in tumor burden. We conclude that vaccination against Gal1 is a promising pro-immunogenic approach for cancer therapy that could potentially enhance the effect of other immunotherapeutic strategies due to its ability to promote CTL influx in tumors.

Platelet-Specific PDGFB Ablation Impairs Tumor Vessel Integrity and Promotes Metastasis.

Platelet-derived growth factor B (PDGFB) plays a crucial role in recruitment of PDGF receptor ß-positive pericytes to blood vessels. The endothelium is an essential source of PDGFB in this process. Platelets constitute a major reservoir of PDGFB and are continuously activated in the tumor microenvironment, exposing tumors to the plethora of growth factors contained in platelet granules. Here, we show that tumor vascular function, as well as pericyte coverage is significantly impaired in mice with conditional knockout of PDGFB in platelets. A lack of PDGFB in platelets led to enhanced hypoxia and epithelial-to-mesenchymal transition in the primary tumors, elevated levels of circulating tumor cells, and increased spontaneous metastasis to the liver or lungs in two mouse models. These findings establish a previously unknown role for platelet-derived PDGFB, whereby it promotes and maintains vascular integrity in the tumor microenvironment by contributing to the recruitment of pericytes. SIGNIFICANCE: Conditional knockout of PDGFB in platelets demonstrates its previously unknown role in the maintenance of tumor vascular integrity and host protection against metastasis.