Tumor Associated Neutrophils (TANs) are engaged into the tumor microenvironment by cytokines and chemokines, can be distinguished according to their activation and cytokine status and effects on tumor cell growing in N1 and N2 TANs. N1 TANs exert an antitumor activity, by direct or indirect cytotoxicity. N2 TANs stimulate immunosuppression, tumor growth, angiogenesis and metastasis by DNA instability, or by cytokines and chemokines release. In tumor patients, either a high number of TANs and Neutrophil-to-Lymphocyte Ratio (NLR) do correlate with poor prognosis, and, so far, TAN counts and NLR can be regarded as biomarkers. Owing to the pivotal role of TANs in stimulating tumor progression, therapeutic strategies to target TANs have been suggested, and two major approaches have been proposed: (a) targeting the CXCL-8/CXCR-1/CXCR-2 axis, thereby blocking TANs or (b) targeting substances produced by polymorpho-nuclear cells that promote tumor growth. Many studies have been accomplished either in vitro and in animal models, whereas clinical studies are restrained, presently, due to the risk of inducing immunosuppression. In this review, we deeply discuss the anti-tumorigenic or pro-tumorigenic activity of TANs. In particular, TANs relevance in tumor prognosis and in vitro therapeutic strategies are widely described. On-going clinical trials, aimed to inhibit neutrophil recruitment into the tumor are also accurately debated.

Neutrophil Extracellular Traps (NETs) are net-like structures composed of DNA-histone complexes and proteins released by activated neutrophils. In addition to their key role in the neutrophil innate immune response, NETs are also involved in autoimmune diseases, like systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and in other non-infectious pathological processes, as coagulation disorders, thrombosis, diabetes, atherosclerosis, vasculitis, and cancer. Recently, a large body of evidence indicates that NETs are involved in cancer progression and metastatic dissemination, both in animal models and cancer patients. Interestingly, a close correlation between cancer cell recruitment of neutrophils in the tumor microenvironment (Tumor Associated Neutrophils. TANs) and NET formation has been also observed either in primary tumors and metastatic sites. Moreover, NETs can also catch circulating cancer cells and promote metastasis. Furthermore, it has been reported that wake dormant cancer cells, causing tumor relapse and metastasis. This review will primarily focus on the pro-tumorigenic activity of NETs in tumors highlighting their ability to serve as a potential target for cancer therapy.

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.

The respective roles of urokinase plasminogen activator (u-PA) and the u-PA receptor in extracellular matrix degradation was investigated. Human pro-u-PA and the human u-PA receptor were expressed independently by two different mouse LB6 cell lines. The matrix degradation capacity of these cell lines individually or in coculture was studied. Although pro-u-PA-producing cells alone degrade the matrix in the presence of plasminogen, u-PA-receptor producing cells do not. Cocultivation of a small fraction of pro-u-PA-producing cells with the receptor-producing cells increases the rate of matrix degradation at least threefold. By immunoprecipitation it was shown that cocultivation of the two cell lines increases the conversion of the inactive pro-u-PA to the active two chain u-PA. The enhancement of matrix degradation and of pro-u-PA activation requires actual binding of pro-u-PA to its receptor because it is inhibited by u-PA-receptor antagonists. The u-PA receptor must be cell associated, as binding of pro-u-PA to a receptor solubilized from the cell surface with phosphatidyl-inositol specific phospholipase C did not enhance the activation of pro-u-PA in the presence of plasminogen. The finding that activity of u-PA is enhanced when it is bound to its receptor, even when the receptor is produced by a different cell, might have important implications for the mechanisms of u-PA-induced extracellular proteolysis in vivo.

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser⁸⁸-Arg-Ser-Arg-Tyr⁹² is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH₂ (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe–dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an αv integrin–dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/αv association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein–expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy. [Mol Cancer Ther 2009;8(9):2708–17]

Several studies have ascertained that uPA and uPAR do participate in tumor progression and metastasis and are involved in cell adhesion, migration, invasion and survival, as well as angiogenesis. Increased levels of uPA and uPAR in tumor tissues, stroma and biological fluids correlate with adverse clinic–pathologic features and poor patient outcomes. After binding to uPAR, uPA activates plasminogen to plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme able to facilitate tumor cell invasion and dissemination to distant sites. Moreover, uPAR activated by uPA regulates most cancer cell activities by interacting with a broad range of cell membrane receptors. These findings make uPA and uPAR not only promising diagnostic and prognostic markers but also attractive targets for developing anticancer therapies. In this review, we debate the uPA/uPAR structure–function relationship as well as give an update on the molecules that interfere with or inhibit uPA/uPAR functions. Additionally, the possible clinical development of these compounds is discussed.