The critical role of angiogenesis in promoting tumor growth and metastasis is strongly established. However, tumors show considerable variation in angiogenic characteristics and in their sensitivity to antiangiogenic therapy. Tumor angiogenesis involves not only cancer cells but also various tumor-associated leukocytes (TALs) and stromal cells. TALs produce chemokines, cytokines, proteases, structural proteins, and microvescicles. Vascular endothelial growth factor (VEGF) and inflammatory chemokines are not only major proangiogenic factors but are also immune modulators, which increase angiogenesis and lead to immune suppression. In our review, we discuss the regulation of angiogenesis by innate immune cells in the tumor microenvironment, specific features, and roles of major players: macrophages, neutrophils, myeloid-derived suppressor and dendritic cells, mast cells, γδT cells, innate lymphoid cells, and natural killer cells. Anti-VEGF or anti-inflammatory drugs could balance an immunosuppressive microenvironment to an immune permissive one. Anti-VEGF as well as anti-inflammatory drugs could therefore represent partners for combinations with immune checkpoint inhibitors, enhancing the effects of immune therapy.

The angiogenic activity of CXC-ELR+ chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-α in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck−/−fgr−/−), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck−/−fgr−/− mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck−/−fgr−/− mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck−/−fgr−/− mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.

IFNs are highly pleiotropic cytokines also endowed with marked antiangiogenic activity. In this study, the mRNA expression profiles of endothelial cells (EC) exposed in vitro to IFN-α, IFN-β, or IFN-γ were determined. We found that in HUVEC as well as in other EC types 175 genes were up-regulated (>2-fold increase) by IFNs, including genes involved in the host response to RNA viruses, inflammation, and apoptosis. Interestingly, 41 genes showed a >5-fold higher induction by IFN-α in EC compared with human fibroblasts; among them, the gene encoding the angiostatic chemokine CXCL11 was selectively induced by IFN-α in EC along with other genes associated with angiogenesis regulation, including CXCL10, TRAIL, and guanylate-binding protein 1. These transcriptional changes were confirmed and extended by quantitative PCR analysis and ELISA; whereas IFN-α and IFN-β exerted virtually identical effects on transcriptome modulation, a differential gene regulation by type I and type II IFN emerged, especially as far as quantitative aspects were concerned. In vivo, IFN-α-producing tumors overexpressed murine CXCL10 and CXCL11, guanylate-binding protein 1, and TRAIL, with evidence of CXCL11 production by tumor-associated EC. Overall, these findings improve our understanding of the antiangiogenic effects of IFNs by showing that these cytokines trigger an antiangiogenic transcriptional program in EC. Moreover, we suggest that quantitative differences in the magnitude of the transcriptional activation of IFN-responsive genes could form the basis for cell-specific transcriptional signatures.

Epidemiological studies are providing strong evidence on beneficial health effects from dietary measures, leading scientists to actively investigate which foods and which specific agents in the diet can prevent diseases. Public health officers and medical experts should collaborate toward the design of disease prevention diets for nutritional intervention. Functional foods are emerging as an instrument for dietary intervention in disease prevention. Functional food products are technologically developed ingredients with specific health benefits. Among promising sources of functional foods and chemopreventive diets of interest, microalgae are gaining worldwide attention, based on their richness in high-value products, including carotenoids, proteins, vitamins, essential amino acids, omega-rich oils and, in general, anti-inflammatory and antioxidant compounds. Beneficial effects of microalgae on human health and/or wellness could in the future be useful in preventing or delaying the onset of cancer and cardiovascular diseases. During the past decades, microalgal biomass was predominately used in the health food market, with more than 75% of the annual microalgal biomass production being employed for the manufacture of powders, tablets, capsules or pastilles. In this review, we report and discuss the present and future role of microalgae as marine sources of functional foods/beverages for human wellbeing, focusing on perspectives in chemoprevention. We dissected this topic by analyzing the different classes of microalgal compounds with health outputs (based on their potential chemoprevention activities), the biodiversity of microalgal species and how to improve their cultivation, exploring the perspective of sustainable food from the sea.

Myeloid-derived suppressor cells (MDSCs) contribute to the induction of an immune suppressive/anergic, tumor permissive environment. MDSCs act as immunosuppression orchestrators also by interacting with several components of both innate and adaptive immunity. Natural killer (NK) cells are innate lymphoid cells functioning as primary effector of immunity, against tumors and virus-infected cells. Apart from the previously described anergy and hypo-functionality of NK cells in different tumors, NK cells in cancer patients show pro-angiogenic phenotype and functions, similar to decidual NK cells. We termed the pro-angiogenic NK cells in the tumor microenvironment “tumor infiltrating NK” (TINKs), and peripheral blood NK cells in cancer patients “tumor associated NK” (TANKs). The contribution of MDSCs in regulating NK cell functions in tumor-bearing host, still represent a poorly explored topic, and even less is known on NK cell regulation of MDSCs. Here, we review whether the crosstalk between MDSCs and NK cells can impact on tumor onset, angiogenesis and progression, focusing on key cellular and molecular interactions. We also propose that the similarity of the properties of tumor associated/tumor infiltrating NK and MDSC with those of decidual NK and decidual MDSCs during pregnancy could hint to a possible onco-fetal origin of these pro-angiogenic leukocytes.

NK cells are effector lymphocytes involved in tumor immunosurveillance; however, in patients with solid malignancies, NK cells have compromised functions. We have previously reported that lung tumor-associated NK cells (TANKs; peripheral blood) and tumor-infiltrating NK cells (TINKs) show proangiogenic, decidual NK-like (dNK) phenotype. In this study, we functionally and molecularly investigated TINKs and TANKs from blood and tissue samples of patients with colorectal cancer (CRC), a neoplasm in which inflammation and angiogenesis have clinical relevance, and compared them to NK cells from controls and patients with nononcologic inflammatory bowel disease. CRC TINKs/TANKs showed decreased expression for the activatory marker NKG2D, impaired degranulation activity, a decidual-like NK polarization toward the CD56CD16CD9CD49 subset. TINKs and TANKs secreted cytokines with proangiogenic activities, and induce endothelial cell proliferation, migration, adhesion, and the formation of capillary-like structures in vitro. dNK cells release specific proangiogenic factors; among which, angiogenin and invasion-associated enzymes related to the MMP9-TIMP1/2 axis. Here, we describe, for the first time, to our knowledge, the expression of angiogenin, MMP2/9, and TIMP by TANKs in patients with CRC. This phenotype could be relevant to the invasive capabilities and proangiogenic functions of CRC-NK cells and become a novel biomarker. STAT3/STAT5 activation was observed in CRC-TANKs, and treatment with pimozide, a STAT5 inhibitor, reduced endothelial cell capability to form capillary-like networks, inhibiting VEGF and angiogenin production without affecting the levels of TIMP1, TIMP2, and MMP9, indicating that STAT5 is involved in cytokine modulation but not invasion-associated molecules. Combination of Stat5 or MMP inhibitors with immunotherapy could help repolarize CRC TINKs and TANKs to anti-tumor antimetastatic ones.-Bruno, A., Bassani, B., D'Urso, D. G., Pitaku, I., Cassinotti, E., Pelosi, G., Boni, L., Dominioni, L., Noonan, D. M., Mortara, L., Albini, A. Angiogenin and the MMP9-TIMP2 axis are up-regulated in proangiogenic, decidual NK-like cells from patients with colorectal cancer.

Angiostatin effectively blocks tumor angiogenesis through still poorly understood mechanisms. Given the close association between immune and vascular regulation, we investigated the effects of angiostatin on angiogenesis-associated leukocytes. Angiostatin inhibited the migration of monocytes and, even more markedly, neutrophils. Angiostatin blocked chemotaxis of neutrophils to CXCR2 chemokine receptor agonists (IL-8, MIP-2, and GROalpha), formyl-Met-Leu-Phe (fMLP), and 12-O-tetradecanoylphorbol 13-acetate, and repressed fMLP-induced mitochondrial activity. Two different angiostatin forms (kringles 1-4 and 1-3) were effective, whereas whole plasminogen had no effect. IL-8, MIP-2, and GROalpha induced intense angiogenic reactions in vivo, but no angiogenic response to these factors was observed in neutropenic mice, demonstrating an essential role for neutrophils. Angiostatin potently inhibited chemokine-induced angiogenesis in vivo, and consistent with in vitro observations, both angiostatin forms were active and whole plasminogen had little effect. Angiostatin inhibition of angiogenesis in vivo was accompanied by a striking reduction in the number of recruited leukocytes. In vivo, the inflammatory agent lipopolysaccharide also induced extensive leukocyte infiltration and angiogenesis that were blocked by angiostatin. Neutrophils expressed mRNAs for ATP synthase and angiomotin, two known angiostatin receptors. These data show that angiostatin directly inhibits neutrophil migration and neutrophil-mediated angiogenesis and indicate that angiostatin might inhibit inflammation.

BackgroundSelected microRNAs (miRNAs) that are abnormally expressed in the serum of patients with lung cancer have recently been proposed as biomarkers of this disease. The measurement of circulating miRNAs, however, requires a highly reliable quantification method. Quantitative real-time PCR (qPCR) is the most commonly used method, but it lacks reliable endogenous reference miRNAs for normalization of results in biofluids. When used in absolute quantification, it must rely on the use of external calibrators. Droplet digital PCR (ddPCR) is a recently introduced technology that overcomes the normalization issue and may facilitate miRNA measurement. Here we compared the performance of absolute qPCR and ddPCR techniques for quantifying selected miRNAs in the serum.ResultsIn the first experiment, three miRNAs, proposed in the literature as lung cancer biomarkers (miR-21, miR-126 and let-7a), were analyzed in a set of 15 human serum samples. Four independent qPCR and four independent ddPCR amplifications were done on the same samples and used to estimate the precision and correlation of miRNA measurements obtained with the two techniques. The precision of the two methods was evaluated by calculating the Coefficient of Variation (CV) of the four independent measurements obtained with each technique. The CV was similar or smaller in ddPCR than in qPCR for all miRNAs tested, and was significantly smaller for let-7a (p = 0.028). Linear regression analysis of the miRNA values obtained with qPCR and ddPCR showed strong correlation (p < 0.001).To validate the correlation obtained with the two techniques in the first experiment, in a second experiment the same miRNAs were measured in a larger cohort (70 human serum samples) by both qPCR and ddPCR. The correlation of miRNA analyses with the two methods was significant for all three miRNAs. Moreover, in our experiments the ddPCR technique had higher throughput than qPCR, at a similar cost-per-sample.ConclusionsAnalyses of serum miRNAs performed with qPCR and ddPCR were largely concordant. Both qPCR and ddPCR can reliably be used to quantify circulating miRNAs, however, ddPCR revealed similar or greater precision and higher throughput of analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0292-7) contains supplementary material, which is available to authorized users.