Cancer produces a variety of collateral effects in patients beyond the malignancy itself, including threats to distal organ functions. However, the basis for such effects, associated with either primary or metastatic tumors, are generally poorly understood. In this study, we show how heart and kidney vascular function is impaired by neutrophils that accumulate in those tissues as a result of tumor formation in two different transgenic mouse models of cancer (RIP1-Tag2 model of insulinoma and MMTV-PyMT model of breast cancer). Neutrophil depletion by systemic administration of an anti-Gr1 antibody improved vascular perfusion and prevented vascular leakage in kidney vessels. We also observed the accumulation of platelet-neutrophil complexes, a signature of neutrophil extracellular traps (NET), in the kidneys of tumor-bearing mice that were completely absent from healthy nontumor-bearing littermates. NET accumulation in the vasculature was associated with upregulation of the proinflammatory adhesion molecules ICAM-1, VCAM-1, and E-selectin, as well as the proinflammatory cytokines IL1b, IL6, and the chemokine CXCL1. Administering DNase I to dissolve NETs, which have a high DNA content, restored perfusion in the kidney and heart to levels seen in nontumor-bearing mice, and also prevented vessel leakage in the blood vasculature of these organs. Taken together, our findings strongly suggest that NETs mediate the negative collateral effects of tumors on distal organs, acting to impair vascular function, and to heighten inflammation at these sites.Cancer Res; 75(13); 2653-62. Ó2015 AACR.

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Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFβ2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFβ signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFβ2 may represent a new target for vascular normalization therapy.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) signaling pathway is in clinical use, but its effect on vascular function and the tumor microenvironment is poorly understood. Here, we investigate cross‐talk between VEGF and proinflammatory TNF‐α signaling in endothelial cells and its impact on leukocyte recruitment. We found that cotreatment with VEGF decreased TNF‐α‐induced Jurkat cell adhesion to human microvascular endothelial cells by 40%. This was associated with inhibition of TNF‐α‐mediated regulation of 86 genes, including 2 T‐lymphocyte‐attracting chemokines, CXCL10 and CXCL11 [TNF‐α concentration 1 ng/ml; 50% inhibition/inhibitory concentration (IC50) VEGF, 3 ng/ml]. Notably, VEGF directly suppressed TNF‐α‐induced gene expression through negative cross‐talk with the NF‐κB‐signaling pathway, leading to an early decrease in IFN regulatory factor 1 (IRF‐1) expression and reduced phosphorylation of signal transducer and activator of transcription 1 (p‐Stat1) at later times. Inhibition of VEGF signaling in B16 melanoma tumor‐bearing mice by sunitinib treatment resulted in up‐regulation of CXCL10 and CXCL11 in tumor vessels, accompanied by up to 18‐fold increased infiltration of CD3+ T‐lymphocytes in B16 tumors. Our results demonstrate a novel role of VEGF in negative regulation of NF‐κB signaling and endothelial activation in the tumor microenvironment and provide evidence that pharmacological inhibition of VEGF signaling enhances T‐lymphocyte recruitment through up‐regulation of chemokines CXCL10 and CXCL11.—Huang, H., Langenkamp, E., Georganaki, M., Loskog, A., Fuchs, P. F., Dieterich, L. C., Kreuger, J., Dimberg, A., VEGF suppresses T‐lymphocyte infiltration in the tumor microenvironment through inhibition of NF‐κB‐induced endothelial activation. FASEB J. 29, 227–238 (2015). http://www.fasebj.org