The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Lymph nodes (LNs) are highly organized secondary lymphoid organs that mediate adaptive immune responses to antigens delivered via afferent lymphatic vessels. Lymphatic endothelial cells (LECs) line intranodal lymphatic sinuses and organize lymph and antigen distribution. LECs also directly regulate T cells, mediating peripheral tolerance to self-antigens, and play a major role in many diseases, including cancer metastasis. However, little is known about the phenotypic and functional heterogeneity of LN LECs. Using single-cell RNA sequencing, we comprehensively defined the transcriptome of LECs in murine skin-draining LNs and identified new markers and functions of distinct LEC subpopulations. We found that LECs residing in the subcapsular sinus (SCS) have an unanticipated function in scavenging of modified low-density lipoprotein (LDL) and also identified a specific cortical LEC subtype implicated in rapid lymphocyte egress from LNs. Our data provide new, to our knowledge, insights into the diversity of LECs in murine LNs and a rich resource for future studies into the regulation of immune responses by LN LECs.

Traditionally, tumor-associated lymphatic vessels have been regarded as passive by-standers, serving simply as a drainage system for interstitial fluid generated within the tumor. However, with growing evidence that tumors actively induce lymphangiogenesis, and that the number of lymphatic vessels closely correlates with metastasis and clinical outcome in various types of cancer, this picture has changed dramatically in recent years. Tumor-associated lymphatic vessels have now emerged as a valid therapeutic target to control metastatic disease, and the first specific anti-lymphangiogenic drugs have recently entered clinical testing. Furthermore, we are just beginning to understand the whole functional spectrum of tumor-associated lymphatic vessels, which not only concerns transport of fluid and metastatic cells, but also includes the regulation of cancer stemness and specific inhibition of immune responses, opening new venues for therapeutic applications. Therefore, we predict that specific targeting of lymphatic vessels and their function will become an important tool for future cancer treatment.

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