Extracellular vesicles can cross the blood–brain barrier (BBB), but little is known about passage. Here, we used multiple-time regression analysis to examine the ability of 10 exosome populations derived from mouse, human, cancerous, and non-cancerous cell lines to cross the BBB. All crossed the BBB, but rates varied over 10-fold. Lipopolysaccharide (LPS), an activator of the innate immune system, enhanced uptake independently of BBB disruption for six exosomes and decreased uptake for one. Wheatgerm agglutinin (WGA) modulated transport of five exosome populations, suggesting passage by adsorptive transcytosis. Mannose 6-phosphate inhibited uptake of J774A.1, demonstrating that its BBB transporter is the mannose 6-phosphate receptor. Uptake rates, patterns, and effects of LPS or WGA were not predicted by exosome source (mouse vs. human) or cancer status of the cell lines. The cell surface proteins CD46, AVβ6, AVβ3, and ICAM-1 were variably expressed but not predictive of transport rate nor responses to LPS or WGA. A brain-to-blood efflux mechanism variably affected CNS retention and explains how CNS-derived exosomes enter blood. In summary, all exosomes tested here readily crossed the BBB, but at varying rates and by a variety of vesicular-mediated mechanisms involving specific transporters, adsorptive transcytosis, and a brain-to-blood efflux system.

A growing body of evidence emphasizes the important role exosomes in different physiological and pathological conditions. Exosomes, virus-size extracellular vesicles (EVs), carry a complex molecular cargo, which is actively processed in the endocytic compartment of parental cells. Exosomes carry and deliver this cargo to recipient cells, serving as an intercellular communication system. The methods for recovery of exosomes from supernatants of cell lines or body fluids are not uniformly established. Yet, studies of the quality and quantity of exosome cargos underlie the concept of “liquid biopsy.” Exosomes are emerging as a potentially useful diagnostic tool and a predictor of disease progression, response to therapy and overall survival. Although many novel approaches to exosome isolation and analysis of their cargos have been introduced, the role of exosomes as diagnostic or prognostic biomarkers of disease remains unconfirmed. This review considers existing challenges to exosome validation as disease biomarkers. Focusing on advantages and limitations of methods for exosome isolation and characterization, approaches are proposed to facilitate further progress in the development of exosomes as biomarkers in human disease.

For solid tumors, such as head and neck squamous cell carcinoma (HNSCC), an adequate blood supply is of critical importance for tumor development and metastasis. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and serve as a communication system between tumor and normal stromal cells. This study evaluates in vitro and in vivo effects mediated by TEX that result in promotion of angiogenesis. TEX produced by PCI-13 (HPV−) and UMSCC47 (HPV+) cell lines or from plasma of HNSCC patients were isolated by mini size exclusion chromatography (mini-SEC). TEX morphology, size, numbers, and molecular profile were characterized, and the angiogenesis-inducing potential was measured in arrays and real-time PCR with human endothelial cells (HUVEC). Uptake of labeled TEX by HUVECs was demonstrated by confocal microscopy. Tube formation, proliferation, migration, and adherence by HUVECs in response to TEX were investigated. The 4-nitroquinoline-1-oxide (4-NQO) oral carcinogenesis mouse model was used to confirm that TEX induce the same results in vivo. TEX were found to be potent inducers of angiogenesis in vitro and in vivo through functional reprogramming and phenotypic modulation of endothelial cells. TEX carried angiogenic proteins and were internalized by HUVECs within 4 hours. TEX stimulated proliferation (P < 0.001), migration (P < 0.05), and tube formation (P < 0.001) by HUVECs and promoted formation of defined vascular structures in vivo. The data suggest that TEX promote angiogenesis and drive HNSCC progression. Future efforts should focus on eliminating or silencing TEX and thereby adding new options for improving existing antiangiogenic therapies. Implications: TEX appear to play an important role in tumor angiogenesis and thus may contribute to tumor growth and metastasis of HNSCC in this context. Mol Cancer Res; 16(11); 1798–808. ©2018 AACR.

Introduction: Tumor-derived exosomes (TEX) and their role in tumor progression by accelerating angiogenesis are of great current interest. A better understanding of the mechanisms underlying TEX-blood vessels cross-talk may lead to improvements in current diagnosis, prognosis and treatment of cancer. Areas covered: For solid tumors, an adequate blood supply is of critical importance for their development, growth and metastasis. TEX, virus-size vesicles which circulate freely throughout body fluids and accumulate in the tumor microenvironment (TME), have been recognized as a new contributor to angiogenesis. TEX serve as a communication system between the tumor and various normal cells and are responsible for functional reprogramming of these cells. The molecular and genetic cargos that TEX deliver to the recipient cells involved in angiogenesis promote its induction and progression. The targeted inhibition of TEX pro-angiogenic functions might be a novel therapeutic approach for control of tumor progression. Expert opinion: TEX circulating in body fluids of cancer patients carry a complex molecular and genetic cargo and are responsible for phenotypic and functional reprogramming of endothelial cells and other normal cells residing in the TME.

Interactions between tumor cells and tumor-associated macrophages (TAMs) are critical for glioblastoma progression. The TAMs represent up to 30% of the glioblastoma mass. The role of TAMs in tumor progression and in the mechanisms underlying tumor growth remain unclear. Using an in vitro model resembling the crosstalk between macrophages and glioblastoma cells, we show that glioblastoma-derived exosomes (GBex) reprogram M1 (mediate pro-inflammatory function) and M2 (mediate anti-inflammatory function) macrophages, converting M1 into TAMs and augmenting pro-tumor functions of M2 macrophages. In turn, these GBex-reprogrammed TAMs, produce exosomes decorated by immunosuppressive and tumor-growth promoting proteins. TAM-derived exosomes disseminate these proteins in the tumor microenvironment (TME) promoting tumor cell migration and proliferation. Mechanisms underlying the promotion of glioblastoma growth involved Arginase-1+ exosomes produced by the reprogrammed TAMs. A selective Arginase-1 inhibitor, nor-NOHA reversed growth-promoting effects of Arginase-1 carried by TAM-derived exosomes. The data suggest that GBex-reprogrammed Arginase-1+ TAMs emerge as a major source of exosomes promoting tumor growth and as a potential therapeutic target in glioblastoma.