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.

Human papillomavirus (HPV)(+) and HPV(−) head and neck cancer (HNC) cells’ interactions with the host immune system are poorly understood. Recently, we identified molecular and functional differences in exosomes produced by HPV(+) vs. HPV(−) cells, suggesting that genetic cargos of exosomes might identify novel biomarkers in HPV-related HNCs. Exosomes were isolated by size exclusion chromatography from supernatants of three HPV(+) and two HPV(−) HNC cell lines. Paired cell lysates and exosomes were analyzed for messenger RNA (mRNA) by qRT-PCR and microRNA (miR) contents by nanostring analysis. The mRNA profiles of HPV(+) vs. HPV(−) cells were distinct, with EGFR, TP53 and HSPA1A/B overexpressed in HPV(+) cells and IL6, FAS and DPP4 in HPV(−) cells. The mRNA profiles of HPV(+) or HPV(−) exosomes resembled the cargo of their parent cells. miR expression profiles in cell lysates identified 8 miRs expressed in HPV(−) cells vs. 14 miRs in HPV(+) cells. miR-205-5p was exclusively expressed in HPV(+) exosomes, and miR-1972 was only detected in HPV(−) exosomes. We showed that HPV(+) and HPV(−) exosomes recapitulated the mRNA expression profiles of their parent cells. Expression of miRs was dependent on the HPV status, and miR-205-5p in HPV(+) and miR-1972 in HPV(−) exosomes emerge as potential discriminating HPV-associated biomarkers.

The potential use of plasma-derived small extracellular vesicles (sEV) as predictors of response to therapy and clinical outcome in chemotherapy-naïve patients with non-small-cell lung cancer (NSCLC) was explored. sEV were isolated by size-exclusion chromatography from the plasma of 79 chemotherapy-naïve NSCLC patients and 12 healthy donors (HD). sEV were characterized with regard to protein content, particle size, counts by qNano, morphology by transmission electron microscopy, and molecular profiles by Western blots. PD-1 and PD-L1 expression on circulating immune cells was analysed by flow cytometry. Pre-treatment levels of total sEV protein (TEP) were correlated with overall (OS) and progression-free survival (PFS). The sEV numbers and protein levels were significantly elevated in the plasma of NSCLC patients compared to HD (p = 0.009 and 0.0001, respectively). Baseline TEP levels were higher in patients who developed progressive disease compared to patients with stable disease (p = 0.007 and 0.001, stage III and IV, respectively). Patient-derived sEV were enriched in immunosuppressive proteins as compared to proteins carried by sEV from HD. TEP levels were positively correlated with CD8⁺PD-1⁺ and CD8⁺PD-L1⁺ circulating T cell percentages and were independently associated with poorer PFS (p < 0.00001) and OS (p < 0.00001). Pre-therapy sEV could be useful as non-invasive biomarkers of response to therapy and clinical outcome in NSCLC.

Exosomes mediate intercellular communication, shuttling messages between cells and tissues. We explored whether exosome tissue sequestration is determined by the exosomes or the tissues using ten radiolabeled exosomes from human or murine, cancerous or noncancerous cell lines. We measured sequestration of these exosomes by the liver, kidney, spleen, and lung after intravenous injection into male CD-1 mice. Except for kidney sequestration of three exosomes, all exosomes were incorporated by all tissues, but sequestration levels varied greatly among exosomes and tissues. Species of origin (mouse vs. human) or source (cancerous vs. noncancerous cells) did not influence tissue sequestration. Sequestration of J774A.1 exosomes by liver involved the mannose-6 phosphate (M6P) receptor. Wheatgerm agglutinin (WGA) or lipopolysaccharide (LPS) treatments enhanced sequestration of exosomes by brain and lung but inhibited sequestration by liver and spleen. Response to LPS was not predictive of response to WGA. Path and heat map analyses included our published results for brain and found distinct clusters among the exosomes and the tissues. In conclusion, we found no evidence for a universal binding site controlling exosome-tissue interactions. Instead, sequestration of exosomes by tissues is differentially regulated by both exosomes and tissues and may be stimulated or inhibited by WGA and inflammation.