Plant-derived exosome-like nanovesicles (EPDENs) have recently been isolated and evaluated as potential bioactive nutraceutical biomolecules. It has been hypothesized that EPDENs may exert their activity on mammalian cells through their specific cargo. In this study, we isolated and purified EPDENs from the strawberry juice of Fragaria x ananassa (cv. Romina), a new cultivar characterized by a high content of anthocyanins, folic acid, flavonols, and vitamin C and an elevated antioxidant capacity. Fragaria-derived EPDENs were purified by a series of centrifugation and filtration steps. EPDENs showed size and morphology similar to mammalian extracellular nanovesicles. The internalization of Fragaria-derived EPDENs by human mesenchymal stromal cells (MSCs) did not negatively affect their viability, and the pretreatment of MSCs with Fragaria-derived EPDENs prevented oxidative stress in a dose-dependent manner. This is possibly due to the presence of vitamin C inside the nanovesicle membrane. The analysis of EPDEN cargo also revealed the presence of small RNAs and miRNAs. These findings suggest that Fragaria-derived EPDENs may be considered nanoshuttles contained in food, with potential health-promoting activity.

Despite the popularity of platelet-rich plasma (PRP) and platelet lysate (PL) in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF) released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC) treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg) showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies.

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets. Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography. Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients. Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.

Background. Exosome-like nanovesicles are biological nanostructures mediating cell-to-cell communication and capable to load selected cargos also in the interaction among different species. Objective. We aimed to explore the content of exosome-like nanovesicles derived from Citrus limon L. and to analyze the effects of their uptake on human cells. Method. We isolated exosome-like nanovesicles from Citrus limon L. juice (EXO-CL) by differential centrifugation. EXOCL were analyzed for short RNA content by advanced sequencing technologies, and for ascorbic acid (vitamin C) and citrate content by enzymatic assays. EXO-CL anti-oxidant and pro-differentiative potential was evaluated in vitro on mesenchymal stromal cells (MSC), a common tool for regenerative strategies for several human tissues. Results. We showed that EXO-CL carry detectable amounts of citrate and vitamin C and, although it was not possible to identify specific miRNAs, we detected short RNA sequences (20-30 bp) with unknown functions and with different distribution size in respect to whole Citrus limon L. juice. In vitro, EXO-CL were uptaken by MSC and had a significant protective effect against oxidative stress. Furthermore, regarding the potential benefit for human bone health, we found that EXO-CL modulate MSC differentiation versus the osteogenic lineage. Conclusion. We demonstrated that incubation with EXO-CL exerts antioxidant activity in human cells. This is most likely due to the direct delivery and uptake of micronutrients by human cells that are well preserved inside the nanovesicle membrane, including the unstable vitamin C. Based on our results, we speculate that fruit-derived nanovesicles have the potential to mediate interspecies influence after food intake.

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