Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids. Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells. Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of NK cells. However, the characteristics and functions of exosomes derived from NK cells remain unknown. In this study, we explored NK cell-derived exosome-mediated antitumor effects against aggressive melanoma in vitro and in vivo.Methods: B16F10 cells were transfected with enhanced firefly luciferase (effluc) and thy1.1 genes, and thy1.1-positive cells were immunoselected using microbeads. The resulting B16F10/effluc cells were characterized using reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and luciferase activity assays. Exosomes derived from NK-92MI cells (NK-92 Exo) were isolated by ultracentrifugation and density gradient ultracentrifugation. NK-92 Exo were characterized by transmission electron microscopy and western blotting. We also performed an enzyme-linked immunosorbent assay to measure cytokines retained in NK-92 Exo cells. The in vitro cytotoxicity of NK-92 Exo against the cancer cells was determined using a bioluminescence imaging system (BLI) and CCK-8 assays. To investigate the possible side effects of NK-92 Exo on healthy cells, we also performed the BLI and CCK-8 assays using the human kidney Phoenix™-Ampho cell line. Flow cytometry and western blotting confirmed that NK-92 Exo induced apoptosis in the B16F10/effluc cells. In vivo, we used a B16F10/effluc cell xenograft model to detect the immunotherapeutic effect of NK-92 Exo. We injected NK-92 Exo into tumors, and tumor growth progression was monitored using the IVIS Lumina imaging system and ultrasound imaging. Tumor mass was monitored after in vivo experiments.Results: RT-PCR and western blotting confirmed effluc gene expression and protein levels in B16F10/effluc cells. B16F10/effluc activity was found to increase with increasing cell numbers, using BLI assay. For NK-92 Exo characterization, western blotting was performed on both ultracentrifuged and density gradient-isolated exosomes. The results confirmed that NK cell-derived exosomes express two typical exosome proteins, namely CD63 and ALIX. We demonstrated by western blot analysis that NK-92 Exo presented two functional NK proteins, namely perforin and FasL. Moreover, we confirmed the membrane expression of FasL. The enzyme-linked immunosorbent assay results indicated that NK-92 Exo can secrete tumor necrosis factor (TNF)-α, which affected the cell proliferation signaling pathway. The antitumor effect of NK-92 Exo against B16F10/effluc cells in vitro was confirmed by BLI (p < 0.001) and CCK-8 assays (p < 0.001). Furthermore, in normal healthy cells, even after 24 h of ...

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Exosomes derived from mesenchymal stem cells (MSCs) have been evaluated for their potential to be used as drug delivery vehicles. Synthetically personalized exosome mimetics (EMs) could be the alternative vesicles for drug delivery. In this study, we aimed to isolate EMs from human MSCs. Cells were mixed with paclitaxel (PTX) and PTX-loaded EMs (PTX-MSC-EMs) were isolated and evaluated for their anticancer effects against breast cancer. EMs were isolated from human bone marrow-derived MSCs. MSCs (4 × 106 cells/mL) were mixed with or without PTX at different concentrations in phosphate-buffered saline (PBS) and serially extruded through 10-, 5-, and 1-μm polycarbonate membrane filters using a mini-extruder. MSCs were centrifuged to remove debris and the supernatant was filtered through a 0.22-μm filter, followed by ultracentrifugation to isolate EMs and drug-loaded EMs. EMs without encapsulated drug (MSC-EMs) and those with encapsulated PTX (PTX-MSC-EMs) were characterized by western blotting, nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). The anticancer effects of MSC-EMs and PTX-MSC-EMs were assessed with breast cancer (MDA-MB-231) cells both in vitro and in vivo using optical imaging. EMs were isolated by the extrusion method and ultracentrifugation. The isolated vesicles were positive for membrane markers (ALIX and CD63) and negative for golgi (GM130) and endoplasmic (calnexin) marker proteins. NTA revealed the size of MSC-EM to be around 149 nm, while TEM confirmed its morphology. PTX-MSC-EMs significantly (p < 0.05) decreased the viability of MDA-MB-231 cells in vitro at increasing concentrations of EM. The in vivo tumor growth was significantly inhibited by PTX-MSC-EMs as compared to control and/or MSC-EMs. Thus, MSC-EMs were successfully isolated using simple procedures and drug-loaded MSC-EMs were shown to be therapeutically efficient for the treatment of breast cancer both in vitro and in vivo. MSC-EMs may be used as drug delivery vehicles for breast cancers.

Hair loss is a common medical problem. In this study, we investigated the proliferation, migration, and growth factor expression of human dermal papilla (DP) cells in the presence or absence of treatment with mesenchymal stem cell extracellular vesicles (MSC-EVs). In addition, we tested the efficacy of MSC-EV treatment on hair growth in an animal model. MSC-EV treatment increased DP cell proliferation and migration, and elevated the levels of Bcl-2, phosphorylated Akt and ERK. In addition; DP cells treated with MSC-EVs displayed increased expression and secretion of VEGF and IGF-1. Intradermal injection of MSC-EVs into C57BL/6 mice promoted the conversion from telogen to anagen and increased expression of wnt3a, wnt5a and versican was demonstrated. The first time our results suggest that MSC-EVs have a potential to activate DP cells, prolonged survival, induce growth factor activation in vitro, and promotes hair growth in vivo.

About 20% of the salivary glands were dysfunctional on SGS 5 years after a single RAI ablation, especially in patients who received higher doses of RAI. While parotid glands are more susceptible to (131)I-related damage, xerostomia was more associated with submandibular gland dysfunction and the prevalence of dysfunctional salivary glands.

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In vivo biodistribution and fate of extracellular vesicles (EVs) are still largely unknown and require reliable in vivo tracking techniques. In this study, in vivo bioluminescence imaging (BLI) using Renilla luciferase (Rluc) was developed and applied to monitoring of EVs derived from thyroid cancer (CAL-62 cells) and breast cancer (MDA-MB-231) in nude mice after intravenous administration and was compared with a dye-based labeling method for EV derived from CAL-62 cells. The EVs were successfully labeled with Rluc and visualized by BLI in mice. In vivo distribution of the EVs, as measured by BLI, was consistent with the results of ex vivo organ analysis. EV-CAL-62/Rluc showed strong signals at lung followed by liver, spleen & kidney (P < 0.05). EV-MDA-MB-231/Rluc showed strong signals at liver followed by lung, spleen & kidney (P < 0.05). EV-CAL-62/Rluc and EV-MDA-MB-231/Rluc stayed in animal till day 9 and 3, respectively; showed a differential distribution. Spontaneous EV-CAL-62/Rluc shown distributed mostly to lung followed by liver, spleen & kidney. The new BLI system used to show spontaneous distribution of EV-CAL-62/Rluc in subcutaneous CAL-62/Rluc bearing mice. Dye (DiR)-labeled EV-CAL-62/Rluc showed a different distribution in vivo & ex vivo compared to EV-CAL-62/Rluc. Fluorescent signals were predominately detected in the liver (P < 0.05) and spleen (P < 0.05) regions. The bioluminescent EVs developed in this study may be used for monitoring of EVs in vivo. This novel reporter-imaging approach to visualization of EVs in real time is expected to pave the way for monitoring of EVs in EV-based treatments.

Recent studies have shown that metabolic tumor volume (MTV) by positron emission tomography ⁄ computed tomography (PET ⁄ CT) is an important prognostic parameter in patients with non-Hodgkin's lymphoma. However, it is unknown whether doxorubicin, bleomycin, vinblastine, dacarbazine (ABVD) alone in early stage Hodgkin's lymphoma would lead to similar disease control as combined modality therapy (CMT) using MTV by PET ⁄ CT. One hundred and twentyseven patients with early stage Hodgkin's lymphoma who underwent PET ⁄ CT at diagnosis were enrolled. The MTV was delineated on PET ⁄ CT by the area ≥SUV max , 2.5 (standardized uptake value [SUV]). Sixty-six patients received six cycles of ABVD only. The other 61 patients received CMT (involved-field radiotherapy after 4-6 cycles of ABVD). The calculated MTV cut-off value was 198 cm 3 . Clinical outcomes were compared according to several prognostic factors (i.e. age ≥50 years, male, performance status ≥2, stage II, B symptoms, ≥4 involved sites, extranodal site, large mediastinal mass, CMT, elevated erythrocyte sedimentation rate and high MTV). Older age (progression-free survival [PFS], P = 0.003; overall survival [OS], P = 0.007), B symptoms (PFS, P = 0.006; OS, P = 0.036) and high MTV (PFS, P = 0.008; OS, P = 0.007) were significant independent prognostic factors. Survival of two high MTV groups treated with ABVD only and CMT were lower than the low MTV groups (PFS, P < 0.012; OS, P < 0.045). ABVD alone was sufficient to control disease in those with low MTV status. However, survival was poor, even if the CMT was assigned a high MTV status. The MTV would be helpful for deciding the therapeutic modality in patients with early stage Hodgkin's lymphoma. (Cancer Sci 2013; 104: 1656-1661