Search

Purpose Natural killer (NK) cells are the key subset of innate-immunity lymphocytes; they possess antitumor activities and are used for cancer immunotherapy. In a previous study, extracellular vehicles (EVs) from NK-92MI cells were isolated and exploited for their ability to kill human cancer cells in vitro and in vivo (multiple injection methods). Here, the potential of NK-cell–derived EVs (NK-EVs) for immunotherapy was improved by priming with interleukin (IL)-15. Methods NK-EVs were isolated from the culture medium without or with IL-15 (NK-EVsIL-15) by ultracentrifugation and were purified via density gradient ultracentrifugation. In addition, NK-EVs and NK-EVsIL-15 were characterized by transmission electron microscopy, nanoparticle-tracking analysis, and western blotting. Flow cytometry, bioluminescence imaging, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed for apoptosis, protein expression, cell proliferation, and cytotoxicity analyses. Furthermore, xenograft tumor–bearing mice were injected with PBS, NK-EVs, or NK-EVsIL-15 intravenously five times. Tumor growth was monitored using calipers and bioluminescence imaging. Toxicity of the nanoparticles was evaluated by measuring the body weight of the mice. Results NK-EVsIL-15 showed significantly higher cytolytic activity toward human cancer cell lines (glioblastoma, breast cancer, and thyroid cancer) and simultaneously increased the expression of molecules associated with NK-cell cytotoxicity. When compared with NK-EVs, NK-EVsIL-15 significantly inhibited the growth of glioblastoma xenograft cells in mice. In addition, both NK-EVs and NK-EVsIL-15 were not significantly toxic to either normal cells or mice. Conclusion IL-15 may improve the immunotherapeutic effects of NK-EVs, thus improving the applications of NK-EVs in the future.

Exosomes are endogenous nanocarriers that can deliver biological information between cells. They are secreted by all cell types, including immune cells such as natural killer (NK) cells. However, mammalian cells release low quantities of exosomes, and the purification of exosomes is difficult. Here, nanovesicles were developed by extrusion of NK cells through filters with progressively smaller pore sizes to obtain exosome mimetics (NK-EM). The anti-tumour effect of the NK-EM was confirmed in vitro and in vivo. The morphological features of the NK-EM were revealed by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blot. In vitro, the cytotoxicity of the NK-EM to cancer cells (glioblastoma, breast carcinoma, anaplastic thyroid cancer and hepatic carcinoma) was assessed using bioluminescence imaging (BLI) and CCK-8 assay. For in vivo study, a xenograft glioblastoma mouse model was established. The anti-tumour activity of NK-EM was confirmed in vivo by the significant decreases of BLI, size and weight (all p .001) of the tumour compared with the control group. Moreover, NK-EM cytotoxicity for glioblastoma cells that related with decreased levels of the cell survival markers p-ERK and p-AKT, and increased levels of apoptosis protein markers cleaved-caspase 3, cytochrome-c and cleaved-PARP was confirmed. All those results suggest that NK-EM exert stronger killing effects to cancer cells compared with the traditional NK-Exo, at the same time, the tumour targeting ability of the NK-EM was obtained in vivo. Therefore, NK-EM might be a promising immunotherapeutic agent for treatment of cancer.

ObjectiveGlioblastoma is a highly aggressive primary brain tumor that is resistant to radiotherapy and chemotherapy. Natural killer (NK) cells have been used to treat incurable cancers. Recent studies have investigated the effectiveness of NK-cell-derived exosomes (NK-Exo) for treating incurable cancers such as melanoma, leukemia, and neuroblastoma; however, NK-Exo have not been used to treat glioblastoma. In the present study, we investigated the antitumor effects of NK-Exo against aggressive glioblastoma both in vitro and in vivo and determined the tumor-targeting ability of NK-Exo by performing fluorescence imaging.MethodsU87/MG cells were transfected with the enhanced firefly luciferase (effluc) and thy1.1 genes; thy1.1-positive cells were selected using microbeads. U87/MG/F cells were assessed by reverse transcription polymerase chain reaction (RT-PCR), western blotting, and luciferase-activity assays. NK-Exo were isolated by ultracentrifugation, purified by density gradient centrifugation, and characterized by transmission electron microscopy, dynamic light scattering (DLS), nanoparticle-tracking analysis (NTA), and western blotting. Cytokine levels in NK-Exo were compared to those in NK cells and NK-cell medium by performing an enzyme-linked immunosorbent assay (ELISA). NK-Exo-induced apoptosis of cancer cells was confirmed by flow cytometry and western blotting. In vivo therapeutic effects and specificity of NK-Exo against glioblastoma were assessed in a xenograft mouse model by fluorescence imaging. Xenograft mice were treated with NK-Exo, which was administered seven times through the tail vein. Tumor growth was monitored by bioluminescence imaging (BLI), and tumor volume was measured by ultrasound imaging. The mice were intraperitoneally injected with dextran sulfate 2 h before NK-Exo injection to decrease the liver uptake and increase the tumor specificity of NK-Exo.ResultsRT-PCR and western blotting confirmed the gene and protein expression of effluc in U87/MG/F cells, with the bioluminescence activity of U87/MG/F cells increasing with an increase in cell number. NTA and DLS results indicated that the size of NK-Exo was ~100 nm, and the western blot results confirmed that NK-Exo expressed exosome markers CD63 and Alix. We confirmed the in vitro cytotoxic effects of NK-Exo on U87/MG/F cells by performing BLI, and the killing effect on U87/MG and U87MG/F cells was measured by CCK-8 and MTT assays (p

Colorectal cancer is the most common cancer in both men and women and the second most common cause of cancer-related deaths. Suicide gene-based therapy with suicide gene-transduced mesenchymal stem cells (MSCs) is a promising therapeutic strategy. A tetracycline-controlled Tet-On inducible system used to regulate gene expression may be a useful tool for gene-based therapies. The aim of this study was to develop therapeutic MSCs with a suicide gene that is induced by an artificial stimulus, to validate therapeutic gene expression, and to monitor the MSC therapy for colon cancer using optical molecular imaging. For our study, we designed the Tet-On system using a retroviral vector and developed a response plasmid RetroX-TRE (tetracycline response element) expressing a mutant form of herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Bone marrow-derived MSCs were transduced using a RetroX-Tet3G (Clontech, CA, USA) regulatory plasmid and RetroX-TRE-HSV1-sr39TK-eGFP-IRES-Fluc2, for a system with a Tet-On (MSC-Tet-TK/Fluc2 or MSC-Tet-TK) or without a Tet-On (MSC-TK/Fluc2 or MSC-TK) function. Suicide gene engineered MSCs were co-cultured with colon cancer cells (CT26/Rluc) in the presence of the prodrug ganciclovir (GCV) after stimulation with or without doxycycline (DOX). Treatment efficiency was monitored by assessing Rluc (CT26/Rluc) and Fluc (MSC-Tet-TK and MSC-TK) activity using optical imaging. The bystander effect of therapeutic MSCs was confirmed in CT26/Rluc cells after GCV treatment. Rluc activity in CT26/Rluc cells decreased significantly with GCV treatment of DOX(+) cells (p < 0.05 and 0.01) whereas no significant changes were observed in DOX(−) cells. In addition, Fluc activity in also decreased significantly with DOX(+) MSC-Tet-TK cells, but no signal was observed in DOX(−) cells. In addition, an MSC-TK bystander effect was also confirmed. We assessed therapy with this system in a colon cancer xenograft model (CT26/Rluc). We successfully transduced cells and developed a Tet-On system with the suicide gene HSV1-sr39TK. Our results confirmed the therapeutic efficiency of a suicide gene with the Tet-On system for colon cancer. In addition, our results provide an innovative therapeutic approach using the Tet-On system to eradicate tumors by administration of MSC-Tet-TK cells with DOX and GCV.

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 co-culture, NK-92 Exo did not exhibit significant side effects. In the in vivo experiments, tumors in the vehicle control group were significantly increased, compared with those in the NK-92 Exo-treated group (p < 0.05). Conclusion: The results of the current study suggest that exosomes derived from NK cells exert cytotoxic effects on melanoma cells and thus warrant further development as a potential immunotherapeutic strategy for cancer.

Mesenchymal stem cells (MSCs) can be used as a therapeutic armor for cancer. Extracellular vesicles (EVs) from MSCs have been evaluated for anticancer effects. In vivo targeting of EVs to the tumor is an essential requirement for successful therapy. Therefore, non-invasive methods of monitoring EVs in animal models are crucial for developing EV-based cancer therapies. The present study to develop bioluminescent EVs using Renilla luciferase (Rluc)-expressing MSCs. The EVs from MSC/Rluc cells (EV-MSC/Rluc) were visualized in a murine lung cancer model. The anticancer effects of EVs on Lewis lung carcinoma (LLC) and other cancer cells were assessed. EV-MSC/Rluc were visualized in vivo in the LLC-efffuc tumor model using optical imaging. The induction of apoptosis was confirmed with Annexin-V and propidium iodide staining. EV-MSC/Rluc and EV-MSCs showed a significant cytotoxic effect against LLC-effluc cells and 4T1; however, no significant effect on CT26, B16F10, TC1 cells. Moreover, EV-MSC/Rluc inhibited LLC tumor growth in vivo. EV-MSC/Rluc-mediated LLC tumor inhibitory mechanism revealed the decreased pERK and increased cleaved caspase 3 and cleaved PARP. We successfully developed luminescent EV-MSC/Rluc that have a therapeutic effect on LLC cells in both in vitro and in vivo. This bioluminescent EV system can be used to optimize EV-based therapy.

Prostate cancer is the most common malignancy and the second leading cause of cancer death in the male population in developing countries. Ukrain is a reaction product of different alkaloids from Chelidoniummajus L. (celandine) conjugated with thiophosphoric acid, which has cytotoxic effects on various malignant cells. In the present study, cell viability was assessed using the dimethyl thiazolyl tetrazolium bromide (MTT)method in PC-3 cells after treatment with Ukrain. The IC(50) value was observed in 10 μg concentration of Ukrain. Bax, Bad, and FasL mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction, and protein expressions of p-Akt, Bcl-2, and caspase 10 were determined by western-blot analysis. Nuclei were stained with 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI). Ukrain significantly increased the pro-apoptotic mRNA expression of Bad, Bax, and FasL; decreased the cell survival protein p-Akt and the anti-apoptotic protein Bcl-2; and increased the protein levels of cleaved poly(ADP)-ribose polymerase (PARP) and caspase-10.The results of this study suggest that Ukrain decreases the cell survival of androgen-independent prostate cancer cells and induces their apoptosis, thus supporting its use as a therapeutic drug for the treatment of prostate cancer