Corrigendum to "In Vivo Tracking of Chemokine Receptor CXCR4-Engineered Mesenchymal Stem Cell Migration by Optical Molecular Imaging".

[This corrects the article DOI: 10.1155/2017/8085637.].

Role of M2-like macrophages in the progression of ovarian cancer.

In this study, we noninvasively assessed whether M2-like macrophages accelerate the progression of ovarian cancer by performing molecular imaging of ovarian cancer cells expressing enhanced firefly luciferase (Effluc) in living mice. First, murine ovarian cancer ID8 cells expressing Effluc (ID8/Effluc cells) were established by retroviral infection. Subsequently, macrophages were isolated from the peritoneal exudate of mice injected with thioglycollate medium and differentiated into M2-like macrophages by adding interleukin 4. To characterize these M2-like macrophages, F4/80 and cluster of differentiation 206 expression levels were determined. Then, the M2-like macrophages were co-cultured with the ID8/Effluc cells and bioluminescence imaging (BLI) of signals from the ID8/Effluc cells was completed. Additionally, migration and wound healing were assessed to evaluate the effects of conditioned medium (CM) from M2-like macrophages on ID8/Effluc cell motility. In the in vivo study, mice were first given either liposome-phosphate-buffered saline or liposome-clodronate (lipo-clodronate). After 24 h, ID8/Effluc cells were intraperitoneally injected into the mice and BLI was completed at the designed time points. Next, histological analysis was conducted to characterize the infiltrated tumor. Flow cytometric analysis revealed high levels of CD206 expression in the differentiated M2-like macrophages. Meanwhile, ID8/Effluc cells co-cultured with these M2-like macrophages proliferated rapidly in an M2-like macrophage, number-dependent manner. The migration of the ID8/Effluc cells was also increased by the application of CM from M2-like macrophages. In vivo BLI revealed that the growth rate of intraperitoneally injected ovarian cancer cells was inhibited following macrophage depletion by treatment with lipo-clodronate. M2-like macrophages accelerated the progression of ovarian cancer, suggesting they are a new therapeutic target for ovarian cancer and that ovarian cancer could be managed by altering the nature of communication between ovarian cancer and macrophages.

Correction: A new bioluminescent reporter system to study the biodistribution of systematically injected tumor-derived bioluminescent extracellular vesicles in mice.

[This corrects the article DOI: 10.18632/oncotarget.22493.].

A Novel Tyrosine Kinase Inhibitor Can Augment Radioactive Iodine Uptake Through Endogenous Sodium/Iodide Symporter Expression in Anaplastic Thyroid Cancer.

Background: Radioactive iodine (RAI) therapy is an important strategy in the treatment of thyroid cancer. However, anaplastic thyroid cancer (ATC), a rare malignancy, exhibits severe dedifferentiation characteristics along with a lack of sodium iodide symporter (NIS) expression and function. Therefore, RAI therapy is ineffective and contributes toward poor prognosis of these patients. Recently, small-molecule tyrosine kinase inhibitors (TKIs) have been used to treat thyroid cancer patients for restoring NIS expression and function and RAI uptake capacity. However, most results reported thus far are associated with differentiated thyroid cancer. In this study, we identified a new TKI and investigated its effects on cell redifferentiation, NIS function, and RAI therapy in ATC. Methods: We identified a new TKI, "5-(5-{4H, 5H,6H-cyclopenta[b]thiophen-2-yl}-1,3,4-oxadiazol-2-yl)-1-methyl-1,2-dihydropyridin-2-one" (CTOM-DHP), using a high-throughput screening system. CTOM-DHP was exposed to 8505C ATC cells at different concentrations and time points. Concentrations of 12.5 and 25 µM and an incubation time of 72 hours were chosen as the conditions for subsequent NIS promoter assays and NIS mRNA and protein expression experiments. In addition, we examined factors related to iodide metabolism after CTOM-DHP treatment as well as the signaling pathways mediating the effects of CTOM-DHP on endogenous NIS expression. RAI uptake and 131I cytotoxicity effects caused by CTOM-DHP pretreatment were also evaluated in vitro and in vivo. Results: Promoter assays as well as mRNA and protein expression analyses confirmed that NIS expression was augmented by treatment of 8505C ATC cells with CTOM-DHP. Moreover, CTOM-DHP treatment robustly increased the expression of other thyroid-specific proteins and thyroid transcription factors related to iodide metabolism. Enhancement of NIS function was demonstrated by an increase in 125I uptake and 131I cytotoxicity. Increased endogenous NIS expression was associated with the inhibition of PI3K/Akt and MAPK signaling pathways. In vivo results also demonstrated an increase in NIS promoter activity and RAI avidity in response to CTOM-DHP treatment. Furthermore, 131I-mediated therapeutic effects preferentially improved in a tumor xenograft mice model. Conclusions: CTOM-DHP, a new TKI identified in this study, enhances endogenous NIS expression and thereby is a promising compound for restoring RAI avidity in ATC.

White blood cell labeling with Technetium-99m (99mTc) using red blood cell extracellular vesicles-mimetics.

BACKGROUND: Extracellular vesicles, have gained increasing attention for their application in drug delivery. Here, we developed a novel method for radiolabeling WBCs with 99mTc using RBC-derived extracellular vesicles -mimetics (EVMs), and monitored in vivo inflammation tracking of 99mTc-WBC using gamma camera in acute inflammation mouse model. METHODS: Engineered EVMs from RBCs were produced by a one-step extrusion method. RBC-EVMs were analyzed by NTA and TEM. Cells were labeled with 99mTc by using 99mTc-RBC-EVMs. Inflammation mice model was prepared and confirmed by 18F-FDG PET/CT. 99mTc-WBCs were injected in mice, and their biodistribution was analyzed by gamma camera. FINDING: The radiochemical purity of 99mTc-RBC-EVMs was 100%. The 99mTc-labeling did't affect the size and morphology. The 99mTc in the cytoplasm of RBC-EVMs was successfully confirmed by high angle annular dark field STEM (scanning transmission electron microscope). Cells were successfully labeled with 99mTc using 99mTc-RBC-EVMs, and the counts per minute was increased in dose- and time-dependent manners. The 18F-FDG PET/CT images confirmed establishment of acute inflammation (left mouse foot). 99mTc-WBCs showed higher uptake in the inflamed foot than non-inflamed foot. INTERPRETATION: This novel method for radiolabeling WBCs using RBC-EVMs. 99mTc labeling may be a feasible method to monitor the in vivo biodistribution of cells.

Enhancement of antitumor potency of extracellular vesicles derived from natural killer cells by IL-15 priming.

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.

A New Approach for Loading Anticancer Drugs Into Mesenchymal Stem Cell-Derived Exosome Mimetics for Cancer Therapy.

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.

New Optical Imaging Reporter-labeled Anaplastic Thyroid Cancer-Derived Extracellular Vesicles as a Platform for In Vivo Tumor Targeting in a Mouse Model.

Extracellular vesicles (EVs), originating from multivesicular bodies by invagination of the endosomal membrane, are communication channels between distant cells. They are natural carriers of exogeneous cellular materials and have been exploited as drug delivery carriers in various diseases. Here, we found that tumor cell-derived EVs can be used as efficient targets in tumors by monitoring with an optical reporter system. Anaplastic thyroid cancer (CAL62) cell-derived EVs with Renilla luciferase (Rluc) were used to target CAL62 tumors in a mouse model. Optical imaging revealed that cancer cell-derived EVs (EV-CAL62/Rluc) targeted the original tumor (CAL62) in mice within 30 min after systemic injection. Furthermore, fluorescence imaging revealed that EV-CAL62/Rluc were internalized into CAL62 tumors in the mice. Ex vivo Optical imaging further confirmed the in vivo finding. Here, we successfully monitored the tumor targeting ability of tumor cell-derived EVs by optical imaging. Based on these results, tumor cell-derived EVs are highly effective natural carriers for drug delivery for cancer therapies.

In vivo Non-invasive Imaging of Radio-Labeled Exosome-Mimetics Derived From Red Blood Cells in Mice.

Exosomes are natural nano-sized membrane vesicles that have garnered recent interest owing to their potential as drug delivery vehicles. Though exosomes are effective drug carriers, their production and in vivo biodistribution are still not completely elucidated. We analyzed the production of exosome mimetics (EMs) from red blood cells (RBCs) and the radio-labeling of the RBC-EMs for in vivo imaging. Engineered EMs from RBCs were produced in large-scale by a one-step extrusion method, and further purified by density-gradient centrifugation. RBC-EMs were labeled with technetium-99m (99mTc). For non-invasive imaging, 99mTc (free) or 99mTc-RBC-EMs were injected in mice, and their biodistribution was analyzed by gamma camera imaging. Animals were sacrificed, and organs were collected for further biodistribution analysis. RBC-EMs have similar characteristics as the RBC exosomes but have a 130-fold higher production yield in terms of particle numbers. Radiochemical purity of 99mTc-RBC-EMs was almost 100% till 2 h reduced to 97% at 3 h. Radio-labeling did not affect the size and morphology of RBC-EMs. In contrast to free 99mTc, in vivo imaging of 99mTc-RBC-EMs in mice showed higher uptake in the liver and spleen, and no uptake in the thyroid. Ex vivo imaging confirmed the in vivo findings. Furthermore, fluorescent imaging confirmed the nuclear imaging findings. Immunofluorescent imaging revealed that the hepatic uptake of RBC-EMs was significantly mediated by kupffer cells (resident hepatic macrophages). Our results demonstrate a simple yet large-scale production method for a novel type of RBC-EMs, which can be effectively labeled with 99mTc, and feasibly monitored in vivo by nuclear imaging. The RBC-EMs may be used as in vivo drug delivery vehicles.

Novel alternatives to extracellular vesicle-based immunotherapy - exosome mimetics derived from natural killer cells.

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.

Migration of mesenchymal stem cells to tumor xenograft models and in vitro drug delivery by doxorubicin.

Mesenchymal stem cells (MSCs) show therapeutic effects in various types of diseases. MSCs have been shown to migrate towards inflamed or cancerous tissues, and visualized after sacrificing the animal. MSCs are able to deliver drugs to target cells, and are an ideal candidate for cancer therapy. The purpose of this study was to track the migration of MSCs in tumor-bearing mice; MSCs were also used as drug delivery vehicles. Human breast cancer cells (MDA-MB-231) and anaplastic thyroid cancer cells (CAL62) were transduced with lentiviral particles, to express the Renilla luciferase and mCherry (mCherry-Rluc) reporter genes. Human bone marrow-derived MSCs were transduced with lentiviral particles, to express the firefly luciferase and enhanced green fluorescence protein (Fluc2-eGFP) reporter genes (MSC/Fluc). Luciferase activity of the transduced cells was measured by bioluminescence imaging (BLI). Further in vitro migration assays were performed to confirm cancer cells conditioned medium dependent MSC and doxorubicin (DOX) treated MSC migration. MSCs were loaded with DOX, and their therapeutic effects against the cancer cells were studied in vitro. In vivo MSC/Fluc migration in mice having thyroid or breast cancer xenografts was evaluated after systemic injection. Rluc activity of CAL62/Rluc (R2=0.911), MDA-MB-231/Rluc (R2=0.934) cells and Fluc activity of MSC/Fluc (R2=0.91) cells increased with increasing cell numbers, as seen by BLI. eGFP expression of MSC/Fluc was confirmed by confocal microscopy. Similar migration potential was observed between MSC/Fluc and naïve MSCs in migration assay. DOX treated MSCs migration was not decreased compared than MSCs. Migration of the systemically injected MSC/Fluc cells into tumor xenografts (thyroid and breast cancer) was visualized in animal models (p<0.05) and confirmed by ex vivo (p<0.05) BLI. Additionally, MSCs delivered DOX to CAL62/Rluc and MDA-MB-231/Rluc cells, thereby decreasing their Rluc activities. In this study, we confirmed the migration of MSCs to tumor sites in cancer xenograft models using both in vivo and ex vivo BLI imaging. DOX-pretreated MSCs showed enhanced cytotoxic effects. Therefore, this noninvasive reporter gene (Fluc2)-based BLI may be useful for visualizing in vivo tracking of MSCs, which can be used as a drug delivery vehicle for cancer therapy.

Targeting and Therapy of Glioblastoma in a Mouse Model Using Exosomes Derived From Natural Killer Cells.

Objective: Glioblastoma 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. Methods: U87/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. Results: RT-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 < 0.001). ELISA results indicated that NK-Exo contained tumor necrosis factor-α and granzyme B. In vivo NK-Exo treatment inhibited tumor growth compared to in control mice (p < 0.001), and pretreatment of xenograft mice with dextran sulfate 2 h before NK-Exo treatment increased the antitumor effect of NK-Exo (p < 0.01) compared to in control and NK-Exo-alone-treated mice. Conclusion: NK-Exo targeted and exerted antitumor effects on glioblastoma cells both in vitro and in vivo, suggesting their utility in treating incurable glioblastoma.

In vivo migration of mesenchymal stem cells to burn injury sites and their therapeutic effects in a living mouse model.

Mesenchymal stem cell (MSC)-based therapy has emerged as a promising therapeutic strategy for tissue regeneration and repair. In this study, we non-invasively monitored the tracking of MSCs toward burn injury sites using MSCs expressing firefly luciferase (Fluc) gene in living mice, and evaluated the effects of the MSCs at the injury site. Murine MSCs co-expressing Fluc and green fluorescent protein (GFP) were established using a retroviral system (referred to as MSC/Fluc). To evaluate the ability of MSC migration toward burn injury sites, cutaneous burn injury was induced in the dorsal skin of mice. MSC/Fluc was intravenously administrated into the mice model and bioluminescence imaging (BLI) was performed to monitor MSC tracking at designated time points. BLI signals of MSC/Fluc appeared in burn injury lesions at 4 days after the cell injection and then gradually decreased. Immunoblotting analysis was conducted to determine the expression of neovascularization-related genes such as TGF-ß1 and VEGF in burnt skin. The levels of TGF-ß1 and VEGF were higher in the MSC/Fluc-treated group than in the burn injury group. Our observations suggested that MSCs might assist burn wound healing and that MSCs expressing Fluc could be a useful tool for optimizing MSC-based therapeutic strategies for burn wound healing.

Regulated Mesenchymal Stem Cells Mediated Colon Cancer Therapy Assessed by Reporter Gene Based Optical Imaging.

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.

Reverting iodine avidity of radioactive-iodine refractory thyroid cancer with a new tyrosine kinase inhibitor (K905-0266) excavated by high-throughput NIS (sodium iodide symporter) enhancer screening platform using dual reporter gene system.

Radioactive-iodine (RAI) therapy is typically unprevailing as anaplastic thyroid cancer (ATC) management, owing to the decrease in the endogenous sodium iodide symporter (NIS) expression. Therefore, new strategies for NIS re-induction are required to improve the efficacy of RAI therapy in ATC. In this study, we developed a novel high-throughput NIS enhancer screening platform using a dual reporter gene system to identify a potent tyrosine kinase inhibitor (TKI) and selected a new hit compound, K905-0266 TKI. The effects of K905-0266 TKI treatment was validated as RAI accumulation, changes in signalling pathway related to thyroid pathogenesis, and cytotoxicity of RAI depending on re-induction of endogenous NIS expression in ATC. Furthermore, we evaluated enhancement of NIS promoter and therapeutic efficacy of RAI in ATC tumour xenograft mice. After K905-0266 TKI treatment, the expression of endogenous NIS was significantly increased, while phosphorylated-ERK was decreased. In addition, the thyroid-metabolising protein expressions were upregulated and increased of RAI accumulation and its therapeutic effects in ATC. Moreover, K905-0266 TKI increased therapeutic efficacy of RAI in ATC tumour in vivo. In conclusion, we successfully established a novel high-throughput NIS enhancer screening platform to excavate a NIS enhancer and identified K905-0266 TKI among TKI candidates and it's proven to increase the endogenous NIS expression and therapeutic efficacy of RAI in ATC. These findings suggest that a novel high-throughput NIS enhancer screening platform is useful for selecting of NIS promoter enhancers. In addition, K905-0266 TKI can be used to re-induce endogenous NIS expression and recover RAI therapy in ATC.

Extracellular vesicles derived from MSCs activates dermal papilla cell in vitro and promotes hair follicle conversion from telogen to anagen in mice.

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.

Development of an athyroid mouse model using 131I ablation after preparation with a low-iodine diet.

We optimized the protocol for thyroid ablation in living mice using radioactive iodine (RAI) and a low-iodine diet (LID). To examine the effect of LID on thyroid ablation, mice were randomly divided into 4 groups: Vehicle, 131I 2.775 MBq, 131I 5.55 MBq, and LID + 131I 2.775 MBq. The LID group was fed a LID for up to 7 days and then mice in the 131I 2.775, 131I 5.55, and LID + 131I 2.775 MBq groups were intravenously administrated with 131I, respectively. Scintigraphy imaging with 99mTc pertechnetate was performed once in 2 weeks for 4 weeks. After establishment of athyroid mice, control or athyroid mice were injected with human anaplastic thyroid cancer cells co-expressing sodium iodine symporter and enhanced firefly luciferase (ARO/NF) to evaluate RAI uptake. Scintigraphy imaging with 99mTc pertechnetate was performed with ARO/NF tumor-bearing mice. Scintigraphy imaging showed decreased thyroid uptake in the LID + 131I 2.775 MBq group compared to other groups. Scintigraphy images showed that tumor uptake was statically higher in athyroid mice than in control mice. These data suggest that these optimized conditions for thyroid ablation could be helpful to establish an in vivo mouse model.

Natural Killer Cell (NK-92MI)-Based Therapy for Pulmonary Metastasis of Anaplastic Thyroid Cancer in a Nude Mouse Model.

OBJECTIVE: Natural killer (NK) cells represent the third largest population of lymphocytes, and they play an important role in immune surveillance against tumors. The lungs are a common metastatic site for anaplastic thyroid cancer (ATC), and metastasis is one of the most frequent causes of mortality in this type of cancer. In the current study, we evaluated the effects of NK cell-based immunotherapy for pulmonary metastasis of ATC and determined how it affects the effector molecules of NK cells. METHODS: Human NK cells (NK-92MI) were retrovirally transduced to express the effluc gene. Human ATC cells (CAL-62) were transduced with the effluc and Rluc genes. The cytotoxicity of NK cells against CAL-62 cells was assessed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay system. Pulmonary metastases of ATC were developed by i.v. injection of CAL-62, and metastasis growth was monitored using bioluminescence imaging (BLI). To treat the metastases, five million NK-92MI cells were injected twice into the caudal vein of nude mice. To assess the targetability of NK cells to ATC tumors, NK-92MI cells expressing the effluc gene (NK/F) were administered through the tail vein of nude mice with a pulmonary metastasis or tumor xenograft. BLI was subsequently performed at 1, 3, 24, and 48 h. RESULTS: NK/F and CAL-62 cells expressing the effluc or Rluc gene (CAL-62/F, CAL-62/R) were successfully established. Expression of the effluc and Rluc genes in NK/F, CAL-62/F, and CAL-62/R cells was verified by RT-polymerase chain reaction, western blotting, and luciferase assay. After coculture of NK-92MI and CAL-62/F cells for 24 h, the BLI signal intensity of CAL-62/F cells proportionally decreased with the number of cocultured NK cells. An ATC pulmonary metastasis mouse model was successfully generated, and NK cells significantly inhibited the growth of the metastasis (p < 0.01). The NK/F cells exhibited targetability to the pulmonary metastasis and tumor xenograft in the mouse model. CONCLUSION: The results of present study suggest that NK cells are able to target ATC tumors and that NK cell-based immunotherapy may serve as an effective therapeutic approach for pulmonary metastases of ATC.

Exosomes Derived From Natural Killer Cells Exert Therapeutic Effect in Melanoma.

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.

In Vivo Tracking of Chemokine Receptor CXCR4-Engineered Mesenchymal Stem Cell Migration by Optical Molecular Imaging.

CXCR4, the stromal cell-derived factor-1 receptor, plays an important role in the migration of hematopoietic progenitor/stem cells to injured and inflamed areas. Noninvasive cell tracking methods could be useful for monitoring cell fate. Therefore, in this study, we evaluated the efficacy of an intravenous infusion of genetically engineered mesenchymal stem cells (MSCs) overexpressing CXC chemokine receptor 4 (CXCR4) to home to the tumor, by optical imaging. We constructed a retroviral vector containing CXCR with dual reporter genes, eGFP and Fluc2, under the control of an EF1α promoter (pBABE-EF1α-CXCR4-eGFP-IRES-Fluc2). We also developed an eGFP-Fluc2 construct in the Retro-X retroviral vector (Retro-X-eGFP-Fluc2). MSCs were transduced with retroviruses to generate CXCR4-overexpressing MSCs (MSC-CXCR4/Fluc2) and MSCs (MSC/Fluc2). CXCR4 mRNA and protein expression was confirmed by RT-PCR and Western blotting, respectively, and it was higher in MSC-CXCR4/Fluc2 than in naive MSCs. eGFP expression was confirmed by confocal microscopy. The transfected MSC-CXCR4/Fluc2 cells showed higher migratory capacity than naive MSCs observed in Transwell migration assay. The in vivo migration of CXCR4-overexpressing MSCs to MDAMB231/Rluc tumor model by BLI imaging was also confirmed. Intravenous delivery of genetically modified MSCs overexpressing CXCR4 with a Fluc2 reporter gene may be a useful, noninvasive BLI imaging tool for tracking cell fate.