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The two-dimensional (2D) monolayer culture as a conventional method has been widely applied in molecular biology fields, but it has limited capability to recapitulate real cell environments, being prone to misinterpretation with poor prediction of in vivo behavior. Recently, the three-dimensional (3D) spheroid culture has been studied extensively. Spheroids are self-assembled cell aggregates that have biomimicry capabilities. The behavior of thyroid cancer under the 3D spheroid culture environment has been studied; however, there are no reports regarding differences in the degree of thyroid cancer cell differentiation under 2D and 3D culture conditions. This study investigated the expression of thyroid differentiation proteins related to iodide-metabolizing mechanisms in thyroid cancer cells under different culture conditions. Four thyroid cancer cell lines and one thyroid follicular epithelial cell line were grown in adherent 2D cell culture and 3D spheroid culture with agarose-coated plates. We observed changes in proliferation, hypoxia, extracellular matrix (ECM), cytoskeleton, thyroid-specific proteins, and thyroid transcription factors. All cell lines were successfully established in the spheroid following cell aggregation. Proliferation considerably decreased, while hypoxia-inducible factor 1-α(HIF1-α) was promoted in 3D spheroids; moreover, 3D spheroids with thyroid cancers showed diminished thyroid differentiation markers, but thyroid follicular epithelial cells revealed either a maintenance or weak decline of protein expression. We verified that the 3D spheroid culture environment can be similar to in vivo conditions because of its alterations in numerous cellular and functional activities, including morphology, cellular proliferation, viability, hypoxia, ECM, cytoskeleton, and thyroid differentiation, compared to the conventional 2D monolayer culture environment. An in vitro experimental study using 3D spheroid culture is ideal for the faster discovery of new drugs. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been demonstrated to deliver therapeutic drugs in preclinical studies. However, their use is limited, as they lack the ability to specifically deliver drugs to tumor tissues in vivo. In the present study, we propose the use of a targeting peptide, IL-4R-binding peptide (IL4RPep-1), to specifically deliver intravenously (i.v.) infused EVs to thyroid tumors. In vivo, a xenograft tumor model was treated with either the control peptide (NSSSVDK) or IL4RPep-1-Flamma; mice were fluorescently imaged (FLI) using an in vivo imaging system at 0–3 h post-treatment. EVs (labeled with DiD dye) were conjugated with IL4RPep-1 through a DOPE-NHS linker and administered to mice intravenously. FLI was performed 0–24 h post-injection, and the animals were sacrificed for further experiments. The morphology and size of EVs, the presence of EV markers such as CD63 and ALIX, and the absence of the markers GM130 and Cyto-C were confirmed. In vivo, FLI indicated an accumulation of i.v. injected IL4RPep-1-Flamma at the tumor site 90 min post-injection. No accumulation of NSSSVDK-Flamma was detected. In vivo, IL4RPep-1-EVs targeted the Cal-62 tumor 2 h post-injection. NSSSVDK-EVs were not even detected in the tumor 24 h post-injection. The quantification of FLI showed a significant accumulation of MSC-EVs in the tumor 2 h, 3 h, and 24 h post-injection. Furthermore, ex vivo imaging and an IF analysis confirmed the in vivo findings. Our results demonstrate the use of the IL4RPep-1 peptide as a targeting moiety of EVs for IL-4R-expressing anaplastic thyroid tumors. [ABSTRACT FROM AUTHOR]

Simple Summary: Thyroid cancers have an excellent prognosis by standard therapy of surgery followed by radioactive-iodine therapy. However, metastatic thyroid cancers do not response to radioactive-iodine therapy by losing iodine avidity. Therefore, reversing iodine avidity to metastatic thyroid cancers gives a new chance of applying radioactive-iodine therapy to the cancers. In the current study, we found that GLI1 knockdown can revert iodine non-avid thyroid cancers to iodine avid cancers by increasing expression of thyroid-specific proteins. Restoration of iodine avidity in thyroid cancers makes the cancers sensitive to radioactive-iodine therapy again. Therefore, the GLI1 can be a potential therapeutic target of radioactive-iodine resistant thyroid cancers. Radioactive-iodine (RAI) therapy is the mainstay for patients with recurrent and metastatic thyroid cancer. However, many patients exhibit dedifferentiation characteristics along with lack of sodium iodide symporter (NIS) functionality, low expression of thyroid-specific proteins, and poor RAI uptake, leading to poor prognosis. Previous studies have demonstrated the effect of GLI family zinc finger 1 (GLI1) inhibition on tumor growth and apoptosis. In this study, we investigated the role of GLI1 in the context of redifferentiation and improvement in the efficacy of RAI therapy for thyroid cancer. We evaluated GLI1 expression in several thyroid cancer cell lines and selected TPC-1 and SW1736 cell lines showing the high expression of GLI. We performed GLI1 knockdown and evaluated the changes of thyroid-specific proteins expression, RAI uptake and I-131-mediated cytotoxicity. The effect of GANT61 (GLI1 inhibitor) on endogenous NIS expression was also assessed. Endogenous NIS expression upregulated by inhibiting GLI1, in addition, increased expression level in plasma membrane. Also, GLI1 knockdown increased expression of thyroid-specific proteins. Restoration of thyroid-specific proteins increased RAI uptake and I-131-mediated cytotoxic effect. Treatment with GANT61 also increased expression of endogenous NIS. Targeting GLI1 can be a potential strategy with redifferentiation for restoring RAI avidity in dedifferentiated thyroid cancers. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A new approach for using extracellular vesicles (EVs) to deliver tyrosine kinase inhibitors (TKIs) to enhance iodine avidity in radioactive iodine-refractory thyroid cancer is needed. We isolated and characterized primary human adipose-derived stem cells (ADSCs) and isolated their EVs. The EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. A new TKI was loaded into the EVs by incubation (37 °C; 10 min) or sonication (18 cycles; 4 s per cycle) with 2 s intervals and a 2 min ice bath every six cycles. TKI loading was confirmed and measured by mass spectrometry. EV uptake into radioactive iodine-refractory thyroid cancer cells (SW1736 cells) was confirmed by microscopy. We treated the SW1736 cells with vehicle, TKI, or TKI-loaded EVs (sonication TKI-loaded EVs [EVsTKI(S)]) and examined the expression of iodide-metabolizing proteins and radioiodine uptake in the SW1736 cells. ADSCs cells showed >99% of typical stem cell markers, such as CD90 and CD105. The EVs displayed a round morphology, had an average size of 211.4 ± 3.83 nm, and were positive for CD81 and Alix and negative for cytochrome c. The mass spectrometry results indicate that the sonication method loaded ~4 times more of the TKI than did the incubation method. The EVsTKI(S) were used for further experiments. Higher expression levels of iodide-metabolizing mRNA and proteins in the EVsTKI(S)-treated SW1736 cells than in TKI-treated SW1736 cells were confirmed. EVsTKI(S) treatment enhanced 125I uptake in the recipient SW1736 cells compared with free-TKI treatment. This is the first study that demonstrated successful delivery of a TKI to radioactive iodine-refractory thyroid cancer cells using EVs as the delivery vehicle. This approach can revert radioiodine-resistant thyroid cancer cells back to radioiodine-sensitive thyroid cancer cells. [ABSTRACT FROM AUTHOR]

A facile and eco-friendly approach was adapted for the preparation of multicolor emitting carbon dots (BL-MECDs) by hydrothermal method using betel leaves. BL-MECDs were synthesized at various reaction times-12 h, 24 h, 36 h, and 48 h at 200 °C. The reaction times were varied to find the optimum reaction condition to obtain BL-MECDs with excellent optical- and chemical properties. BL-MECDs were characterized by various analytical techniques. The surface morphology, degree of graphitization, and their composition were examined by field emission scanning electron microscope, Raman spectrum, and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. The optical properties were evaluated via ultraviolet–visible and fluorescence spectroscopy techniques. The result suggests that BL-MECDs prepared at 36 h have enriched functional groups and excellent fluorescence emission with acceptable quantum yield (15%). Thus, BL-MECDs prepared at 36 h were utilized for further studies. X-ray photoelectron spectroscopy confirmed the existence of functional groups such as carboxyl, carbonyl, and hydroxyl groups. Moreover, the obtained BL-MECDs exhibited excellent prolonged stability and photostability even after 150 days of storage in atmospheric condition and by continuous irradiation of about 100 min with 365 nm UV-lamb, respectively. Further, BL-MECDs showed excellent biocompatibility and negligible cytotoxicity and thus explored for cell imaging for normal mouse fibroblast cell line (L929) and human anaplastic thyroid cancer cells line (SW1736). Cellular fluorescence imaging suggesting that BL-MECDs have great potential in bioimaging applications. [Display omitted] • Novel multicolor emitting CDs (BL-MECDs) were prepared from betel leaves. • BL-MECDs exhibit excitation-dependent emission with QY of 15%. • BL-MECDs showed excellent photostability and low toxicity against L929/SW1736 cells. • BL-MECDs effectively internalized into the cells and emitted strong fluorescent signals. • BL-MECDs can be a great potential in the bioimaging of cells in vitro / in vivo. [ABSTRACT FROM AUTHOR]