Tumor burdens monitored by bioluminescence imaging 14, 24, and 34 days after tumor inoculation.

<p>(A and B) The tumor signals in the I-131 and NK groups were significantly lower than those in the control group at 34 days. The combined group showed the lowest stationary tumor signal over time, which was significantly lower than that in the I-131 and NK groups at 34 days. * p<0.05 compared with I-131 and NK groups,** p<0.005 compared with control group.</p

<i>In vitro</i> Cytotoxicity assay using Calcein-AM.

<p>NK cell cytotoxocity is significantly increased according to effector : target cell (E : T) ratio in the control and I-131 groups. NK cell cytotoxicities were significantly higher in irradiated cells than in non-irradiated cells at all E : T ratios of 2.5 : 1, 5 : 1, and 10 : 1. * p<0.05, ** p<0.005 compared with control.</p

Characterization image of MDA-231/NF cells.

<p>(A–C) Tc-99m pertechnetate SPECT/CT images show focal tracer uptake (arrow) in the right flank of the MDA-231/NF tumor xenograft. There is also noted tracer uptake at the stomach (arrow head). (D–F) <i>In vivo</i> bioluminescence imaging for a mouse with MDA-231/NF cell implantation. (D) MDA-231/NF cells were implanted subcutaneously into the right hind-flank (1×10⁵; arrow head), left hind-flank (3×10⁵; double arrow head), and right fore-flank (9×10⁵; arrow) of the mouse. (E) Bioluminescence signals from implanted MDA-231/NF cells are clearly visualized in the right hind-flank (arrow head), left hind-flank (double arrow head), and right fore-flank (arrow). (F) The signal intensity from tumor cells increased with increasing cell number. * p<0.05 compared with 1×10⁵ cells, *** p<0.001 compared with 1×10⁵ and 3×10⁵ cells.</p

RT-PCR analysis of human sodium/iodide symporter (hNIS) and enhanced firefly luciferase (effluc) gene expression in MDA-231, MDA-231/NF cells and human thyroid tissue.

<p>RT-PCR revealed hNIS mRNA expression in MDA-231/NF cells and human thyroid tissue, and effluc mRNA expression in MDA-231/NF cells. RT-PCR fragments have lengths of 583 bp and 316 bp for hNIS and effluc in MDA-231/NF cells; however, these bands do not appear in MDA-231 cells.</p

<i>In vitro</i> I-125 uptake assay and luciferase assay in MDA-231 and MDA-231/NF cells.

<p>(A) I-125 uptake by MDA-231/NF cells increased according to cell number. I-125 uptake by MDA-231 cells remained at the basal level. *** p<0.001 compared with MDA-231 and MDA-231/NF cells blocked by KClO_4. (B) Bioluminescence signals of MDA-231/NF cells increased according to cell number. Bioluminescence signal of MDA-231 cells remained at the basal level. CPM: count per minute, RLU: Relative Light Units, *** p<0.001 compared with MDA-231 cells.</p

Phenotype analysis in MDA-231/NF cells by flow cytometry.

<p>(A, B and C) The levels of DR5, Fas, and MIC A/B expression in tumor cells that received I-131 were significantly higher than in tumor cells received. (D) I-131 therapy resulted in decreased expression of HLA-A,B,C, but without statistical significance. * p<0.05, ** p<0.005, *** p<0.001 compared with control.</p

Genetically engineered suicide gene in mesenchymal stem cells using a Tet-On system for anaplastic thyroid cancer

<div><p>Anaplastic thyroid cancer (ATC) is the most aggressive malignancy of the thyroid, during which undifferentiated tumors arise from the thyroid follicular epithelium. ATC has a very poor prognosis due to its aggressive behavior and poor response to conventional therapies. Gene-directed enzyme/prodrug therapy using genetically engineered mesenchymal stromal cells (MSC) is a promising therapeutic strategy. The doxycycline (DOX)-controlled Tet inducible system is the most widely utilized regulatory system and could be a useful tool for therapeutic gene-based therapies. For example, use a synthetic “tetracycline-on” switch system to control the expression of the therapeutic gene thymidine kinase, which converts prodrugs to active drugs. The aim of this study was to develop therapeutic MSCs, harboring an inducible suicide gene, and to validate therapeutic gene expression using optical molecular imaging of ATC. We designed the Tet-On system using a retroviral vector expressing herpes simplex virus thymidine kinase (HSV1-sr39TK) with dual reporters (eGFP-Fluc2). Mouse bone marrow-derived mesenchymal stromal cells (BM-MSC) were transduced using this system with (MSC-Tet-TK/Fluc2) or without (MSC-TK/Fluc) the Tet-On system. Transduced cells were screened and characterized. Engineered MSCs were co-cultured with ATC (CAL62/Rluc) cells in the presence of the prodrug ganciclovir (GCV) and stimulated with DOX. The efficiency of cell killing monitored by assessing Rluc (CAL62/Rluc) and Fluc (MSC-Tet-TK/Fluc and MSC-TK/Fluc) activities using IVIS imaging. Fluc activity increased in MSC-Tet-TK/Fluc cells in a dose dependent manner following DOX treatment (R2 = 0.95), whereas no signal was observed in untreated cells. eGFP could also be visualized after induction with DOX, and the HSV1-TK protein could be detected by western blotting. In MSC-TK/Fluc cells, the Fluc activity increased with increasing cell number (R2 = 0.98), and eGFP could be visualized by fluorescence microscopy. The Fluc activity and cell viability of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells decreased significantly following GCV treatment. A bystander effect of the therapeutic cells confirmed in co-cultures of CAL62 cells, an anaplastic thyroid cancer cell line, with either MSC-Tet-TK/Fluc cells or MSC-TK/Fluc cells. The Rluc activity in MSC-Tet-TK/Fluc co-cultures, derived from the CAL62/Rluc cells, decreased significantly with GCV treatment of DOX treated cultures, whereas no significant changes were observed in untreated cultures. In addition, the Fluc activity of MSC-Tet-TK/Fluc cells also decreased significantly with DOX treatment whereas no signal was present in untreated cultures. A bystander effect also be demonstrated in co-cultures with MSC-TK/Fluc cells and CAL62/Rluc; both the Rluc activity and the Fluc activity were significantly decreased following GCV treatment. We have successfully developed a Tet-On system of gene-directed enzyme/prodrug delivery using MSCs. We confirmed the therapeutic bystander effect in CAL62/Rluc cells with respect to MSC-Tet-TK/Fluc and MSC-TK/Fluc cells after GCV treatment with and without DOX. Our results confirm the therapeutic efficiency of a suicide gene, with or without the Tet-On system, for ATC therapy. In addition, our findings provide an innovative therapeutic approach for using the Tet-On system to eradicate tumors by simple, repeated administration of MSC-Tet-TK/Fluc cells with DOX and GCV.</p></div

Cell viability of MSC, MSC-Tet-TK/Fluc, and MSC-TK/Fluc cells using a CCK-8 assay.

<p>(A) Cell viability of naïve MSCs treated with GCV for 48 h. (B) Cell viability of MSCs treated with DOX for 48 h. (C) Cell viability of MSC-Tet-TK/Fluc cells treated after GCV treatment for 48 h with and without DOX. (D) Cell viability of MSC-TK/Fluc cells after GCV treatment for 48 h. The values are expressed as the mean ± standard deviation (SD), of three experiments. NS: denotes no significant change. * indicates significance at p<0.05 (by Student's t test).</p

Fluc activity of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells.

<p>(A). Fluc activity of MSC-Tet-TK/Fluc cells treated with GCV for 48 h after induction with and without DOX. (B) Fluc activity of MSC-TK/Fluc cells treated with GCV for 48 h. (C) Quantitation of the Fluc2 activity of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells. The values are expressed as the mean ± standard deviation (SD), of three individual experiments and measurements. * indicates significance level at *p<0.05, **p<0.01., ***p<0.001 (by Student's t test).</p

Transduction of MSCs, with triple fusion (TF) reporter genes without the Tet-On system.

<p>(A) Scheme for the TF reporter gene plasmid pCMV-pTRE3G-sr39tk-eGFP-Fluc, which has a minimal promoter. (B) Fluc activity detected by BLI imaging and quantitative analysis of Fluc signal as a function of MSC-TK/Fluc cell number. (C) eGFP expression in MSC-TK/Fluc shown by confocal microscopy.</p