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

Abstract

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.

Fig 1. Transduction of MSCs, with triple fusion (TF) reporter genes.

(A) Schematic of the TF reporter gene construct pTRE3G-sr39tk-eGFP-Fluc and the plasmid expressing the Tet-On 3G activator. (B) Confocal microscopy image of eGFP in MSC-Tet-TK/Fluc cells after induction with DOX (2 μg/mL) for 24 h. (C) Fluc activity in stably transduced MSCs (MSC-Tet-TK/Fluc) after DOX treatment for 24 h and detected using BLI quantitative imaging of Fluc activity. (D) Western Blot analysis of HSV1-TK protein after 24 h induction of MSC-Tet-TK/Fluc cells with DOX (0,1,2 and 4 μg/ml).

Fig 2. Transduction of MSCs, with triple fusion (TF) reporter genes without the Tet-On system.

(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.

Fig 3. ³H Penciclovir uptake assay in MSCs, MSC-Tet-TK/Fluc, and MSC-TK/Fluc cells.

The penciclovir uptake of naïve MSCs, MSC-TK/Fluc, untreated and DOX-treated (2 μg/mL) MSC-Tet-TK/Fluc cells over 1, 2, and 4 h time periods.

Fig 4. Cell viability of MSC, MSC-Tet-TK/Fluc, and MSC-TK/Fluc cells using a CCK-8 assay.

(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).

Fig 5. Fluc activity of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells.

(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).

Fig 6. Bystander effect of suicide gene transduced cells.

(A) Rluc activity of CAL62/Rluc cells co-cultured with naïve MSCs (1:1) followed by GCV treatment (0.25 to 8μM) for 48 h. (B) Rluc activity of CAL62/Rluc cells co-cultured (1:1) with MSC-Tet-TK/Fluc cells followed by GCV treatment for 48 h, with and without DOX (2 μg/mL) treatment (C) Rluc activity of CAL62/Rluc cells co-cultured (1:1) with MSC-TK/Fluc cells after GCV treatment for 48 h. The values are expressed as the means ± 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).

Fig 7. Fluc activity of MSC-Tet-TK/Fluc and MSC-TK/Fluc cells.

(A) Fluc2 activity of MSC-Tet-TK/Fluc cells co-cultured (1:1) with CAL62/Rluc cells and treated with GCV for 48 h. (B) Fluc2 activity of MSC-TK/Fluc cells co-cultured (1:1) with CAL62/Rluc cells and treated with GCV for 48 h. 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).

Fig 8. Rluc activity of CAL62/Rluc cells after therapeutic genes co-injected with CAL62/Rluc cells with and without DOX and GCV for four days treatment.

(A) BLI of Rluc activity and quantitative Rluc activity of MSC-Tet-TK group. CAL62/Rluc cells co-injected with MSC (Left) and MSC-Tet-TK (Right). (B) BLI of Rluc activity and quantitative Rluc activity of MSC-TK group. CAL62/Rluc cells co-injected with MSC (Left) and MSC-TK (Right) side of the mice. Three mice data's were presented as means ± standard deviation (SD). The P value <0.05 was considered statistically significant by Student t-test.