335 GENE THERAPEUTIC EFFECT OF ENGINEERED STEM CELLS EXPRESSING CYTOSINE DEAMINASE AND INTERFERON-B AGAINST ENDOMETRIAL CANCER IN A XENOGRAFT MOUSE MODEL

Abstract

To generate an animal model of endometrial cancer, Ishikawa cells (2 × 10⁶ cells) were implanted via subcutaneous injection into SCID mice. In order to evaluate the therapeutic effect on tumour growth, human neural stem cells (HB1.F3), transduced with genes expressing cytosine deaminase (CD) and interferon-β (IFN-b), were labelled with the membrane dye stained with CM-DiI and were injected nearby into the tumour masses (250 mm³) in these SCID mice (n = 24). Two days after stem cell injections, 5-fluorocytosine (5-FC) was intraperitoneally injected (500 mg kg^–1 per day) for 14 consecutive days. Mice were sacrificed at 48 h after the last treatment with 5-FC delivered by intraperitoneal injection for 14 days. Results demonstrated that tumour mass growth in mice treated with stem cells and 5-FC was significantly inhibited (450 mm³ ± 52) in comparison to that of nontreated mice (1400 mm³ ± 124; P < 0.01). We confirmed that injected stem cells migrated to the tumour masses by visualising the red-coloured cells in the endometrial cancer masses. The RT-PCR analysis showed that the VEGF gene was highly expressed in endometrial cancer cells, while migration-induced uPA, SDF-1, SCF, MCP-1, IL-6, and IL-8 genes were moderately expressed in these tumour cells. Histological analysis of tumour masses showed that the aggressive nature of endometrial tumour masses, determined by labelled stem cells, was inhibited in treated mice. A further analysis showed that expression of proliferating nuclear antigen (PCNA) was significantly decreased in tumour masses from treated mice. To evaluate the effect of 5-FU on Ishikawa cells, 5-FU (1.0 mg mL^–1) was added to in vitro-cultured cells and the levels of IFN-a/b receptor 2 (IFNAR2) and BAX, a proapoptotic genes, were detected by RT-PCR. In these cells, IFNAR2 and BAX genes were significantly increased by the addition of 5-FU. Taken together, these results indicate that co-expression of CD and IFN-b significantly inhibited the growth of endometrial tumour masses in the presence of 5-FC, and 5-FU and IFN-b inhibits tumour growth by inhibition of DNA synthesis and enhancing the apoptotic cascade, respectively. We provide evidence to support the efficacy of therapeutic stem cell-based immuno-therapy involving the targeted expression of CD and IFN-b genes at endometrial cancer sites.

This work was supported by a National Research Foundation of Korea (NRF) grant (2013R1A1A2059092) funded by the Ministry of Education, Science and Technology (MEST) of the Republic of Korea government.