The tumor-tropic properties of neural stem cells (NSCs) led to the development of a novel strategy for delivering therapeutic genes to tumors in the brain. to 5-FC or 5-FU was determined for the F3.CD cells and compared with that of the parental F3 cells. The F3.CD cells were sensitive to 5-FC exposure at concentrations >0.2 mmol/l (< 0.05, Figure 1a,b), which confirmed the enzymatic conversion of the prodrug 5-FC to the cytotoxic 5-FU. After 24 hours of 5-FC treatment, the F3.CD cells showed signs of mild toxicity, and after 48 hours, the toxic effect was very clear (Supplementary Figure S2). Figure 1 Cytosine deaminase (CD) Rabbit Polyclonal to Smad2 (phospho-Thr220) from HB1.F3.CD (F3.CD) cells effectively converts the prodrug 5-fluorocytosine (5-FC) to 5- fluorouracil (5-FU). (a) F3 and F3.CD cells were treated with 0.5 mmol/l 5-FC or 5-FU for 4 days. (b) The cytotoxic effects of 5-FU and … MDA-MB-435 (a human breast cancer cell line), PC14PE6 (a human lung adenocarcinoma cell line), and K1735 cells (a mouse melanoma cell line) were not affected by 5-FC treatment even at 0.5 mmol/l, consistent with the lack of CD expression in these cells (data not shown). In contrast, all the cell lines were sensitive to 5-FU (Figure 1c,d). Among the tumor cell lines, the lowest sensitivity was observed with the MDA-MB-435 cells. F3.CD coculture inhibits tumor cell growth To confirm the bystander effect of the CD produced by the F3.CD cells, a coculture system was used to determine the effect on viability of cocultured tumor cells. In coculture with F3.CD cells, 0.5 mmol/l 5-FC significantly inhibited the growth of MDA-MB-435, PC14PE6, and K1735 cells (< 0.05, Figure 2). This effect was not observed with the parental F3 863029-99-6 cells. Similarly, without addition of 5-FC, the F3.CD coculture had no effect on the tumor cell growth (data not shown). These results show that F3. CD 863029-99-6 cells convert sufficient amounts of 5-FC to 5-FU to effectively kill MDA-MB-435, PC14PE6, and K1735 cells therapeutic efficacy of F3.CD cells Next, the migrating potential and therapeutic efficacy of the F3.CD cells were determined. F3.CD cells were injected into the brain of MDA-MB-435 tumor-bearing animals, contralateral to the tumor cell implantation. The distribution of F3.CD cells and tumor volumes were determined in brain tissue 2 days after the last 5-FC treatment (Figure 3a). A 863029-99-6 large number of CD-immunoreactive F3.CD cells were identified in the tumor bed and at the tumor-normal parenchyma interface. Some migrating F3.CD cells were found in the corpus callosum (Figure 3b). Histological analysis showed significantly reduced tumor volumes in the brains of 863029-99-6 5-FC-treated F3.CD animals: (Figure 3c; group 4, mean SD = 7.89 4.18 mm3) compared the control groups [group 1, Hanks’ balanced salt solution (HBSS) injection + saline, 19.81 6.86 mm3; group 2, F3.CD injection + saline, 17.14 5.24 mm3; group 3, HBSS injection + 5-FC, 20.94 2.55 mm3]. The tumor sizes were reduced up to 60%. No abnormalities in the brain parenchyma surrounding the tumors were in treated animals. Figure 3 Treatment with F3.CD cells and 5-fluorocytosine (5-FC) has a significant therapeutic effect. (a) Timeline for the metastatic brain tumor animal model and subsequent treatment using F3.CD cells and 5-FC. (b) F3.CD cells were injected into the brain … We confirmed the therapeutic efficacy of the F3.CD cells using another brain metastasis animal model, which was made by injection of MDA-MB-435 cells into the right internal carotid artery (ICA) of immune-deficient mouse. F3.CD cells were implanted into left brain hemisphere stereotactically and 5-FC was administrated systemically (Figure 863029-99-6 4a). Both tumor volumes and numbers of tumor masses of treated mice were reduced significantly [Figure 4b,c; group 2, sum of tumor volumes = 1.46 2.67 mm3 (mean SD), number of tumor masses in.