Evaluation of the therapeutic effect of cytosine deaminase-expressing mesenchymal stem cells and 5-fluorocytosine on glioma using molecular imaging

Abstract

Introduction: Despite advances in chemotherapy and radiation after tumor resection, the high propensity of glioma cells to invade the surrounding normal brain tissue reduces the survival rate of patients The therapeutic use of mesenchymal stem cells (MSCs) has recently been extensively investigated in cancer therapy because of these cells tumor targeting properties. In this study, the efficacy of combination therapy using MSCs expressing cytosine deaminase (CD) and prodrug 5-FC in a mouse glioma model was evaluated using various molecular imaging modalities. Methods: Human glioma cell lines (U373, U87MG) and primary glioblastoma cells from a patient (GBM28) were stably transfected with fluorescent and luciferase reporter genes for tumor cell imaging. Glioma cells were treated with standard therapy (ionizing radiation, Temozolomide (TMZ)) or 5-FU in a dose dependent manner. Microarray and real-time PCR were performed to determine the expression levels of radio-resistant and 5-FU metabolism related genes. Therapeutic MSC/CDs were established by the transfection of CD genes into human bone-marrow derived MSCs. Next, in vitro and in vivo 19F-magnetic resonance spectroscopy (19F-MRS) was performed to estimate the conversion of 5-FC to 5-FU by the MSC/CDs. To validate the in vitro anticancer effect of MSC/CDs with 5-FC, luciferase-expressing glioma cells were co-cultured with MSC/CDs. For in vivo monitoring of the therapeutic effect of MSC/CDs with 5-FC, U87MG/Luc cells were inoculated into the mouse cranium. Consequently, therapeutic MSC/CD were transplanted into the mouse cranium. Tumor growth was measured using bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and positron emission tomography (PET). Results: Glioma cells and MSCs were successfully monitored using fluorescence and BLI. U87MG cells were killed following exposure to ionizing radiation (3 Gy), with a survival rate of 82.8% ± 9.59%, while U373 and GBM28 ells had a survival rate of 57.9% ± 6.88% and 48.0% ± 6.0%. In contrast, 36.3% ± 8.9% of U87MG cells survived after 5-FU treatment (4 uM), whereas 68.6% ± 15% of U373 cells survived. Unlike in ionizing radiation studies, U87MG cells were found to be two times more sensitive to 5-FU than U373 cells. Microarray analysis demonstrated that U87MG cells highly express DNA repair-related genes compared to U373 cells. The expression of rate-limiting enzymes of 5-FU metabolism in U87MG cells is low. In vitro and in vivo studies of 19F-MRS revealed the effective conversion of 5-FC to 5-FU by MSC/CDs. Based on a co-culture experiment with MSC/CDs and glioma cells, MSC/CDs showed an anticancer effect on neighboring cancer cells in proportion to increasing the 5-FC dosage and the MSC/CD ratio. The in vitro anticancer effects of MSC/CD therapy with 5-FC on U87MG cells were two times more effective than on U373 cells. In a glioma orthotopic model, a BLI/MRI/PET molecular imaging system revealed 70% inhibition of tumor growth by MSC/CD with 5-FC therapy. Furthermore, no tumorigenesis or pathological abnormalities were observed at the MSC/CD transplantation site. Conclusions: Taken together, the efficacy of a new stem cell mediated enzyme/prodrug therapy was determined using various imaging modalities. In particular, BLI was able to accurately verify the therapeutic effect of MSCs in vitro and in vivo. In addition, the different response of therapeutic efficacy on glioma cells was revealed using microarray analysis. Therefore, the suggested MSC/CD and prodrug 5-FC therapy could be an effective anticancer therapy option for radio-resistant and 5-FU-sensitive glioma treatment.