Stem cells carrying a suicide gene have emerged as therapeutic candidates for their cytotoxic bystander effects on neighboring
cancers, while being non-toxic to other parts of the body. However, traditional cytotoxicity assays are unable to adequately assess the
therapeutic effects of bystander cells. Here, we report a method to assess bystander effects of therapeutic stem cells against 3-dimensionally grown
glioma cells in real time. U87
glioma cells were stably transduced to express a green fluorescence
protein and co-cultivated with mesenchymal stem cells engineered to carry a bacterial
cytosine deaminase gene (MSC/CD). Following addition of a 5-fluorocytine (5-FC)
prodrug to the co-culture, fluorescence from U87 cells was obtained and analyzed in real time. Notably, the IC50 of 5-FC was higher when U87 cells were grown 3-dimensionally in soft
agar medium for 3 weeks, as compared to those grown for one week in two-dimensional monolayer cultures. Additionally, more MSC/CD cells were required to maintain a similar level of efficacy. Since three-dimensional growth of
glioma cells under our co-culture condition mimics the long-term expansion of
cancer cells in vivo, our method can extend to an in vitro assay system to assess stem cell-mediated anti-
cancer effects before advancing into preclinical animal studies.