(15b) Mesenchymal Stem Cells Anchored with Thymidine Phosphorylase for Doxifluridine-Mediated Cancer Therapy

Introduction:Many tumors express thymidine phosphorylase (TYMP) with various levels, however due to tumor heterogeneity the amount of TYMP is usually not enough to convert prodrug doxifluridine to toxic drug fluorouracil (5-FU). Since human mesenchymal stem cells (hMSCs) has unique features of tumor-tropism and low immunogenicity, the purpose of this study is to use mesenchymal stem cells as carriers to deliver TYMP to cancer cells and then trigger their death by administrating doxifluridine.

Materials and Methods:First, TYMP gene sequence and core streptavidin (coreSA) were constructed into pET-30a plasmid. After bacterial transformation and colony screening, TYMP-coreSA fusion protein was expressed by IPTG induction and purified by immobilized metal affinity chromatography. The concentration and enzyme activity of purified TYMP-coreSA fusion protein were determined separately by Bradford assay and prodrug conversion assay. The characterized TYMP-coreSA was further anchored on the cell membrane of biotinylated hMSCs via biotin-streptavidin binding. hMSCs anchored with TYMP-coreSA were then co-cultured with adenocarcinoma A549 cells and treated with 200 uM prodrug doxifluridine over the course of five days. Two ratios of TYMP-coreSA-tethered hMSCs to A549 cells in the co-cultured wells were tested for anticancer efficacy.

Results and Discussion:The expression and purification of TYMP-coreSA fusion protein was visualized with a clear band in the target 75 kDa region by SDS-PAGE and Western blot analysis. TYMP-coreSA level and prodrug conversion were confirmed by enzyme activity assay. For the co-culture of TYMP-coreSA-decorated hMSCs and A549 cells, our results showed that 2:1 ratio led to A549 cell death on day 2, in comparison with cell death on day 3 for 1:1 ratio. Moreover, the eradication of A549 cancer cells was shown after 5 days for 2:1 ratio of TYMP-coreSA-tethered MSCs to A549 cells for prodrug therapy.

Conclusions: Harnessing hMSCs as cell carriers for the delivery of TYMP enzyme to cancer cells could lead to significant cell death post-treatment of prodrug doxifluridine.