Mesenchymal stem cells anchored with thymidine phosphorylase for doxifluridine-mediated cancer therapy

Abstract

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 (5′-DFUR) to toxic drug 5-fluorouracil (5-FU). Since human mesenchymal stem cells (hMSCs) have 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. First, the TYMP gene sequence and core streptavidin (core SA) were constructed into pET-30a(+) plasmid. After bacterial transformation and colony screening, TYMP-SA fusion protein was expressed by IPTG induction and purified by immobilized metal affinity chromatography and characterized by SDS-PAGE and western blot with a clear band at 75 kDa. The characterized TYMP-SA was further anchored on the cell membrane of biotinylated hMSCs via biotin-streptavidin binding. hMSCs anchored with TYMP-SA were then co-cultured with adenocarcinoma A549 cells (with different ratios) and treated with 100 μM prodrug doxifluridine over the course of four days. Our results showed that a 2 : 1 ratio led to the eradication of A549 cells at the end of the experiment with less than 5% confluency, in comparison with the 1 : 1 and 1 : 2 ratios which still had about 13% and 20% confluency respectively. In conclusion, harnessing hMSCs as cell carriers for the delivery of TYMP enzyme to cancer cells could lead to significant cell death post-treatment of the prodrug doxifluridine.