目的:靶向癌症治疗的一个主要障碍是确定实体瘤特异性和大量表达的合适靶标。某些细菌菌株选择性地定殖实体瘤并且可以将遗传编码的货物分子递送至肿瘤细胞。这里,我们设计了细菌在肿瘤中表达单体链霉亲和素(mSA),并通过使用生物素化成像探针可视化肿瘤相关mSA的存在开发了一种新型的肿瘤预靶向系统。
方法:我们构建了表达与麦芽糖结合蛋白融合的mSA的质粒,并优化了核糖体结合位点序列以提高溶解度和表达水平。用重组质粒转化大肠杆菌MG1655,其表达由pBAD启动子驱动。在将细菌注射到带有CT26小鼠结肠癌细胞的小鼠中4天后,用L-阿拉伯糖诱导mSA的表达。在施用生物素化荧光染料后,通过光学成像观察mSA在肿瘤组织中的选择性积累。还进行了活细菌细胞的计数。
结果:与常规系统相比,新的表达系统导致mSA的表达明显更高,并持续与生物素结合。mSA表达组的肿瘤组织成像信号明显强于非表达组(P=0.0005)。此外,用L-阿拉伯糖多次诱导后,肿瘤组织中的荧光信号再次被检测到。肿瘤组织中的细菌计数在有和没有L-阿拉伯糖的条件之间没有显着差异(P=0.45)。肿瘤组织的Western印迹分析证实了mSA与生物素的表达和结合。
结论:我们成功地改造了携带表达mSA的重组质粒的肿瘤靶向细菌,目标是,并表达在,肿瘤组织。这些数据证明了当与生物素化成像探针或治疗剂组合时这种新型肿瘤预靶向系统的潜力。
OBJECTIVE: A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains selectively colonize solid tumors and can deliver genetically encoded cargo molecules to the tumor cells. Here, we engineered bacteria to express monomeric streptavidin (mSA) in tumors, and developed a novel tumor pre-targeting system by visualizing the presence of tumor-associated mSA using a biotinylated imaging probe.
METHODS: We constructed a plasmid expressing mSA fused to maltose-binding protein and optimized the ribosome binding site sequence to increase solubility and expression levels. E. coli MG1655 was transformed with the recombinant plasmid, expression of which is driven by the pBAD promotor. Expression of mSA was induced by L-
arabinose 4 days after injection of bacteria into mice bearing CT26 mouse colon carcinoma cells. Selective accumulation of mSA in tumor tissues was visualized by optical imaging after administration of a biotinylated fluorescent dye. Counting of viable bacterial cells was also performed.
RESULTS: Compared with a conventional system, the novel expression system resulted in significantly higher expression of mSA and sustained binding to biotin. Imaging signals in tumor tissues were significantly stronger in the mSA-expressing group than in non-expressing group (P = 0.0005). Furthermore, the fluorescent signal in tumor tissues became detectable again after multiple inductions with L-
arabinose. The bacterial counts in tumor tissues showed no significant differences between conditions with and without L-
arabinose (P = 0.45). Western blot analysis of tumor tissues confirmed expression and binding of mSA to biotin.
CONCLUSIONS: We successfully engineered tumor-targeting bacteria carrying a recombinant plasmid expressing mSA, which was targeted to, and expressed in, tumor tissues. These data demonstrate the potential of this novel tumor pre-targeting system when combined with biotinylated imaging probes or therapeutic agents.