Chan-Hyams Jasmine V E, Ackerley David F
School of Biological Sciences, Victoria University of Wellington, Wellington, 6012, New Zealand.
Centre for Biodiscovery, Victoria University of Wellington, Wellington, 6012, New Zealand.
MethodsX. 2020 Jan 23;7:100797. doi: 10.1016/j.mex.2020.100797. eCollection 2020.
Bacterial-directed enzyme-prodrug therapy (BDEPT) uses tumour-tropic bacteria armed with a genetically-encoded prodrug-converting enzyme to sensitise tumours to a systemically-administered prodrug. A strong bystander effect (i.e., efficient bacteria-to-tumour transfer of activated prodrug metabolites) is critical to maximise tumour cell killing and avoid bacterial self-sterilisation. To investigate the bystander effect in bacteria we developed a sensitive screen that utilised two strains grown in co-culture. The first of these was an activator strain that overexpressed the nitroreductase NfsA, and the second was a nitroreductase null recipient strain bearing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by genotoxic prodrug metabolites can only occur following their transfer from the activator to the recipient cells. This can be monitored both in fluorescence based microtitre plate assays and by flow-cytometry, enabling modelling of the abilities of diverse nitroaromatic prodrug metabolites to exit a Gram negative vector.
细菌导向酶-前药疗法（BDEPT）利用携带基因编码前药转化酶的肿瘤靶向细菌，使肿瘤对全身给药的前药敏感。强大的旁观者效应（即活化的前药代谢物从细菌到肿瘤的有效转移）对于最大限度地杀伤肿瘤细胞和避免细菌自我杀菌至关重要。为了研究细菌中的旁观者效应，我们开发了一种灵敏的筛选方法，该方法利用共培养的两种菌株。其中第一种是过表达硝基还原酶NfsA的激活菌株，第二种是携带SOS-GFP DNA损伤响应基因构建体的硝基还原酶缺失受体菌株。在这个系统中，遗传毒性前药代谢物诱导GFP表达只能在它们从激活菌株转移到受体细胞后发生。这可以在基于荧光的微量滴定板分析和流式细胞术中进行监测，从而能够模拟不同硝基芳香族前药代谢物从革兰氏阴性载体中释放出来的能力。
