Thermal-responsive ultrasound activatable in situ production of therapeutics for real-time imaging and targeted tumor therapy

Rationale: Engineered bacteria with tumor-targeting capabilities and genetic modification potential have emerged as promising vectors for targeted tumor therapy through the effective production and release of therapeutic molecules.Controlled gene expression and real-time monitoring of therapeutic protein expression in tumors are of great significance for optimizing the therapeutic effective of engineered bacteria production as well as anti-tumor effects.Methods: We constructed engineered E. coli MG1655 cells using recombinant thermal plasmid pBV220 carrying the optical imaging reporter gene miRFP720 and the therapeutic gene Cytolysin A, which could be controlled expressed synchronously by thermal effect produced by focused ultrasound.The concentrations of miRFP720 and Cytolysin A expressed by engineered bacteria after focused ultrasound irradiation were detected in vitro and in vivo with western blot analysis and enzyme-linked immunosorbent assay (ELISA) analysis.The immune cell infiltration rate and inflammation factors after treatment were detected by flow cytometry and ELISA analysis.The anti-tumor efficacy of engineered bacteria alone or combined with anti-Programmed cell death 1 ligand 1 (aPD-L1) immunotherapy were evaluated on subcutaneous tumor models and orthotopic glioblastoma models.Results: Upon ultrasound-controlled activation, the expression level of therapeutic molecule Cytolysin A as well as the optical imaging probe miRFP720 were enhanced.Importantly, the expression level of Cytolysin A could be monitored in vivo non-invasively by the signal intensity produced by miRFP720, which provided guidance for optimized ultrasound-mediated therapeutic production in situ.Furthermore, with optimized ultrasound activation, Cytolysin A not only exerted potent anti-tumor effects but also induced immunogenic cell death, enhancing the therapeutic efficiency of aPD-L1 immunotherapy for deep site tumor treatment. Conclusion:In this study, a real-time imaging guided ultrasound activatable tumor targeted therapy mode was established.Utilizing the thermal effect of focused ultrasound, Cytolysin A-miRFP720 engineered bacteria can concurrently express the therapeutic molecule Cytolysin A and the imaging probe miRFP720, which optimized the expression level of therapeutic production by engineered bacteria through imaging guided ultrasound irradiation mode, and realized enhanced the therapeutic efficiency of local tumor anti-tumor efficiency as well as aPD-L1 immunotherapy.