Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.
Keywords: microbubble; transfection; ultrasound.
Conflict of interest statement
The authors declare no competing interest.
Enhanced microbubble contrast agent oscillation following 250 kHz insonation.Sci Rep. 2018 Nov 5;8(1):16347. doi: 10.1038/s41598-018-34494-5. Sci Rep. 2018. PMID: 30397280 Free PMC article.
[Ultrasound-mediated microbubble destruction enhances exogenous gene expression in NIH3T3 cells in vitro].Hua Xi Kou Qiang Yi Xue Za Zhi. 2008 Apr;26(2):198-200, 205. Hua Xi Kou Qiang Yi Xue Za Zhi. 2008. PMID: 18605465 Chinese.
Ultrasound-mediated gene delivery with cationic versus neutral microbubbles: effect of DNA and microbubble dose on in vivo transfection efficiency.Theranostics. 2012;2(11):1078-91. doi: 10.7150/thno.4240. Epub 2012 Nov 8. Theranostics. 2012. PMID: 23227124 Free PMC article.
[Enhancement of gene transfection efficiency and therapeutic effect of ultrasound-targeted microbubble destruction in vivo with cationic microbubble].Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2018 Feb 15;32(2):228-236. doi: 10.7507/1002-1892.201706058. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2018. PMID: 29806417 Chinese.
Advances in low-frequency ultrasound combined with microbubbles in targeted tumor therapy.J Zhejiang Univ Sci B. 2019 Apr.;20(4):291-299. doi: 10.1631/jzus.B1800508. J Zhejiang Univ Sci B. 2019. PMID: 30932374 Free PMC article. Review.