Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites

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, reach-ing an effective expansion ratio of 35 for a peak negative pres-sure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid con-struct, 20% of tumor cells remained viable, and similar to 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-beta, producing 150 pg/10(6) cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid con-trols and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-beta). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal ad-ministration of checkpoint inhibition, a single application of IFN-beta plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.