Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites
Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites
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DOI：
10.1073/pnas.1914906117
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发表时间：
2020-06-09
影响因子：
11.1
通讯作者：
Ferrara, Katherine W.
中科院分区：
文献类型：
--
作者：
Ilovitsh, Tali;Feng, Yi;Ferrara, Katherine W.
已经证明在实体瘤中难以实现稳健的细胞毒性T细胞浸润。我们着手开发一种灵活的方案，以有效地抑制肿瘤和基质细胞产生免疫激活细胞因子，从而在减瘤的同时增强T细胞浸润。通过将超声与肿瘤靶向微泡相结合，产生膜孔，并促进可控的局部转染。在这里，我们应用了比之前应用的低得多的传输频率（250 kHz）。在500 kPa负压下，微泡的有效膨胀比为35。结合低频超声与肿瘤靶向微泡和DNA质粒构建体，20%的肿瘤细胞保持存活，并且在体外和体内用报告基因转染了这些剩余细胞的约20%。体内转染的大多数细胞是粘蛋白1 + /CD 45-肿瘤细胞。然后用编码IFN-β的质粒DNA转染肿瘤和基质细胞，体外产生150 pg/10（6）个细胞，与无超声或无质粒对照相比增加150倍，与靶向微泡和超声（无IFN-β）治疗相比增加50倍。这种分泌的增强超过了先前报道的其他体外治疗的四倍至五倍增加。结合腹膜内注射检查点抑制剂，单次应用IFN-β质粒转染减少了体内肿瘤生长，并在局部和远处肿瘤部位招募了有效的免疫细胞。
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.