Dong Wei, Wang Guihu, Li Senyang, Wang Qian, Li Wenjuan, Liu Heyuan, Liang Yingxue, Zhou Zhe, He Xinrui, Guo Wenlei, Yuan Jianing, Chai Yichao, Geng Jing, Li Zongfang
National and Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, the Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, People's Republic of China.
Shaanxi Provincial Clinical Medical Research Center for Liver and Spleen Diseases, CHESS-Shaanxi Consortium, the Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, People's Republic of China.
Immunotargets Ther. 2025 Aug 24;14:901-930. doi: 10.2147/ITT.S534444. eCollection 2025.
Splenic immunomodulation triggered by ultrasound shows a significant anti-inflammatory effect against various inflammatory diseases, whose mechanism is mainly attributable to the activation of cholinergic anti-inflammatory pathway (CAP). However, the potential role and underlying mechanism of splenic ultrasound stimulation in cancer management have been rarely reported and superficially defined.
Following optimization of ultrasonic parameters, this study evaluated the anti-tumor efficacy of splenic sonication across multiple tumor models (eg, orthotopic H22 hepatocellular carcinoma (HCC), orthotopic Hepa1-6 HCC, and subcutaneous 4T1 breast cancer), and applied flow cytometry to quantify dynamic alterations in immune cell populations. Furthermore, in orthotopic H22 HCC models, this study employed fluorescence-activated cell sorting, RNA sequencing, splenic nerve blockade via absolute ethanol ablation, and in vitro Ca²⁺ flux assays to delineate the mechanisms underlying ultrasound-mediated splenic anti-tumor immunity.
This study first assessed the therapeutic effect of focused ultrasound precisely targeting the spleen (FUS sti. spleen) on various tumors at specific ultrasonic doses. It fully demonstrated that FUS directly stimulated splenic immune cell proliferation and activation (especially NK and CD8 T cells) rather than CAP excitation to modulate splenic immune function. Particularly, NK cells are much more indispensable and important in responding to FUS stimulation for cancer suppression than CD8 T cells. RNA sequencing of NK and CD8 T cells, as well as in vitro experiments revealed that FUS firstly regulated calcium-related signaling pathways to further modulate others, such as PI3K-AKT, Rap1, and Hippo pathways to promote immune cell proliferation, migration and activation to suppress cancer cell deterioration. Particularly, FUS sti. spleen and FUS intervention on the tumor synergistically induced the best tumor suppression than each of the two taken individually.
FUS sti. spleen facilitated immunocyte proliferation and activation through altering calcium-dependent signaling rather than CAP excitation to modulate anti-tumor immunity, indicating substantial clinical translation potential.
超声引发的脾脏免疫调节对多种炎症性疾病显示出显著的抗炎作用，其机制主要归因于胆碱能抗炎通路（CAP）的激活。然而，脾脏超声刺激在癌症治疗中的潜在作用和潜在机制鲜有报道且定义模糊。
在优化超声参数后，本研究评估了脾脏超声处理对多种肿瘤模型（如原位H22肝癌（HCC）、原位Hepa1-6肝癌和皮下4T1乳腺癌）的抗肿瘤疗效，并应用流式细胞术量化免疫细胞群体的动态变化。此外，在原位H22肝癌模型中，本研究采用荧光激活细胞分选、RNA测序、通过无水乙醇消融进行脾脏神经阻滞以及体外Ca²⁺通量测定来阐明超声介导的脾脏抗肿瘤免疫的潜在机制。
本研究首次评估了在特定超声剂量下精确靶向脾脏的聚焦超声（FUS刺激脾脏）对各种肿瘤的治疗效果。充分证明FUS直接刺激脾脏免疫细胞增殖和激活（尤其是NK细胞和CD8⁺ T细胞），而非通过激发CAP来调节脾脏免疫功能。特别是，在响应FUS刺激以抑制癌症方面，NK细胞比CD8⁺ T细胞更不可或缺且更重要。对NK细胞和CD8⁺ T细胞的RNA测序以及体外实验表明，FUS首先调节钙相关信号通路，进而调节其他通路，如PI3K-AKT、Rap1和Hippo通路，以促进免疫细胞增殖、迁移和激活，从而抑制癌细胞恶化。特别是，FUS刺激脾脏和FUS干预肿瘤协同诱导的肿瘤抑制效果优于单独使用两者中的任何一种。
FUS刺激脾脏通过改变钙依赖性信号通路促进免疫细胞增殖和激活，而非通过激发CAP来调节抗肿瘤免疫，显示出巨大的临床转化潜力。
