Li Jixi, Wen Linqing, Guo Yujun, Yao Dan, Sun Bihang, Mou Hai, Chen Nianzhi, Zhou Kun, Wang Yan, Chen Wenzhi
State Key Laboratory of Ultrasound in Medicine and Engineering, College of Biomedical Engineering, Chongqing Medical University, Chongqing, 400016, China.
Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Cancer Immunol Immunother. 2025 Aug 6;74(9):281. doi: 10.1007/s00262-025-04137-6.
Despite achieving complete remission (CR) through surgery, chemoradiotherapy, targeted therapy, and other treatment modalities, breast cancer, particularly triple-negative breast cancer (TNBC) remains at a high risk of recurrence and metastasis. Enhancing anti-tumor immunity to eliminate residual tumor cells may reduce TNBC relapse. Our previous research indicated low-intensity pulsed ultrasound (LIPUS) can activate the anti‑tumor immunity. Here, we explore the potential of the suitable strategy with LIPUS, and the combination with an immune checkpoint inhibitor, anti-PD-1 antibody (αPD-1) to anti‑tumor immunity in TNBC.
In the xenografted mouse model, different times (7, 14, 21, and 28d) and organs (spleen, bone marrow, spleen + bone marrow) of LIPUS were received to experimental groups. In 4T-1 low tumor burden model, after LIPUS irradiated different organs for 28d or combined with αPD-1, the tumor volume, lung metastasis, and survival time were observed. Flow cytometry, immunohistochemistry, immunofluorescence, multiplex immunofluorescence, and ELISA were performed to elucidate detailed impacts of LIPUS or combination treatment on anti‑tumor immunity. RNA-seq was used to preliminarily explore the biological mechanism of LIPUS.
LIPUS inhibited the growth of 4T-1 xenografted, and the irradiation time was the main factor. In 4T-1 low tumor burden model, further studies had shown that LIPUS of spleen + bone marrow could best active anti‑tumor immunity primarily through CD8 cytotoxic T lymphocytes (CD8CTLs) and myeloid-derived suppressor cells (MDSCs), but not reduce the rate of tumor formation. LIPUS combined with αPD-1 not only reduce tumor formation rate but also further enhance anti‑tumor immunity mainly by means of CD8CTLs, as well as led to significant changes in cytokines. The RNA-seq results also suggested the anti-tumor immune biological processes and signaling pathways were generated after LIPUS irradiating immune organs.
Irradiating the spleen and bone marrow with LIPUS for 28 days may be an applicable treatment strategy, as it induces an increase in CD8CTLs and a decrease in MDSCs, as well as an upregulation of mRNA levels in multiple immunomodulatory pathways. LIPUS combined with αPD-1 elicits a superior anti-tumor immune response and inhibits tumor formation in a 4T-1 low tumor burden model. This study provides a novel approach for treating TNBC after achieving CR by combining LIPUS with αPD-1.
尽管通过手术、放化疗、靶向治疗等多种治疗方式可实现完全缓解（CR），但乳腺癌，尤其是三阴性乳腺癌（TNBC）仍具有较高的复发和转移风险。增强抗肿瘤免疫力以清除残留肿瘤细胞可能会降低TNBC的复发率。我们之前的研究表明，低强度脉冲超声（LIPUS）可激活抗肿瘤免疫力。在此，我们探索LIPUS联合免疫检查点抑制剂抗程序性死亡蛋白1抗体（αPD-1）在TNBC中抗肿瘤免疫的潜在适宜策略。
在异种移植小鼠模型中，将不同时间（7、14、21和28天）及不同器官（脾脏、骨髓、脾脏+骨髓）接受LIPUS照射的小鼠分为实验组。在4T-1低肿瘤负荷模型中，LIPUS照射不同器官28天后或联合αPD-1，观察肿瘤体积、肺转移情况及生存时间。采用流式细胞术、免疫组化、免疫荧光、多重免疫荧光及酶联免疫吸附测定（ELISA）等方法，以阐明LIPUS或联合治疗对抗肿瘤免疫的具体影响。利用RNA测序（RNA-seq）初步探索LIPUS的生物学机制。
LIPUS可抑制4T-1异种移植瘤的生长，照射时间是主要影响因素。在4T-1低肿瘤负荷模型中，进一步研究表明，脾脏+骨髓联合照射LIPUS能主要通过细胞毒性T淋巴细胞（CD8CTLs）和骨髓来源的抑制性细胞（MDSCs）最佳地激活抗肿瘤免疫力，但不能降低肿瘤形成率。LIPUS联合αPD-1不仅可降低肿瘤形成率，还能主要通过CD8CTLs进一步增强抗肿瘤免疫力，并导致细胞因子发生显著变化。RNA-seq结果还提示，LIPUS照射免疫器官后可产生抗肿瘤免疫生物学过程和信号通路。
用LIPUS照射脾脏和骨髓28天可能是一种可行的治疗策略，因为它可诱导CD8CTLs增加、MDSCs减少，并上调多种免疫调节途径中的mRNA水平。在4T-1低肿瘤负荷模型中，LIPUS联合αPD-1可引发更强的抗肿瘤免疫反应并抑制肿瘤形成。本研究为LIPUS联合αPD-1治疗CR后的TNBC提供了一种新方法。
