Metastasis represents the leading cause of death in patients with breast cancer. Traditional Chinese medicine is particularly appreciated for metastatic diseases in Asian countries due to its benefits for survival period prolongation and immune balance modulation. However, the underlying molecular mechanisms remain largely unknown. This study aimed to explore the antimetastatic effect and immunomodulatory function of a clinical formula Aiduqing (ADQ). Naive CD4+ T cells, regulatory T cells (Tregs), and CD8+ T cells were sorted by flow cytometry. Then, breast cancer cells and these immune cells were co-cultured in vitro or co-injected into mice in vivo to simulate their coexistence. Flow cytometry, ELISA, qPCR, double luciferase reporter gene assay, and chromatin immunoprecipitation assay were conducted to investigate the immunomodulatory and antimetastatic mechanisms of ADQ. ADQ treatment by oral gavage significantly suppressed 4T1-Luc xenograft growth and lung metastasis in the orthotopic breast cancer mouse model, without noticeable hepatotoxicity, nephrotoxicity, or hematotoxicity. Meanwhile, ADQ remodeled the immunosuppressive tumor microenvironment (TME) by increasing the infiltration of tumor-infiltrating lymphocytes (TILs) and cytotoxic CD8+ T cells, and decreasing the infiltration of Tregs, naive CD4+ T cells, and tumor-associated macrophages (TAMs). Molecular mechanism studies revealed that ADQ remarkably inhibited CXCL1 expression and secretion from TAMs and thus suppressed the chemotaxis and differentiation of naive CD4+ T cells into Tregs, leading to the enhanced cytotoxic effects of CD8+ T cells. Mechanistically, TAM-derived CXCL1 promoted the differentiation of naive CD4+ T cells into Tregs by transcriptionally activating the NF-κB/FOXP3 signaling. Lastly, mouse 4T1-Luc xenograft experiments validated that ADQ formula inhibited breast cancer immune escape and lung metastasis by suppressing the TAM/CXCL1/Treg pathway. This study not only provides preclinical evidence supporting the application of ADQ in inhibiting breast cancer metastasis but also sheds novel insights into TAM/CXCL1/NF-κB/FOXP3 signaling as a promising therapeutic target for Treg modulation and breast cancer immunotherapy.

中文翻译：

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爱毒清方通过抑制TAM/CXCL1诱导的Treg分化和浸润抑制乳腺癌转移

转移是乳腺癌患者死亡的主要原因。中医药在亚洲国家对转移性疾病特别受欢迎，因为它有利于延长生存期和调节免疫平衡。然而，潜在的分子机制仍然很大程度上未知。本研究旨在探讨临床方剂爱毒清（ADQ）的抗转移作用和免疫调节功能。通过流式细胞术对初始 CD4+ T 细胞、调节性 T 细胞 (Tregs) 和 CD8+ T 细胞进行分选。然后，将乳腺癌细胞和这些免疫细胞在体外共培养或在体内共注射到小鼠体内以模拟它们的共存。流式细胞仪、ELISA、qPCR、双荧光素酶报告基因检测、并进行染色质免疫沉淀测定以研究ADQ的免疫调节和抗转移机制。在原位乳腺癌小鼠模型中，通过口服强饲法进行的 ADQ 治疗显着抑制了 4T1-Luc 异种移植物的生长和肺转移，而没有明显的肝毒性、肾毒性或血液毒性。同时，ADQ 通过增加肿瘤浸润淋巴细胞 (TIL) 和细胞毒性 CD8+ T 细胞的浸润，并减少 Tregs、幼稚 CD4+ T 细胞和肿瘤相关巨噬细胞 (TAM) 的浸润来重塑免疫抑制性肿瘤微环境 (TME)。分子机制研究表明，ADQ 显着抑制 TAM 中 CXCL1 的表达和分泌，从而抑制幼稚 CD4+ T 细胞趋化和分化为 Tregs，导致 CD8+ T 细胞的细胞毒作用增强。从机制上讲，TAM 衍生的 CXCL1 通过转录激活 NF-κB/FOXP3 信号传导促进幼稚 CD4+ T 细胞分化为 Tregs。最后，小鼠 4T1-Luc 异种移植实验证实 ADQ 配方通过抑制 TAM/CXCL1/Treg 通路抑制乳腺癌免疫逃逸和肺转移。该研究不仅提供了支持 ADQ 在抑制乳腺癌转移中的应用的临床前证据，而且还为 TAM/CXCL1/NF-κB/FOXP3 信号转导作为 Treg 调节和乳腺癌免疫治疗的有希望的治疗靶点提供了新的见解。小鼠 4T1-Luc 异种移植实验证实 ADQ 配方通过抑制 TAM/CXCL1/Treg 通路抑制乳腺癌免疫逃逸和肺转移。该研究不仅提供了支持 ADQ 在抑制乳腺癌转移中的应用的临床前证据，而且还为 TAM/CXCL1/NF-κB/FOXP3 信号转导作为 Treg 调节和乳腺癌免疫治疗的有希望的治疗靶点提供了新的见解。小鼠 4T1-Luc 异种移植实验证实 ADQ 配方通过抑制 TAM/CXCL1/Treg 通路抑制乳腺癌免疫逃逸和肺转移。该研究不仅提供了支持 ADQ 在抑制乳腺癌转移中的应用的临床前证据，而且还为 TAM/CXCL1/NF-κB/FOXP3 信号转导作为 Treg 调节和乳腺癌免疫治疗的有希望的治疗靶点提供了新的见解。