Recent findings suggested that premetastatic niche (PMN) is a prerequisite in mediating cancer metastasis. Previously we demonstrated that XIAOPI formula could inhibit breast cancer lung metastasis via inhibiting tumor associated macrophages (TAMs)-secreted CXCL1. Herein, we aimed to explore the effects of XIAOPI formula on preventing breast cancer PMN formation and its underlying molecular mechanisms. CXCL1 expression of TAMs was detected by qPCR and Western blotting assay. The influences of XIAOPI formula on the proliferation of TAMs and 4 T1 in the co-culture system were tested by CCK8 or EdU staining. Transwell experiment was applied to determine the effects of XIAOPI formula on the invasion ability of HSPCs and 4 T1. Breast cancer xenografts were built by inoculating 4 T1 cells into the mammary pads of Balb/c mice and lung metastasis was monitored by luciferase imaging. Immune fluorescence assay was used to test the epithelial-mesenchymal transition process and PMN formation in the lung tissues. The effects of XIAOPI formula on TAMs phenotype, hematopoietic stem/progenitor cells (HSPCs) and myeloid-derived suppressor cells (MDSCs) were determined by flow cytometry. It was found that XIAOPI formula could inhibit the proliferation and polarization of M2 phenotype macrophages, and reduce CXCL1 expression in a dose-dependent manner. However, M1 phenotype macrophages were not significantly affected by XIAOPI formula. TAMs/CXCL1 signaling was subsequently found to stimulate the recruitment of c-Kit+/Sca-1+ HSPCs and their differentiation into CD11b+/Gr-1+ MDSCs, which were symbolic events accounting for PMN formation. Moreover, XIAOPI formula was effective in inhibiting HSPCs activation and suppressing the proliferation and metastasis of breast cancer cells 4 T1 induced by HSPCs and TAMs co-culture system, implying that XIAOPI was effective in preventing PMN formation in vitro. Breast cancer xenograft experiments further demonstrated that XIAOPI formula could inhibit breast cancer PMN formation and subsequent lung metastasis in vivo. The populations of HSPCs in the bone marrow and MDSCs in the lung tissues were all remarkably declined by XIAOPI formula treatment. However, the inhibitory effects of XIAOPI formula could be relieved by CXCL1 overexpression in the TAMs. Taken together, our study provided preclinical evidence supporting the application of XIAOPI formula in preventing breast cancer PMN formation, and highlighted TAMs/CXCL1 as a potential therapeutic strategy for PMN targeting therapy.

中文翻译：

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XIAOPI公式通过抑制TAM / CXCL1信号传导来抑制乳腺癌的转移前生态位形成。

最近的发现表明，转移前的利基（PMN）是介导癌症转移的先决条件。以前，我们证明XIAOPI配方可以通过抑制分泌肿瘤相关巨噬细胞（TAMs）的CXCL1抑制乳腺癌的肺转移。在本文中，我们旨在探讨XIAOPI配方对预防乳腺癌PMN形成的作用及其潜在的分子机制。通过qPCR和Western blotting检测TAMs的CXCL1表达。通过CCK8或EdU染色测试了XIAOPI配方对共培养系统中TAMs和4 T1增殖的影响。应用Transwell实验确定XIAOPI配方对HSPCs和4 T1侵袭能力的影响。通过将4个T1细胞接种到Balb / c小鼠的乳腺垫中来构建乳腺癌异种移植物，并通过荧光素酶成像监测肺转移。免疫荧光测定法用于测试肺组织中的上皮-间质转化过程和PMN形成。通过流式细胞术确定了XIAOPI配方对TAMs表型，造血干/祖细胞（HSPCs）和骨髓源性抑制细胞（MDSCs）的影响。结果发现，XIAOPI制剂可以抑制M2表型巨噬细胞的增殖和极化，并以剂量​​依赖的方式降低CXCL1的表达。但是，XIAOPI公式并未显着影响M1表型巨噬细胞。随后发现TAM / CXCL1信号刺激了c-Kit + / Sca-1 + HSPC的募集及其向CD11b + / Gr-1 + MDSC的分化，这是解释PMN形成的象征性事件。此外，XIAOPI配方可有效抑制HSPCs和TAMs共培养系统诱导的HSPCs活化并抑制乳腺癌细胞4 T1的增殖和转移，这表明XIAOPI配方可有效预防体外PMN的形成。乳腺癌异种移植实验进一步证明，XIAOPI配方可在体内抑制乳腺癌PMN的形成和随后的肺转移。通过XIAOPI配方奶粉处理，骨髓中的HSPC数量和肺组织中的MDSC数量均显着下降。然而，XIAOPI配方的抑制作用可以通过TAM中CXCL1的过表达来缓解。在一起