Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata, Kita-ku, Okayama, 700-8558, Japan.
Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata, Kita-ku, Okayama, 700-8558, Japan.
Breast Cancer Res. 2022 Sep 12;24(1):60. doi: 10.1186/s13058-022-01557-5.
Patients with triple-negative breast cancer (TNBC) often have poorer prognosis than those with other subtypes because of its aggressive behaviors. Cancer cells are heterogeneous, and only a few highly metastatic subclones metastasize. Although the majority of subclones may not metastasize, they could contribute by releasing factors that increase the capacity of highly metastatic cells and/or provide a favorable tumor microenvironment (TME). Here, we analyzed the interclonal communication in TNBC which leads to efficient cancer progression, particularly lung metastasis, using the polyclonal murine 4T1 BC model.
We isolated two 4T1 subclones, LM.4T1 and HM.4T1 cells with a low and a high metastatic potential, respectively, and examined the effects of LM.4T1 cells on the behaviors of HM.4T1 cells using the cell scratch assay, sphere-forming assay, sphere invasion assay, RT-qPCR, and western blotting in vitro. We also examined the contribution of LM.4T1 cells to the lung metastasis of HM.4T1 cells and TME in vivo. To identify a critical factor which may be responsible for the effects by LM.4T1 cells, we analyzed the data obtained from the GEO database.
Co-injection of LM.4T1 cells significantly augmented lung metastases by HM.4T1 cells. LM.4T1-derived exosomes promoted the migration and invasion of HM.4T1 cells in vitro, and blocking the secretion of exosome abrogated their effects on HM.4T1 cells. Analyses of data obtained from the GEO database suggested that Wnt7a might be a critical factor responsible for the enhancing effects. In fact, a higher level of Wnt7a was detected in LM.4T1 cells, especially in exosomes, than in HM.4T1 cells, and deletion of Wnt7a in LM.4T1 cells significantly decreased the lung metastasis of HM.4T1 cells. Further, treatment with Wnt7a increased the spheroid formation by HM.4T1 cells via activation of the PI3K/Akt/mTOR signaling pathway. Finally, infiltration of αSMA-positive fibroblasts and angiogenesis was more prominent in tumors of LM.4T1 cells and deletion of Wnt7a in LM.4T1 cells markedly reduced angiogenesis.
We demonstrated, for the first time, that a low metastatic subclone can enhance lung metastasis of highly metastatic subclone via exosomal Wnt7a and propose Wnt7a as a molecular target to treat TNBC patients.
三阴性乳腺癌（TNBC）患者的预后通常比其他亚型差，因为其具有侵袭性。癌细胞是异质性的，只有少数高转移性亚克隆转移。尽管大多数亚克隆可能不会转移，但它们可能通过释放增加高转移性细胞能力的因子和/或提供有利的肿瘤微环境（TME）来做出贡献。在这里，我们使用多克隆鼠 4T1BC 模型分析了导致 TNBC 有效进展，特别是肺转移的克隆间通讯。
我们分离了两个具有低和高转移潜能的 4T1 亚克隆，LM.4T1 和 HM.4T1 细胞，并使用细胞划痕实验、球体形成实验、球体侵袭实验、RT-qPCR 和 Western blot 在体外研究了 LM.4T1 细胞对 HM.4T1 细胞行为的影响。我们还研究了 LM.4T1 细胞对 HM.4T1 细胞肺转移和体内 TME 的贡献。为了确定可能导致 LM.4T1 细胞作用的关键因素，我们分析了 GEO 数据库中获得的数据。
共注射 LM.4T1 细胞显著增强了 HM.4T1 细胞的肺转移。LM.4T1 衍生的外泌体促进了 HM.4T1 细胞的迁移和侵袭，阻断外泌体的分泌可消除其对 HM.4T1 细胞的作用。从 GEO 数据库中分析的数据表明，Wnt7a 可能是负责增强作用的关键因素。事实上，与 HM.4T1 细胞相比，LM.4T1 细胞，尤其是外泌体中的 Wnt7a 水平更高，并且在 LM.4T1 细胞中删除 Wnt7a 可显著降低 HM.4T1 细胞的肺转移。此外，Wnt7a 处理通过激活 PI3K/Akt/mTOR 信号通路增加了 HM.4T1 细胞的球体形成。最后，LM.4T1 细胞肿瘤中αSMA 阳性成纤维细胞和血管生成的浸润更为明显，而在 LM.4T1 细胞中删除 Wnt7a 则明显减少了血管生成。
我们首次证明低转移性亚克隆可以通过外泌体 Wnt7a 增强高转移性亚克隆的肺转移，并提出 Wnt7a 作为治疗三阴性乳腺癌患者的分子靶标。
