肝癌干细胞来源外泌体递送miR-140激活肿瘤相关巨噬细胞促进肝癌在肺内定植的机制研究

Circulating tumor cells can recruit and activate tumor-associated macrophages (TAMs) to create a suitable microenvironment for their colonization. However, the underlying mechanism of this process remains obscure. Our previous studies proved that malignant transformed liver stem cells, which were induced by Notch1 up-regulation (WBN1), possessed cancer stem cell properties and could generate poorly differentiated liver cancer in situ and developed lung metastases in an isogenic orthotopic rat model. Moreover, TAMs were recruited and activated by WBN1 cells to promote colonization of WBN1 cells in the lung. Our recent studies showed that WBN1-derived exosomal miR-140 were markedly increased compared with the control. NF-κB p65 signaling pathway was activated in TAMs that were co-cultured with WBN1 cells. Furthermore, secreted IL-6 of these TAMs was significantly increased and in turn promoted WBN1 cells proliferation and invasion. Given the above, we assumed that liver-cancer-stem-cell derived exosomes may deliver miR-140 to activate TAMs and then promote colonization of cancer cells in the lung. In this project, studies in vitro and in vivo will be performed and the expression levels of miR-140/NF-κB p65/IL-6 will be analyzed in serum exosomes and tissue samples from liver cancer patients with lung metastasis. Our goal is to reveal the mechanisms of liver-cancer-metastasis-to-lung and hopefully provides preclinical evidence for the treatment of such clinical settings.

循环肿瘤细胞可募集并激活肿瘤相关巨噬细胞为其最终在转移靶器官定植创造适宜的转移前微环境，机制未明。我们前期研究证实Notch1诱导恶性转化的肝干细胞（WBN1）具有癌干细胞特性，在大鼠肝脏可形成肝癌，且通过募集并激活肿瘤相关巨噬细胞促进其在肺内定植。进一步研究发现WBN1可分泌高表达miR-140的外泌体，激活巨噬细胞NF-κB p65通路增加IL-6分泌，进而促进WBN1增殖和侵袭。据此我们假设：肝癌干细胞来源外泌体递送miR-140激活肿瘤相关巨噬细胞促进肝癌在肺内定植。本项目拟①通过体内外实验研究WBN1外泌体miR-140对肿瘤相关巨噬细胞的激活作用及对肝癌肺内定植的促进作用，并验证NF-κB p65/IL-6调节途径；②分析肝癌肺转移患者血清外泌体及组织样本中miR-140、NF-κB p65和IL-6的表达及相关性。本研究旨在揭示肝癌肺转移的机制，为针对肝癌转移的治疗提供依据。

癌干细胞是恶性肿瘤发生、转移和复发的根源。本项目基于前期在成体肝干细胞研究的基础上，进一步探索了Notch1/外泌体miR-140在成体肝干细胞肿瘤发生、侵袭、转移中的作用。我们首先利用生物信息学方法分析TCGA、CEO数据库中Notch1在肝癌中的表达及预后作用，发现Notch1高表达与肝癌患者预后不良密切相关，且Notch1-c-myc信号轴调控细胞干性促进肝癌进展，并在我院240例肝癌组织中进一步验证；利用慢病毒稳定上调大鼠成体肝干细胞WB-F344中Notch1的表达后，经过选择性培养后恶性转化为肿瘤干细胞样细胞，表达CD44、CD133等干细胞标记，敲低c-myc可以抑制Notch1驱动的肿瘤干细胞样特性。Notch1介导恶性转化的大鼠成体肝干细胞具有极强的远处转移能力，在同基因型大鼠肝脏原位接种后形成低分化肝癌，呈现出间质样的细胞表型，并发生肺转移，而利用氯磷酸盐清除体内的巨噬细胞可以显著抑制肺转移的发生。进一步研究发现，肿瘤来源外泌体可携带miR-140传递至巨噬细胞，通过下调SIRT-1，激活NF-κB信号通路，表达IL-6、IL-10等M2型巨噬细胞标记，使其向肿瘤相关巨噬细胞转化；被激活的肿瘤相关巨噬细胞可反过来促进肿瘤的侵袭与转移。我们的结果表明Notch1可调控肝成体干细胞发生恶性转化，并维持肿瘤干细胞自我更新，且通过外泌体miR-140调控巨噬细胞表型，增强肿瘤肺转移。Notch1/miR-140信号轴可作为抑制肝癌肺转移的潜在治疗靶标。此外，我们对高/低转移潜能肝癌干细胞来源外泌体调节巨噬细胞功能与机制的进一步探讨，并对肝癌纳米靶向载体进行了一系列研究。外泌体调节巨噬细胞重塑转移前免疫微环境是我们下一步研究的方向。