Notch1上调CCL2促进肝癌干细胞在肺内定植的研究

There are three stages for circulating tumor cells forming tumor masses in target organs - extravasation, micrometastasis and colonization. Circulating tumor cells can recruit and activate tumor-associated macrophages (TAMs) to create a suitable microenvironment for their colonization. Our previous studies proved that malignant transformed liver stem cells, which were induced by Notch1 up-regulation (WBN1), generate poorly differentiated liver cancer and developed lung metastases in an unique isogenic orthotopic rat model. Moreover, TAMs were recruited and activated by WBN1 to help them extravasation. However, the underlying mechanism of the final stage, colonization, remains obscure. Our further studies showed that Notch1 could up-regulate the expression of CCL2, a known chemokine factor that contributes lung metastasis of breast cancer by recruiting and activating TAMs. Given the above, we assumed that Notch1 could up-regulate CCL2 to activate TAMs and then promoting colonization of liver cancer stem cells in the lung. In this project, gain/loss of function studies of Notch1/CCL2 in vitro and in vivo, especially in the unique liver cancer in situ/lung metastasis rat model, will be performed. This study can reveal the mechanisms of liver-cancer-metastasis-to-lung and hopefully provides preclinical evidence for the treatment of such clinical settings.

循环肿瘤细胞必须经过血管外渗、形成微转移和定植三个阶段才可在靶器官形成转移灶，在定植阶段肿瘤细胞可募集并激活肿瘤相关巨噬细胞为其定植创造适宜的转移前微环境。我们前期研究发现：Notch1诱导恶性转化的肝干细胞（肝癌干细胞WBN1）在大鼠肝脏可形成肝癌，且均伴肺转移；WBN1可通过募集并激活肿瘤相关巨噬细胞促进其自身外渗出血管。然而，WBN1外渗出血管后在肺定植的机制是什么？进一步实验发现，Notch1可上调趋化因子CCL2的表达。已知CCL2可通过激活肿瘤相关巨噬细胞促进乳腺癌细胞在肺内定植；据此我们假设：Notch1可上调CCL2激活肿瘤相关巨噬细胞促进肝癌干细胞在肺内定植。本项目拟通过慢病毒载体调控Notch1/CCL2的表达及肝癌肺转移动物模型、体外细胞实验深入探讨Notch1是否通过上调CCL2促进肝癌干细胞在肺内定植。本研究有助于揭示肝癌肺转移的机制，为针对肝癌转移的治疗提供依据

循环肿瘤细胞必须经过血管外渗、形成微转移和定植三个阶段才可在靶器官形成转移灶，在定植阶段肿瘤细胞可募集并激活肿瘤相关巨噬细胞为其定植创造适宜的转移前微环境。本项目是在前期“Notch1诱导恶性转化的肝干细胞（肝癌干细胞WBN1）在大鼠肝脏可形成肝癌，且均伴肺转移”的研究基础上，进一步探索“Notch1是否通过上调CCL2激活肿瘤相关巨噬细胞促进肝癌干细胞在肺内定植”。我们首先通过荷瘤动物实验证实沉默Notch1/CCL2可减少具有高转移能力的肝癌（干）细胞肺转移发生；然后通过体外实验证实Notch1/CCL2可激活肿瘤相关巨噬细胞进而促进肝癌（干）细胞增殖、侵袭、逃避凋亡等；最后通过临床样本研究证实，在肝癌组织中Notch1的表达与肿瘤体积、TNM分期、肺转移率及患者预后密切相关。此外我们还发现，癌基因c-myc及血管细胞粘附因子-1（VCAM1）在上述过程中也发挥重要作用。上述结果表明，Notch1、CCL2以及肿瘤相关巨噬细胞在促进肝癌（干）细胞在肺内定植进而形成肺转移过程中发挥重要作用。上述成果利于我们更好的理解肝癌肺转移的不同阶段、不同步骤以及在相关过程中起关键作用的基因、信号通路及上下游调控机制，有助于揭示肝癌肺转移的机制，为针对肝癌肺转移的治疗提供实验依据。