基于脂代谢信号通路重编程鉴定CTCs用于肝癌早期诊断的研究

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. More than 50% of new cases of HCC in the world are in China every year. Early diagnosis and monitoring of recurrence and metastasis are particularly important for improving the 5-year survival rate of HCC. The forming of circulating tumor cells (CTCs) which are liquid biopsies representing the primary tumor, is a necessary step for the metastasis of malignant tumors through the bloodstream, and also a marker for monitoring minimal residual disease after tumor treatment. Studies have shown that peripheral blood CTCs counts are closely related to the condition of HCC patients, cancer staging and prognosis of patients. However, currently, most CTCs enrichment and separation platforms only distinguish CTCs from blood cells by cell physical characteristics or use several types of cell surface markers to detect CTCs subpopulations. It is difficult to identify functional CTCs which are stable presence in the blood circulation system and form metastases in the distance. In recent years, as one of the important features of tumors, the relationship between abnormal lipid metabolism and tumorigenesis, development, invasion and metastasis has been gradually revealed. The liver acts as an important organ of lipid metabolism, and abnormal metabolic changes are throughout the liver lesions. This study intends to study the lipid metabolism signaling pathway of CTCs in HCC and find a subset of CTCs with abnormal lipid metabolism, which provides a new way to study the mechanism of recurrence and metastasis of HCC, and explores the potential value in clinical application of reprogramming of lipid metabolism in CTCs as an early diagnosis marker for HCC recurrence and metastasis.

肝癌是我国高发的恶性肿瘤之一，每年全球超过50%的肝癌新发病例在我国。对其进行早期诊断和监测复发与转移，对提高肝癌5年生存率尤为重要。循环肿瘤细胞(CTCs)作为一种代表原发肿瘤的液体活检标本，是恶性肿瘤经血道转移的必经步骤，也是监测肿瘤治疗后微小残留病变的标志物。然而，当前大多数CTCs富集和分离平台只通过细胞物理学特性将CTCs从血液细胞中区分或利用几类细胞表面标志物对CTCs亚群进行检测，很难鉴定出能够稳定存在于血液循环系统并在远处形成转移灶的有功能的CTCs。近年来，作为肿瘤重要特征之一，脂代谢异常与肿瘤发生、发展、侵袭和转移的关系被逐渐揭示。本研究拟通过对肝癌CTCs脂代谢信号通路的研究，发现脂代谢异常的CTCs亚群，为建立新的CTCs鉴定方法和研究肝癌复发转移机制提供思路，并探讨CTCs脂代谢重编程作为肝癌复发和转移早期诊断标志的临床应用价值。

循环肿瘤细胞（CTCs）被认为是肿瘤转移和微小残留癌灶的前驱事件，然而并非全部脱落入血的CTCs可以在外周血循环过程中存活并成功形成转移灶。过去常规用于分离CTCs的标志物主要靶向细胞上皮和间质表型，考虑到脂代谢异常在原发性肝癌（HCC）恶性进程中的重要作用，本研究提供了一种通过靶向肝脏特异性脂质代谢标志物组合来鉴定功能性HCC-CTC的新方法,并初步探讨了脂代谢异常CTCs的检出对肝癌预后诊断的临床应用价值。本研究：①共收集临床样本89例，其中包括30例健康外周血样本作为对照组样本，与HCC细胞系比较鉴定了肝癌细胞在脂类物质代谢和信号转导通路中的差异化表达基因；②基于多个数据库进一步筛选了差异化表达基因在外周血白细胞亚型、各类肿瘤细胞系、肝癌组织以及正常组织样本中的表达情况，并最终确立了两种肝脏特异性脂代谢标志物（载脂蛋白B，APOB和白蛋白，ALB）和白细胞标志物（C型蛋白酪氨酸磷酸酶受体，CD45）的多标志物组合用以鉴定HCC-CTCs；③利用原位杂交滚环扩增技术结合共聚焦显微镜评估对样本进行可视化检测；④考虑到CTCs在外周血的痕量存在，设计靶向多序列的多组挂锁探针以放大检测信号，优化了研究方案；⑤对34例HCC样本和25例肝硬化样本中脂代谢标志物阳性CTCs进行了量化和表征。研究结果表明：①靶向APOB、ALB和CD45的多标记和多组探针引物组合可用于鉴定外周血中HCC细胞；②53％（18/34）的HCC患者样本被检出APOB和/或ALB的脂代谢异常CTC（CTC数目：1-7个）；③在18例CTCs阳性的患者中，4例样本被检出有CTC细胞团簇，这4例患者中，3例发生了肝癌远处转移，1例为肝癌切除术后复发；④25例肝脏良性疾病患者外周血样本中未检测到APOB和/或ALB阳性细胞；⑤2年随访资料显示，34例肝癌患者中共有16例因肝癌死亡，这16例患者中有14例检出脂代谢异常CTCs，占全部脂代谢异常CTC阳性样本的78%（14/18），初步提示脂质代谢标志物阳性CTCs的检出与肝癌患者预后不良这一事件有关。