肝癌循环肿瘤细胞异质性及其转移复发的相关性研究

Circulating tumor cells (CTCs) are the cells in peripheral blood that detached from the primary or metastasis cancer lesions, which have long been considered a reflection of tumor aggressiveness. Our previous studies have firstly found that remaining cells enriched from lung cancers, ovarian cancers and pancreatic cancers could be classified into 5 patterns by using simultaneously immunohistochemical staining of CK and CD45 and fluorescence in situ hybridization with the centromere of chromosome 8 (CEP8) probe method we established: CK+CD45-DAPI+CEP8=2, CK+CD45-DAPI+CEP8>2, CK-CD45-DAPI+ CE P8>2, CK-CD45-DAPI+CEP8=2, and CK+/-CD45+DAPI+CEP8=2 or >2. Patterns of CK+CD45-DAPI+CEP8=2, CK+CD45-DAPI+CEP8>2 and CK-CD45-DAPI+CEP8>2 were considered as CTCs, and CK-CD45-CEP8=2 and CK+/-CD45+DAPI+CEP8=2 or >2 were considered as indeterminate cells. Moreover, CTCs positive I-II phase pancreatic cancer patients showed metastasis and worse survival rate. Hence, we will further explore the relationship between CTCs heterogeneity and tumor aggressiveness in hepatocellular carcinoma by using the same methods. CTCs identified were then collected by laser capture micro-dissection followed by whole genome amplification to explore tumor markers for tumor metastasis and recurrence, which may supply a new judgment for its early diagnosis and prognosis evaluation.

循环肿瘤细胞（Circulating tumor cells, CTCs）由原发或转移灶脱落进入外周血的过程中发生上皮间质转化，能够反映肿瘤是否发生侵袭转移。前期我们首次通过建立免疫荧光化学染色联合荧光原位杂交技术鉴定CTCs，发现肺癌、卵巢癌及胰腺癌患者外周血CTCs具有异质性，可分为五个亚群：CK+CD45-CEP8=2、CK+CD45-CEP8＞2、CK-CD45-CEP8＞2、CK-CD45-CEP8=2、CK+/-CD45+CEP8=2/＞2，且I-II期异质性表型阳性的胰腺癌患者较阴性者预后差，推测异质性CTCs与疾病的恶性进展有关。本课题将进一步采用此方法探讨肝癌患者外周血中CTCs的异质性，并通过激光显微切割分离纯化单个亚群细胞并全基因组扩增分析异质性CTCs与肝癌转移复发相关的分子表达水平，以期从新角度解析肝癌的发生发展转移，同时也为肝癌早期诊断提供参考依据。

循环肿瘤细胞的异质性与肿瘤的侵袭转移有关，因此本研究通过CD45-CEP1/8法鉴定肝癌循环肿瘤细胞，证实肝癌患者外周血存在异质性CTCs，且同一患者术后CTCs较术前CTCs肿瘤负荷量明显减低。在胃癌、结直肠癌患者中，我们在原有FISH检测的基础上进一步引入间质性标志物vimentin将CTCs分为7个亚群，FISH+/CK-/vimentin-、FISH+/CK+/vimentin-、FISH+/CK-/vimentin+、FISH-/CK+/vimentin+、FISH-/CK+/vimentin-、FISH-/CK-/vimentin+、FISH+/CK+/vimentin+。结果发现胃癌、结直肠癌患者外周血均存在上皮性、上皮间质转化（EMT）以及间质性的异质性CTCs。通过分析各亚群细胞与患者肿瘤侵袭的关系，尚未证实哪一个亚群与患者肿瘤进展有关。单细胞检测技术突破有望明确异质性CTCs的分子分型以及疾病进展的分子机制提供了新的参考指标，也有望为寻找新的治疗靶点带来启发。