Characterisation Of A Novel Cell Line(CSQT-2) With High Metastatic Activity Derived From Portal Vein Tumour Thrombus Of Hepatocellular Carcinoma

[Introduction]Hepatocellular carcinoma (HCC) is one of the most common cancers in China. Portal vein tumour thrombus (PVTT), the characteristic of HCC, is highly associated with the progression and metastasis of HCC. Although great progress has been made in its 5-year survival rate due to the advances in early detection and surgical technique, the 1-year survival rate of PVTT is still low, with a higher metastasis and recurrence rate. Recently, more and more hypothesis of PVTT were rised by different groups of scientists, but with these still couldn't make a reasonable explanation to the mechanism of PVTT, which is due to that there is no method to study the biological and physiological characteristics of this disease in lack of appropriate cell model. Therefore, a new cell line derived from portal vein tumor thrombus is important for the study of PVTT.[Aims]In order to study the mechanism of PVTT, we planned to establish a cell line derived from portal vein tumour thrombus of hepatocellular carcinoma. Moreover, we would investigate the basic characterizations of this cell line, including migration, metastasis etc.[Methods]1. Establishment of PVTT cell line (CSQT-2) derived from human portal vein tumor thrombus.In order to establish a PVTT cell line derived from human portal vein tumor thrombus, we take advantage of series of methods, such as (Surgical Orthotopic Implantation of Histologically Intact Tumor Tissue) SOI and so on.2. Characteristics of human PVTT cell line CSQT-2In order to study the Characteristics of human PVTT cell line CSQT-2, we take advantage of different assays of in vitro and in vivo methods, such as CSQT-2 cell morphology was observed under light and electron microscope. Cell growth curve was plotted with MTT assay. Studies of cell cycle and stage were performed by flow-cytometry. Xenograft was performed by inoculating CSQT-2 cells into the flanks of the nude mice. IVIS was used to detecte the metastasis of CSQT-2.[Results]A successive cell line named CSQT-2 was established with the methods of SOI and so on. The cell line exhibited aggressive phenotypes in terms of cell growth, survival, migration, xenograft and metastasis. Transmission electron microscopy showed cells were in various sizes, mainly irregular type. There were abundant microvilli on the cell surface. Desmosomes and gap junction could be observed among cells, also with a few junctional complexs. There was a tendency to form structure of gland and strand alike. Structure of bile canaliculus alike was formed. Glycogen granules were rich in cytoplasm and formed lake-shaped while mitochondria and rough endoplasmic reticulum were moderate. Dissociated ribosome was rich. Cell growth curve showed biological characteristics of the common malignant epithelial tumor. Population doubling time was 48 hours. The cell DNA was Tetraploid and 51.51% cells were in G0/G1 stage,13.82% G2/M,34.67% S. In nude mice, CSQT-2 cells showed stronger subcutaneous tumourigenecity ability, tumors could be formed with a thousand cells in 6 weeks. Moreover, an orthotopic transplantation assay demonstrated that PVTT can be generated in nude mice when CSQT-2 cells were inoculated in the liver. The expression of CD133, CD90 and EpCAM were tested via FACS in both P5 and P60 generation of CSQT-2. It was found that in the early passages,8.1% of P5 expressed CD133, and 4.1% CD90, however, none of that could be detected in P60. In contrast to CD 133 and CD90 expression, EpCAM positivity in CSQT-2 cells dramatically increased with more passages, from 24.8% to 99.8%.[Conclusion]1. CSQT-2 cell line established with the methods of SOI etc. can grow stably and proliferate immortally.2. CSQT-2 has its own charaeteristics compared with other HCC cell lines. First prominent of the cell line is its rapid growth, fast attachment and shorter doubling time; second characteristic is its favorable tumorigenesity. In nude mice, CSQT-2 cells showed stronger subcutaneous tumourigenecity ability. The third one lies in that PVTT can be generated in nude mice when CSQT-2 cells were inoculated in the liver and that it exhibits a typical migratory tendency in the vascular branches of portal vein. The fourth one is that CSQT-2 owned its characteristics in expression of tumor markers. Therefore, CSQT-2 is a novel cell line with high metastatic activity derived from human portal vein tumor thrombus and it may provide a suitable model with which to investigate the molecular mechanisms of PVTT-related HCC.