Establishment And Characterization Of Human Hepatocellular Carcinoma Cell Lines Derived From Portal Vein Tumor Thrombus (PVTT)

BackgroundHepatocellular carcinoma (HCC) is the second most common cause of death from cancer in China. Generally speaking, although great progress has been made in its diagnosis and treatments due to the advances in early detection and surgical technique, the overall prognosis for HCC patients remains dismal because most of these patients also have early portal vein invasion at the first time of diagnosis. Metastasis such as portal vein tumor thrombus (PVTT) remains one of the major challenges before hepatocellular carcinoma is finally conquered. Many studies show that portal vein tumor thrombus metastasis not only lies in final liver cancer patients but exists in the early stages liver cancer patients. A gradual increase in survival of liver cancer patients will not be occured unless PVTT would treat appropriately. At present, there is no cell line which has some characteristics of PVTT. Therefore, a new cell line derived from portal vein tumor thrombus is important for the study of PVTT.ObjectiveTo establish a cell line derived from portal vein tumor thrombus and study its biology characteristics.Methods1. Establishment of HCC cell line derived from human portal vein tumor thrombusThese tissues were enzymatic dissociated by collagenase and inoculated in vitro respectively, The single HCC cell were obtained by natural purification and anchoring repeatedly. The cells were passaged with trypsin. Growth status was observed under light microscope. The cells were freezed with temperature down slowly in liquid nitrogen and resuscitated quickly at 37℃.2. Characteristics of human HCC cell line CSQT-1 CSQT-1 cell morphology was observed under light and electron microscope. The survival rate was measured by counting cells stained with protamine blue. Cell adhesion rate was measured by counting cell number at 15min,30min,1h,2h and 4h.Cell growth curve was plotted with MTT assay. Studies of cell cycle and stage were performed by flow-cytometry. Chromosomes was made by traditional method and analyzed by photographed. Cloning efficiency was measured by counting clone number growing in plate. Expressions of tumor markers such as AFP and CD133 were detected by Eliza or flow-cytometry. Xenograft was performed by inoculating CSQT-1 cells into the flanks of the nude mice s. c. and the tumors were observed, thus Xenograft rate was counted. Cell contamination was detected by transmission electron microscopy.3. To construct a nude mice model for PVTT and establish a new HCC cell line from xenograft in nude miceThe human PVTT tissues were implantated into liver subcapsule of nude mice, when it reached 1.5-2.0cm in diameter and was passaged in the same way in other nude mice to construct SOI (Surgical Orthotopic Implantation) models. Xenograft in the liver was obtained after 5 passages and inoculated into a plate after dissociated by collagenase I to establish a new cell line CSQT-2. The liver of the nude mice were embedded by paraffin to detect micro-PVTT by HE stain.Results1. Cells grew instably at the early stage of primary culture and needed a high density when passaged. Population doubling time was not the same. But the cells grew very stably after 15th passage. Cells were mainly polygonal epithelial like invarious size over 90%. Cells grew in overlap and piled up in high density(>2×106/mL). Passaging time was 2-3 days average.2. Transmission electron microscopy showed cells were in various size, mainly irregular type. There were abundant microvilli on the cell surface. Desmosomes and gap junction could be seen between cells, also with a few junctional complexs. There was a tendency to form structure of gland and strand alike. Strueture 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. The survival rate was 95% after resuscitating cells freezed in liquid nitrogen. Cell growth curve showed biological characteristics of the common malignant epithelial tumor. Population doubling time was 48hours.The cells adhension rate was over 90% after 4 hours growing in the plate, and its cloning efficieney was 20%. The cell DNA was Tetraploid and 64.0% cells were in G1/G0 stage,19.0% G2/M,10.0% S. Chromosomes was mainly Tetraplont. The AFP was negative. CSQT-1 has the ability of tumorigenicity to nude mice. No mycoplasma contamination was detected.3. A reproducible nude mice model for PVTT was established by SOI. A new HCC cell line CSQT-2 was established and micro-PVTT was found in the liver of the nude mice by HE stain.Conclusion1. CSQT-1 cell line which was from human portal vein tumor thrombus tissue can grow stably and has ability to proliferate immortally. The cell line keeps some characteristics of its original tissue.AFP and HBsAg are negative. There is a tendeney to form structure of gland and strip alike. Structure of bile canaliculus alike is formed.2. CSQT-1 and CSQT-2 has its own charaeteristics compared with other HCC cell lines. The difference are shown as: the first prominent characteristic of the cell line is that it derived from portal vein tumor thrombus tissue, the second one is its rapid growth,fast attachment and shorter doubling time; the third one lies in that AFP and HBsAg are negative,while AFP and HBsAg are positive in most of other HCC cell lines; the fourth one is its favorable tumorigenesity and the transplanted tumors grow larger with time; the fifth one is that the cell line has a biological propertics of high concordance and little variation in different passages with no obvious variation of its proportion of CD133 positive cells at different passages; the sixth one is that cell lines from PVTT have ability of formation micro-PVTT in nude mice. Therefore, the CSQT-1 and CSQT-2 are novel HCC cell lines and they will be a valuable model for the studies of PVTT in vitro and in vivo,especially for the researchs of correlation between CD133 positive related tumor stem cell with PVTT formation.Potential applications and noveltyHuman hepatocellular carcinoma cell lines derived from human portal vein tumor thrombus have been established, which provided an ideal models and platform for the study on formation mechanisms of PVTT.