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Orthotopic Implantation of Human Hepatocellular Carcinoma in Mice

Analysis of Tumor Progression and Establishment of the BCLC-9 Cell Line

¹ BCLC Group, Liver Unit, Digestive Disease Institute, Hospital Clínic, Institut d’Investigació Biomèdica August Pi i Sunyer (IDIBAPS), ² Laboratory of Pathology, Hospital Clínic, IDIBAPS, and ³ Cell Biology and Pathology Department, Faculty of Medicine, IDIBAPS, University of Barcelona, Barcelona, Spain; ⁴ Laboratory of Translational Research, Institut Català d’Oncologia, L’Hospitalet del Llobregat, Barcelona, Spain; and ⁵ Genetic Service and ⁶ Hematopathology Unit, Hospital Clínic, Barcelona, Spain

Purpose: To allow the longitudinal investigation of molecular events associated with the progression of human hepatocellular carcinoma (HCC), we sought to develop a murine model by orthotopic implantation of tumor fragments obtained from patients diagnosed at early stage.

Experimental Design: Tumor pieces (2 x 2 mm) were implanted on the liver surface of nu/nu mice. After xenograft growing, subsequent passages were performed to achieve long-term implant viability. Isolation of tumoral hepatocytes was done to establish new cell lines. HCC characteristics, proliferation rate, apoptotic index (terminal deoxynucleotidyl transferase-mediated nick end labeling), and expression of cell-cycle regulators (cyclins E and A, p21^Cip1, p27^Kip1, p16^INK4a, pRb, and p53) were assessed by Western Blot and immunohistochemistry, to correlate them with tumor progression.

Results: Five (50%) of the 10 primary HCCs resulted in small slow-growing liver implants. Three of them are viable after 48 months, whereas the remaining two survived for 15 and 13 months. Xenografts throughout passages exhibited a more aggressive phenotype with a poorer degree of differentiation, intense proliferation, moderate apoptosis, cell-cycle deregulation, p53 alterations, microvascular invasion, and dissemination. In one single passage, we observed critical growth delay, which was associated with significant p27^kip1 overexpression. We established the anchor-free growing BCLC-9 cell line from one xenograft. This has gains of chromosomes 7, 5p, 6q, and 9q, is hepatitis B virus-DNA positive, does not secrete -fetoprotein, and has TP53 missense mutations in codons 192 and 242.

Conclusions: The orthotopic implantation of early HCC fragments in nude mice provides a useful model to investigate the mechanisms of human HCC evolution and to establish new cell lines.