Orthotopic implantation of human hepatocellular carcinoma in mice: analysis of tumor progression and establishment of the BCLC-9 cell line.
OBJECTIVE
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.


METHODS
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 alpha-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.
Armengol
Carolina
C
BCLC Group, Liver Unit, Digestive Disease Institute, Hospital Clínic, Institut d'Investigació Biomèdica August Pi i Sunyer (IDIBAPS), Barcelona, Spain.
Tarafa
Gemma
G
Boix
Loreto
L
Solé
Manel
M
Queralt
Rosa
R
Costa
Dolors
D
Bachs
Oriol
O
Bruix
Jordi
J
Capellá
Gabriel
G
D000818:Animals; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D045744:Cell Line, Tumor; D002470:Cell Survival; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D018183:Transplantation Chimera; D014183:Transplantation, Heterologous