[Establishment of human hepatocellular carcinoma cell line with spontaneous pulmonary metastasis through in vivo selection].
OBJECTIVE
To establish a hepatocellular carcinoma (HCC) cell line from lung metastatic lesions of human HCC in nude mice so as to provide suitable model for the study of lung-metastasis-related molecular mechanisms.


METHODS
HCC clone cells MHCC97-H were inoculated subcutaneously into nude mice. The pulmonary metastatic lesions were harvested from the moribund animals and then re-implanted into the nude mice for the second round of selection. The same procedure was repeated twice. New cell line from the third round of lung metastasis was thus established and the following parameters were studied: morphology, in vitro growth curve, plate efficiency, migration, karyotype, flow cytometry, immunocytochemistry for AFP, albumin and cytokeratin 8 (CK8), flourescent PCR for HBV DNA, tumorigenicity and spontaneous metastasis via subcutaneous injection and orthotopic implantation of tumor tissue.


RESULTS
The cell line established from nude mouse lung metastasis was designated as HCCLM3, consisting of polygonal epithelial cells with hypotriploid karyotype, its main range of chromosome being 55 - 58. The cell population doubling time was 34.9 hours, plate efficiency 32.4 +/- 3.2%, and cell migration rate 20 +/- 2 microm/h. The cells were positive for AFP, albumin, CK8, and P16 and negative for P53, nm23 and HBsAg. Fluorescent PCR showed HBV DNA integration into cellular genome. When 5 x 10(6) cells were injected subcutaneously into nude mice, the tumorigenicity was 100% with a latency period of 11 +/- 1 d. Five weeks after subcutaneous injection, the pulmonary metastatic rate was 100%, the median number of lung metastases being 121 per mouse. After orthotopic implantation of tumor tissue into nude mouse liver for 35 d, wide spread regional and distant metastases occurred, the metastatic rate was 100% for abdominal wall, 80% for organs in abdominal cavity, 100% for liver, 70% for diaphragm, and 100% for lung. The median number of lung metastatic lesions was 268 per mouse.


CONCLUSIONS
A new HCC cell line has been established, which is characterized by high pulmonary metastasis via both subcutaneous and orthotopic inoculation. It provides a new model for the study of liver cancer metastasis.
Liver Cancer Institute and Zhong Shan Hospital of Fudan University, Shanghai, 200032, China.
Yan
Y
Li
Zhaoyou
Z
Tang
Shenglong
S
Ye
Yinkun
Y
Liu
Jie
J
Chen
Qiong
Q
Xue
Xiaowu
X
Huang
Jun
J
Chen
Weihua
W
Bao
Jiong
J
Yang
Dongmei
D
Gao
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000473:pathology* / Q000556:secondary; D018929:Cell Culture Techniques / Q000379:methods; D004195:Disease Models, Animal; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology*; D008175:Lung Neoplasms / Q000556:secondary*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D014407:Tumor Cells, Cultured