Isolation And Characterization Of Human Hepatocellular Carcinoma HepG2 Heterogeneous Sublines With Different Metastatic Potentials

Objective: To isolate and characterize heterogeneous sublines from human hepatocellular carcinoma cell line (HepG-2), discuss the mechanism of HCC heterogeneity.Methods: (1) Heterogeneous sublines were isolated with limited dilution method from human hepatocellular carcinoma cell line (HepG-2) (2) The morphological character of these sublines were observed by inverted phase contrast microscope, transmission electron microscope, and scanning electron microscope. (3) The growth rate of these two sublines was examined by counting cell in vitro, and cell cycle and DNA content were investigated by flow cytometry. (4) The movability of these two sublines was examined by the agarose drop method. (5) The oncogenic and metastatic potential of these two heterogeneous sublines were studied by the subcutaneous transplantation model and the intra-spleen inoculation metastasizing into liver model respectively.Results: (1) The HepG2-D3 and HepG2-F4 heterogeneous sublines have been isolated and named from human hepatocellular carcinoma cell line (HepG2). (2) HepG2-F4 shows a sharp shape, smooth on its surface, and has many swelling deformed mitochondria in its cytoplasm. Whereas, HepG2-D3 has a multi-angle shape, abundant microvilli which form pseudopodia on its surface, and many lysosomes in its cytoplasm (3) When HepG2-F4 was compared with HepG2-D3, tumor cell doubling time was 18 hours vs. 26 hours, maximum proliferation power was 16.7 vs. 12.6, and proliferation cycle (S+G2M) was 53.7% vs. 37.6%. (4) The movability of HepG2-D3 is obviously higher than HepG2-F4(p < 0.01). (5) HepG2-F4 can grow in the subcutaneous of nude mice (3/3), but can't survive when transplanted in spleen (0/10). HepG2-D3 can survive and metastasize into liver diffusely (10/10) when transplanted in spleen. Conclusion: (1) Two heterogeneous sublines have been isolated from HepG-2 andshow different characters on their morphology, proliferation rate, and movability in vitro, et al. (2) HepG2-D3 has a higher metastatic potential, but HepG2-F4 can't metastasis in vivo. (3) The intra-spleen inoculation metastasizing into liver model can be used on screening higher metastatic potential hepatocellular carcinoma. (4) These two heterogeneous sublines may be valuable for further study on the molecular mechanisms of the heterogeneity and metastasis in hepatocellular carcinoma.