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Zhang T.,Cancer Diagnasis and Treatment Center | Li D.,Cancer Diagnasis and Treatment Center | Qi Z.,Cancer Diagnasis and Treatment Center | Peng J.,Cancer Diagnasis and Treatment Center | And 7 more authors.
Chinese Journal of Cancer Biotherapy | Year: 2013

Objective: To build a method for enrichment, culture and identification of a cell subset which has the characteristics of liver cancer stem cells (CSC) in vivo. Methods: Huh7 liver cancer cells were subjected to enrichment culture in spheric culture condition with cancer stem cell enrichment medium to obtain liver cancer stem cell-like cells. The stem cell-like cells were continuously cultured and proliferated to form liver cancer stem cell sphere in vitro. Flow cytometry assay was performed to detect the expression of the bio-marker moleculars of CSC such as EpCAM (also named CD326), CD90 and CD133. Colony formation assay and tumorigenicity experiments in nude mice were used to detect the colony formation abilities and tumorigenicity of Huh7 cells and Huh7 stem cell-like cells, respectively. Results: After enrichment culture for 3-7 days, Huh7 cells formed cell spheres. The liver cancer stem cell-like sphere-forming cells exhibited self-renewal ability and multiplication capacity. The proportion of EpCAM positive cells was significantly increased compared to uh7 cells ([ 99. 6 ±0. 31 J% vs [0. 12 ±0. 05 J%,P <0. 01). owever, there is no significant difference in the expression of CD90 ([0. 11% ±0.06]% vs[0.09% ±0.07],P>0.05) and CD 133 ([0. 17% ±0.08]% vs [0. 15% ±0. 05 \,P >0. 05) between the Huh7 cells and sphere-forming cells. The number of colonies formed by enriched sphere-forming cells was significantly more than that formed by Huh7 cells ([188. 67 ± 12. 5 J vs [79 ± 16. 7 J,P < 0. 01). Compared with the nude mice inoculation of Huh7 cells in the control group, tumor-forming time of nude mice noc-ulation of Huh7 stem cell-like cells in the experimental group was obviously shorter (11 vs 30 d), and showed a higher ratio of tumor formation(100% vs 16. 67%) when all the mice of two groups were seeded with 5 X 10 cells. Both tumor volume ([171.90 ±10.94] vs [86.39 ±11.21] mm3, P<0.01) and weight ([2. 98 ± 0. 82] vs [0.32 ±0. 17]g, P < 0. 01) of nude mice in the experimental group was respectively bigger and heavier than that of the control group when the seeding cell number was 5 × 10 cells. Conclusion: Liver cancer stem cell-like cells could be enriched and transformed from Huh7 cells with sphere-forming culture method in vitro, and these cells have more powerful tumorigenicity than Huh7 cells do.

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