title: Survival advantages of multicellular spheroids vs. monolayers of HepG2 cells in vitro.
abstract: Mammalian cells grow in three-dimensions (3-D) in vivo. Commonly used two-dimensional (2-D) cell cultures are inadequate to recreate the biological microenvironment of tumor cells. The potentially different outcomes from 2-D and 3-D culture systems may have a significant impact on the relevance of experimental findings. The purpose of this study was to characterize the human hepatoma cell line HepG2 in 2-D and 3-D cultures. HepG2 cells in 2-D and 3-D cultures were treated with cisplatin, 5-fluorouracil, and adriamycin and were analyzed by scanning electron microscopy and transmission electron microscopy. Cell cycle progression and apoptosis were detected by flow cytometry. Inhibition of cell proliferation was quantified by MTT assay. The expression of E-cadherin, CD44v6, VEGF, KDR, endostatin, Bax, and cytochrome-c were analyzed by immunohistochemical (IHC) staining. As compared to the 2-D monolayer culture, the 3-D multicellular spheroids (MCS) of HepG2 cells featured a greater fraction of cells in G1 phase and were organized with more abundant cell-cell adhesion. In addition, cells in MCS were significantly less apoptotic in maintenance culture media and were more resistant to drug-induced apoptosis. E-cadherin, CD44v6, VEGF, KDR, endostatin, and cytochrome-c levels were increased in MCS as compared to 2-D cell cultures. In conclusion, MCS conferred differentiated phenotypes including increased cell-cell adhesion and G1 phase cell cycle arrest, enhanced cellular resistance to apoptosis, and upregulated angiogenic potential. Based on our data, a multicellular morphological hierarchy may sustain the growth/survival advantages of cancer cells in vivo. Therefore, a 3-D culture system should be the preferred technique for cancer biology investigation.
authors.affiliation: Department of Medical Oncology, The First Affiliated Hospital of The School of Medicine of Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, P.R. China. chunli5158@163.com
authors.forename: Chun-Li
authors.initials: CL
authors.lastname: Li
authors.forename: Tao
authors.initials: T
authors.lastname: Tian
authors.forename: Ke-Jun
authors.initials: KJ
authors.lastname: Nan
authors.forename: Na
authors.initials: N
authors.lastname: Zhao
authors.forename: Ya-Huan
authors.initials: YH
authors.lastname: Guo
authors.forename: Jie
authors.initials: J
authors.lastname: Cui
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Wang-Gang
authors.initials: WG
authors.lastname: Zhang
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D015820:Cadherins / Q000096:biosynthesis; D045744:Cell Line, Tumor; D002470:Cell Survival; D002945:Cisplatin / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D066298:In Vitro Techniques; D018874:Spheroids, Cellular / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis
chemical_list: D000970:Antineoplastic Agents; C112697:CD44v6 antigen; D015820:Cadherins; D018960:Hyaluronan Receptors; D013778:Tetrazolium Salts; D013844:Thiazoles; D042461:Vascular Endothelial Growth Factor A; D004317:Doxorubicin; D040301:Vascular Endothelial Growth Factor Receptor-2; C022616:thiazolyl blue; D002945:Cisplatin; D005472:Fluorouracil