[Cultivation and characterization of tumor spheres generated from human colorectal cancer cell lines].
OBJECTIVE
To study on the cultivation method for tumor spheres from colorectal cancer cell lines and identify whether resulting Colo205 spheroid cells display cancer stem cell characteristics.


METHODS
Lovo, Colo205 and SW480 cells were seeded in serum free medium (SFM) with EGF and bFGF. Flow cytometry analysis, cell invasion assay and xenograft experiment were applied to examine the cell surface marker expression pattern, cell invasive ability and in vivo tumorigenicity of both Colo205 spheres and parental cells. CD44 expression of tumor spheroid cells was also analyzed after cultured with serum supplemented medium by flow cytometry. CD44, Musashi-1 and Oct4 mRNA were detected in these two cells by RT-PCR.


RESULTS
Tumor spheres could be generated from three colorectal cancer cell lines in SFM. The formation and proliferation of tumor spheres were benefited from fresh SFM, cell dissociation reagent Accutase and the floating status of cancer cells. The overwhelming majority of spheroid cells were CD44+ cells. But CD44+ cells were gradually decreased when spheres cultured with serum supplemented medium. Colo205 spheres have higher Musashi-1 and Oct4 mRNA expression, tumor-initiating capability and invasive ability compared with those of parental cells.


CONCLUSIONS
Tumor spheres in which enrich cancer stem cells can be generated and matained from colorectal cancer cell lines in SFM on floating-culture condition.
Zhou
Ji-Tao
JT
Department of Abdominal Cancer, Cancer Center, West China Hospital, Sichuan University, Chengdu, China.
Gong
Ri-Xiang
RX
Chen
Xiang-Zheng
XZ
Zhou
Hai-Jun
HJ
Zhu
Ya-Jie
YJ
Liu
Su-Rui
SR
Huang
Juan
J
Bi
Feng
F
D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000473:pathology*; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000166:cytology* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism
D018960:Hyaluronan Receptors; C491297:MSI1 protein, human; D009419:Nerve Tissue Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins