Isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma.
OBJECTIVE
To explore the method of isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma cell line PANC-1.


METHODS
The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 (1:1 volume) (DMEM-F12) supplemented with 20% fetal bovine serum (FBS). Subpopulation cells with properties of tumor stem cells were isolated from pancreatic adenocarcinoma cell line PANC-1 according to the cell surface markers CD44 and CD24 by flow cytometry. The proliferative capability of these cells in vitro were estimated by 3-[4,5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) method. And the tumor growth of different subpopulation cells which were injected into the hypodermisof right and left armpit of nude mice was studied, and expression of CD44 and CD24 of the CD44(+)CD24(+) cell-formed nodules and PANC-1 cells were detected by avidin-biotin-peroxidase complex (ABC) immunohistochemical staining.


RESULTS
The 5.1%-17.5% of sorted PANC-1 cells expressed the cell surface marker CD44, 57.8% -70.1% expressed CD24, only 2.1%-3.5% of cells were CD44(+) CD24(+). Compared with CD44(-)CD24(-) cells, CD44(+)CD24(+) cells had a lower growth rate in vitro. Implantation of 10(4) CD44(-)CD24(-) cells in nude mice showed no evident tumor growth at wk 12. In contrast, large tumors were found in nude mice implanted with 10(3) CD44(+)CD24(+) cells at wk 4 (2/8), a 20-fold increase in tumorigenic potential (P < 0.05 or P < 0.01). There was no obvious histological difference between the cells of the CD44(+)CD24(+) cell-formed nodules and PANC-1 cells.


CONCLUSIONS
CD44 and CD24 may be used as the cell surface markers for isolation of pancreatic cancer stem cells from pancreatic adenocarcinoma cell line PANC-1. Subpopulation cells CD44(+)CD24(+) have properties of tumor stem cells. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, it may be a very promising target for new drug development.
Huang
Peng
P
Department of Pancreatic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province, Wuhan, China. hpeng2003@sina.com
Wang
Chun-You
CY
Gou
Shan-Miao
SM
Wu
He-Shui
HS
Liu
Tao
T
Xiong
Jiang-Xin
JX
D000230:Adenocarcinoma / Q000276:immunology / Q000473:pathology*; D000818:Animals; D051927:CD24 Antigen / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000276:immunology / Q000473:pathology*; D010190:Pancreatic Neoplasms / Q000276:immunology / Q000473:pathology*; D013778:Tetrazolium Salts; D013844:Thiazoles; D014183:Transplantation, Heterologous
D051927:CD24 Antigen; D018960:Hyaluronan Receptors; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue