Isolation of cancer stem cells from pancreatic cancer cell line and identification of their biological characteristics
10.3969/j.issn.1001-1978.2014.04.031
- VernacularTitle:胰腺癌干细胞的分离培养及生物学特性鉴定
- Author:
Junying ZENG
;
Xing HU
;
Xianjin WU
;
Shenghua LI
;
Jianhui PI
- Publication Type:Journal Article
- Keywords:
pancreatic cancer;
cancer stem cells;
CD24 +CD44 + ESA+;
RON;
epithelial-mesenchymal transition;
drug resistance
- From:
Chinese Pharmacological Bulletin
2014;(4):583-587
- CountryChina
- Language:Chinese
-
Abstract:
Aim To isolate cancer stem cells from human pan-creatic cancer cell line L3. 6pl and to identify their biological characteristics. Methods L3. 6pl cells were cultivated in com-mercial low adhesion plate with serum-free stem cell culture me-dium ( MEM/F12 1:1 ) supplemented with B27. The cancer stem cells reformed into floating spheres were isolated. The method of tumor sphere formation was used to isolate/enrich and characterize the cancer stem cells in pancreatic carcinoma cell line L3. 6pl. Cancer stem cell spheres were collected and sorted using magnetic cell sorting ( magnetic activated cell sorting, MACS) technology, with the cell surface markers of CD24 +CD44 + ESA+. Self-renewal and EMT-related oncogene expres-sion were measured with Western blot. Cancer stem cells differ-entiation potential and the expression of cancer stem cell related signs were checked with Immunofluorescence assay. To deter-mine tumorigenesis in vivo, Xenograft assay in NOD-SCID mice were performed respectively, then immunohistochemistry proto-oncogene c-Met and RON expression were also checked. West-ern blot was used to detect the changes of stemness relative tran-scriptional factors and epithelial markers expressed in spheres before and after differentiation. Drug resistance of pancreatic cancer stem cells to gemcitabine or paclitaxel was measured with MTT assay. Results CD24 + CD44 + ESA+ cells were signifi-cantly tumorigenic, and cultured in serum-free conditions to form spheroids, which had the characteristics of stem cells with self-renewal, EMT and drug-resistant capabilities, and had a posi-tive correlation with the c-Met, RON protein expression. Con-clusion Human pancreatic stem cells are successfully isolated, which provides a useful model for individualized therapy and e-valuation of the therapeutic efficacy for pancreatic cancer pa-tients.
