title: [Isolation of gastric cancer stem cells based on colony morphology and detection of their sensitivity to 5-fluorouracil].
abstract: OBJECTIVE
  To investigate the sensitivity of gastric cancer stem cells (GCSCs) to 5-fluorouracil (5-FU), and to explore the cytological mechanism of drug-resistance of gastric cancer.
  
  
  METHODS
  Immunohistochemical staining was employed to examine expression of stem cell marker CD44 and drug-resistance protein thymidylate synthase (TS) in 71 cases with gastric cancer. Based on morphology of cell colonies derived from AGS gastric cell line, colonies consisting of GCSCs were isolated, and expression of CD44 and TS, as well as self-renewal capacity of GCSCs were detected. Sensitivity of GCSCs to 5-FU was examined through CCK-8 assay.
  
  
  RESULTS
  The positive rates of CD44 and TS were 59.2% (42/71) and 56.3% (40/71) in gastric cancer, and expression of CD44 was associated with that of TS (χ(2)=12.76, P<0.01; Kappa=0.41). Serial sections indicated that CD44+ cancer cells simultaneously expressed TS. AGS developed morphologically diverse colonies, and the GCSCs colonies exhibited a tight and regular shape, which were called holoclone. Holoclones expressed CD44 and TS strongly, possessing capacity of robust self-renewal and forming a lot of second passage clones after incubation. Subclones expressed CD44 and TS weakly, forming less second passage clones. Paraclones almost did not express CD44 and TS, forming no second passage clone after incubation. Affected by 5-Fu, three holoclones showed less growth inhibition compared with another colony type and wild-type AGS cells. Furthermore, IC50 of 5-FU for three holoclones was (113.43±5.81), (272.68±25.75) and (118.14±17.75) μmol/L respectively, which were significantly higher than that of one subcolony type [(16.97±1.01) μmol/L] and AGS cells [(27.52±0.59) μmol/L] (all P<0.05).
  
  
  CONCLUSIONS
  GCSCs possess lower sensitivity to 5-FU, and may play a critical role in drug-resistance of gastric cancer.
authors.affiliation: Department of Gastroenterology, 101 Hospital of PLA, Wuxi, China.
authors.forename: Zhi-hua
authors.initials: ZH
authors.lastname: Zhou
authors.forename: Jian-dong
authors.initials: JD
authors.lastname: Zhang
authors.forename: Gui-fang
authors.initials: GF
authors.lastname: Xu
authors.forename: Hai-bin
authors.initials: HB
authors.lastname: Zhao
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D019008:Drug Resistance, Neoplasm; D005260:Female; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000473:pathology*; D013274:Stomach Neoplasms / Q000473:pathology*; D014407:Tumor Cells, Cultured; D055815:Young Adult
chemical_list: D005472:Fluorouracil