Isolation Of Gastric Cancer Stem Cells From Free Cancer Cells In Peritoneal Washes And Its Significance

[Background]:Cancer stem cells are reported to exist in most tissues, including the brain,bone marrow,colon etc.They are thought to possess both the self-renewal capacity andasymmetrical division capacity to generate progenitor cells which differentiate into other cells. Only CSCs are able to sustain and propagate the tumors and give rise to invasive lesions and metastase.The microenvironmental stem cell compartment is termed the niche and it maintains optimal conditions for cells with stem cell capacity. Due to improvements in biotechnology it is now possible to stain and sort CSCs expressing specific surface markers. This is usually done via fluorescence activated cell sorting or magnetic bead isolation.However, these traditional experiment includes enzymatic digestion and machinery isolation which may disrupt the ideal local environment for the growth of cancer cells. Objective:first to isolate CD44positive cells from a human gastric cancer cell line AGS by magetic activated cell sorting and culture in serum free medium in vitro.then to isolate the gastric cancer stem cells from free cancer cells in peritoneal washes. Methods:In the first part, the CD44positive cells were isolated from AGS by magetic activated cell sorting,then were cultured in serum free medium with epidermal growth factor(EGF), basic fibroblast growth factor(bFGF)to obtain tumor cell spheres in vitro. In the Second part,45patients with gastric cancer were devided into two groups according to peritoneal metastasis.The expressions of CD44and CK20were examined by flow cytometry assay and immunohistochemical staining. Resalts (1)99.2%of CD44positive cells were isolated by magetic activated cell sorting.(2)31%of CD44positive cells formed floating cell spheres successfully, while CD44netagive cells could not. The floating sphere-forming cells possessed the potentials of self-renewal.Added FBS into the SFM, the sphere-forming cells could differentiate to CD44netagive cells.(3) CD44positive cells in gastic cancer tissues was36.9%and significantly correlated to the depth of tumor invasion, peritoneal metastasis and lymph node metastasis.(4) There was no difference between the expressions of CD44in gastric cancer tissues and peritoneal metastasis (P=0.133)。(5) The expression of CD44+CK20+was significantly higher in peritoneal washes from gastric cancer patients with peritoneal metastasis than without (P=0.00)。Conclusions(1)MACS could isolate tumor cells efficiently, and more important, the sorted cells could culture in vitro.(2) CD44positive cells possessd some properties of cancer stem cells.(3) Free cancer cells in peritoneal washes from gastric cancer patients contains gastric CSCs.(4) It is possible to directly isolate gastic CSCs from peritoneal washes from gastric cancer patients which may offer a novel method to the diagnosis and treatment of gastic cancer.