Stem-like Properties Of Lewis Lung Carcinoma Cancer Stem Cells Regulated By Twist2

Purpose: We explored a new method for isolation and identification of CSCs which could be passaged continuously from LLC-P and its self-renewal regulating mechanisms.Methods: The LLC-SE culture can be established through 8 consecutive rounds of collecting floating cells that become independent of attachment for growth and form spheroids from the LLC-P.For stem cell properties verification,the expressions of pluripotent genes Bmi1(B-cell-specific moloney murine leukemia virus insertion site 1),Cd133(cluster of differentiation-133),Aldh1a1(aldehyde dehydrogenase family 1,subfamily A1),Oct4(octamer-binding transcription factor 4),Sox2(sex-determining region Y box 2),Nanog and Klf4(Kruppellike factor 4)were measured by RT-PCR.The abilities of proliferation and spheroid formation were assessed by 6-wells plate agar spheroid formation assay.The ability of single cell cloning was analyzed by 96-wells plate single cell cloning assay.In vivo tumorigenicity was measured by nude mice subcutaneous transplantation which was considered as the classical model for stem cell property assessment.And tumor metastasis ability was measured by left lung orthotopic implanting in syngeneic C57 mice.We use RT-PCR to detect the differential expression of genes and use siRNA to explore the self-renewal regulating mechanisms of LLC-SE.Furthermore,LLC-SD and LLC-ASD were clarified through 5 consecutive single cell cloning assay.Symmetric and asymmetric cell division was verified by BrdU marking.Results: The results of RT-PCR showed that the expression of Bmi1 in LLC-SE was much higher than that in LLC-P.The results of spheroid formation assay and cloning assay showed that spheroid formation ability and single cell cloning ability of LLC-SE were better than that of LLC-P.The results of in vivo experiments showed that tumorigenicity and metastasis ability of LLC-SE were better than that of LLC-P.The results of RT-PCR showed that the expression of Twist2 in LLC-SE was much higher than that in LLC-P.When Twist2 was knocked down,spheroid formation ability and single cell cloning ability of LLC-SE reduced.And the tumorigenicity of LLC-SE changed as well.Outcome:In this work,we developed a new method for the isolation of long lasting CSCs from cultured tumor cells.This method will allow the generation of cellular models for mechanistic characterization of CSCs.LLC-SD and LLC-ASD which can perform symmetric and asymmetric cell division were successfully established with 5 consecutive single cell cloning assay.And the regulating mechanism of stem cell properties was preliminary explored.