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Isolation of Lewis Lung Carcinoma Cancer Stem Cells by Consecutive Spheroid Enrichment and Characterization of Their Stem Cell Properties
Liu Yongli, Kong Liangsheng, Sun Zhiwei, Wang Jianyu*, Xing Ruoxi*
Laboratory of Translational Cancer Stem Cell Research, Institute of Life Sciences,
Chongqing Medical University, Chongqing 400016, China
Abstract: We explored a new method for isolation and identification of cancer stem cells (CSCs) which could be passaged continuously from parental Lewis lung carcinoma cells (LLC-Parental). The LLC-spheroid enrichment (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-Parental cells. 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 (Kruppel-like factor 4) were measured by Real-time 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. The results of Real-time PCR showed that the expression of Bmi1 in LLC-SE was much higher than that in LLC-Parental. 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-Parental. The results of in vivo experiments showed that tumorigenicity and metastasis ability of LLC-SE were better than that of LLC-Parental. 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.
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