Abstract: Objective To establish an asymmetric dividing cell line(LLC-ASD cells) derived from mouse Lewis lung carcinoma cancer cells( LLC-parental cells), and to investigate its stemness features in order to lay a foundation for depth studying the function of asymmetric dividing in the cancer biology. Methods In order to obtain asymmetrically dividing LLC cells (LLC-ASD cells) derived from LLC-Parental cells, 8 times of consecutive culture, enrichment and collection of floating spheriod forming cells followed by 5 times of consecutive single cell cloning were conducted. Immunofluorescence assay was used to visualize and quantify the rate of asymmetric division in LLC-ASD cells labeled by BrdU. For comparing the stem characteristics of LLC-Parental and LLC-ASD, RT-qPCR, clonogenic assay in 6-well plate,single cell spheroid formation assay with agar in 6-well plate and 96-well-plate single cell cloning assay were conducted. In vivo, LLC-parental cells and LLC-ASD cells were subcutaneously transplanted in nude mice to determine the effect of the difference in stem cell like properties on tumorigeneicy. The same lung transplantation into tumor experiment in mice were used to compare the differences in cancer biology characteristics. Results Asysmmetric dividing cells were found in LLC-ASD cell culture through the BrdU immunofluorescence assay and the rate of asymmetric division in the anaphase cells was as high as 50%。According to the clonogenic assay in 6-well plate,single cell spheroid formation assay with agar in 6-well plate and 96-well-plate single cell cloning assay in LLC-ASD cells, the results showed that they were more prominent than those in the LLC-Parental cells（P <0.05). In vivo, the tumor metastatic ability of LLC-ASD was enhanced than that of LLC-parental when transplanted to the C57 mice. Further, the tumorigenic ability of LLC-ASD cells was also increased compared to that of LLC-parental cells when subcutaneously transplanted to the nude mice. Conclusions The asymmetric dividing cell line derived from mouse Lewis lung carcinoma cancer cell line (LLC-ASD cells) is established which exhibits stemness properties. The establishment and characterization of this model will facilitate the studies of the function of asymmetric cell dividing in cancer biology .

Key words: asymmetric dividing , cancer stem cells, mouse Lewis lung cancer