Identification Of Biological Characteristics Of Xuanwei Lung Adenocarcinoma Stem Cells

[Objective] Identification of biological characteristics of lung adenocarcinoma stem cells from the original cloning cultured SPs cells which were separated by flow cytometry, and analysis the relationship between morphology and sternness among different cells.[Method] To divide the subsets which were separated originally and cloning cutured into groups:The cells in serum medium include SPs and SPcm subsets, SPs-C11 subsets looks like long spindle and SPs-E8 subsets looks like irregular polygon which were separated from SPs cells. SPcm-G10 cells looks like irregular polygon and SPcm-B10 cells looks like long spindle which were separated from SPcm cells. SPs-E8-tu cells were cultured in serum free medium. The identification of biological characteristics of lung adenocarcinoma stem cells of SPs subsets were divided into two parts, one in vitro and the other in vivo. The experiments in vitro include:1. To detect the related molecular signatrues of cancer stem cells by flow cytometry and to study their expression; 2. To test the retention capacity of BrdU of different subsets to understand their division mode; 3. To investigate the expression of lung stem cell molecular signatrues SCGB1A1/proSP-C by using immunofluorescence technique. The experiment in vivo includes:4. To study the oncogenicity of different subsets on NOD/SCID mice by injecting them with different number of cells, and then observing the oncogenicity and minimum numbers of cells.5. SPSS17.0 statistical software was used for data analysis.[Results] 1. After dyeing the cells using the antibodies of fluorescein, according to the flow cytometry analysis, the expression of the molecular signatures of cancer stem cells of different subsets were different. For the two group of cells in serum medium, SPs-C11 cells were higher than SPcm-B10 cells at the level of the expression of Nanog, hTERT, CD34, CD221. There was significant difference among the groups, P<0.01. SPs-E8 cells were higher than SPcm-G10 cells at the level of the expression of hTERT, CD221, P<0.01. But at the level of the other proteins, there was no significant difference, P>0.05.2. BrdU experiment showed that SPs and SPcm were negative expression in serum medium.3. Immunofluorescence experiment:After detecting SCGB1A1 and proSP-C which were lung stem cell molecular signatrues, we found the expressions of the two proteins of SPs-C11 in serum medium were higher than SPcm-B10, SPs-E8、SPcm-G10 were negative expression, while the expression of SCGB1A1、proSP-C of SPs-E8-tu cells in serum free medium were higher than SPs-C11 in serum medium in terms of rate and strength.4. Nude mice experiment:After injecting the two group cells in serum medium at the concentration of 1×107and 2×107, for both SPs-C11 and SPcm-B10, minimum quantity of tumorigenic cells was 1×107, but the size of SPs-C11 was bigger than SPcm-B10; After injecting the serum free medium cells at the concentration of 0.5×106, it grew big tumor measuring 17×12×8mm3. On the process of observation until 12 weeks, SPs-E8 and SPcm-G10 didn’t form tumor at injected area while the others form tumors successfully. After excising the tumors from the mice, we found the shape of the tumors were irregular, nodular mass,the pathology report showed that it’s lung adenocarcinoma.[Conclusions] 1. SPs cells remained more biological characteristics of stem cells than SPcm cells, especially for the SPs-C11 subsets looks like long spindle which highly expressed molecular signatrues of cancer stem cells including Nanog、hTERT、CD34、CD221; However, as the cells repeatedly subculture, SPs and SPcm had already differentiated at different level under serum medium.2. Cell morphology had nothing to do with the biological characteristics of Xuanwei lung adenocarcinoma stem cells.3. The method of serum free medium was better than that of serum medium in maintaining lung adenocarcinoma stem cell characteristics.4. We acquired subsets of SPs-E8-tu which were rich in Xuanwei lung adenocarcinoma stem cells.