Screening Of Lung Cancer Stem Cell Surface Markers And Researching Enrichment Methods Of Lung Cancer Stem Cell

Objective:In this study, CD133, CD24, CD44 and CD221 in different types of human lung cancer cell lines expression was detected and screening potential stem cell surface markers of lung cancer by flow cytometry. using serum-free suspension culture into spheresl and cisplatin-induced accumulation Lung cancer cell lines of lung cancer stem cells, and detection of the screened surface marker molecules in which the expression, in order to obtain effective separation of cancer stem cell surface markers, for the following human lung cancer stem cell selection and function of experimental basis.Method:Detect CD 133,CD24,CD44 and CD221 in lung adenocarcinoma cell line A549, Squamous cell carcinoma cell line YTMLC-9, large cell carcinoma cell was limited by the line L9981 with high metastatic potential and cell line NL9980 with low metastatic potential by flow cytometry, screen the possible lung cancer stem cells mark molecules. With L9981 and A549 for research object, lung cancer cells sphere of L9981 was found by serum-free suspension culture and cisplatin induction. Then the morphological differences of sphere cells and adherent L9981 cells cultured in serum-containing mediums are observed., Cell proliferation was analyzed by Vi-cell viability analyzer; invasion ability was tested by transwell assay; and in vivo tumorigenicity of the two groups of cells was studied in nude mouse. And identified the cell line A549 with the same method, detected the expression level of CD133 and CD24 in the lung cancer stem cell subsets, explored the possibility of CD133 and CD24 as molecular markers of lung cancer stem cell.Rusult:1. Single parameter examination results show that the expression levels of CD221 and CD44 are much higher in lung cancer cell lines YTMLC-9, A549, L9981 and NL9980,while CD 133 and CD24'expression levels are much lower in all kinds of Lung cancer cell lines. The results showed that the expression level of CD133-CD24-, CD133-CD24+, CD221-CD24-and CD221+CD24-is higher in every cell lines, while CD133+CD24-, CD133+CD44+, CD221-CD24+, CD221+CD24+is low.2. A549 cells form into the ball hardly in serum-free suspension culture, while L9981 cells form into ball significantly. The L9981 cell in suspension culture compared with the cells adherent to the wall grow slower, at least 1 week to lots of growth. But the ball once formed, it maintains more longer than the cells adherent to the wall. After disposalof DDP in L9981 and A549 cells, the morphous of the cells of resisitance and the volume are different,and the volumn increased, the light quality is poor.3.The doubling time of A549 and L9981 were (46.05±1.95)h and (54.28±1.41) hours in RPMI1640/FBS, while (33.00±1.44h and (34.49±1.81) hours in suspension serum-free media combined with DDP.In addition,compared to the A549 and L9981 cultured in RPMI1640/FBS,the invasion ability and tumorigenic cap acity increased 5 and 20 folds in suspension serum-free media combined with DDP,a nd the difference demonstrated satistical significance(P<0.05).4.The flow cytometry was employed to detect the CD133 and CD24 protein expressio n.In comparison with the cells cultured in RPMI1640/FBS,the results showed that the expression of CD 133 protein increased by a large margin,while the CD24 expression had a marked decrease in suspension serum-free media combined with DDP, and ther e were statistical differenece(P<0.05).In vivo study showed that after being injected s ubcutaneously with A549 cells and L9981 cells cultured in different medium, there w ere obvious difference in tumor formation rate of mice.On biopsy, this was an infiltrat ing lung cancer.Besides,we also detected the CD133 and CD24 protein expression usi ng immunohistochemistry, and the results showed the percentage of the CD133 was 2 %,while the CD24 protein had no expression.Conclusion:1 According to the rule that the proportion of tumor stem cells is low in tumor cell population, CD133-CD24- and CD 133-CD24+ might be preliminarily predicted to be surface immune molecular marks of non-small cell lung cancer stem cells, at least which is near to lung cancer stem cell marks.2. After L9981 and A549 cells were treated with suspension medium containing growth factor and cisplatin, L9981 cells had significant sphere effect. The proliferation, invasion and grafting tumor abilities of L9981 sphere cells were higher than L9981 adherent cells (P<0.05). However, A549 cells had weak sphere effect. The proliferation, invasion and grafting tumor abilities of L9981 treated higher than A549 adherent cells (P<0.05). These showed that lung cancer sphere cells contained many lung cancer stem cells or lung cancer stem cell like cells.3. CD133+ high expression and CD24+ low expression in lung cancer stem cell like cells sub-group suggested that CD133+CD24- may be a potential surface mark or one of some stage in lung cancer stem cell development.