Identifying And Characterizing Side Population Of Yunnan Lung Cancer Cell Lines

[Objective] To isolate and characterize the stem cell-like cancer cells in Yunnan lung cancer cell lines using flow cytometry (FCM) and Hoechst 33342 dye efflux assay.[Methods] Several lung cancer cell lines recently established from female subjects in Xuanwei, Yunnan province and previously established Yunnan lung cancer cell lines GLC-82 and YTMLC-90 were examined for the presence of SP cells by Flow cytometry (FCM). A cell line was selected to sort the SP cells by Hoechst 33342 dye efflux assay, which were then examined for their characteristics associated with cancer stem cells and malignancy by a series of experiments. Both SP cells and non-SP cells were harvested for further studies respectively. The characterizations of stem-like cancer cells between SP cells and non-SP cells were examined using soft agar culture, ECM gel invasion assay (transwell), DNA ploidy and phase distribution of cell cycles. In the meanwhile, mRNA expression level of putative stem cell marker genes (ABCG2, MDR1, hTERT, SFTPC, and SCGB1A1) were measured using real time RT-PCR and several molecules on cytomembrane were detected by FCM, which were known as the markers associated with tumor initiating cells, including ABCG2, MDR1, CD 133, CD44, CD45 and CD34. miRNAs are a class of small non-coding RNA species that regulate gene expression, many exhibit tissue-specific expression and are misregulated in cancer, so microRNA expression profile was examined using GeneChip miRNA Array, too. In addition, the sorted SP cells were cultured for SP re-analysis to know the repopulating ability to regenerate SP and non-SP cells. Then, sorted SP cells were choosen separately to cloned culture. Subsequently, the following experiments on cellular morphology, soft agarose colony forming efficiency (CFE), growth curve, DNA ploidy and phase distribution of cell cycles, the sensitivity of chemotherapeutic drugs and cells accumulated BrdU and mRNA expression level of putative stem cell marker genes (same as the former) were carried out. Next, xenograft experiments were conducted in Balb/c-nude mice.[Results] Two new cancer cell lines (XLS-06 and XLA-07) were established in this study. It was found that SP phenotype is observed in all four human lung cancer cell lines (XLS-06, XLA-07, GLC-82 and YTMLC-90), They all contained a distinct fraction of SP cells, and their respective contribution to XLS-06, XLA-07, GLC-82 and YTMLC-90 were 0.3%,2.6%,0.9% and 0.1% of gated cells, which decreased significantly in the presence of verapamil. In comparison with non-SP cells, SP cells showed some different futures, for example, more cells were laid in G0/G1 of cell cycle, invasiveness in vitro and CFE were higher, the expression level of genes' mRNA (ABCG2, MDR1, hTERT, SFTPC, and SCGB1A1) and of proteins (ABCG2, MDR1, and CD 133) also increased significantly. Moreover, the miRNA expression profiling was observed to be obviously different between SP cells and non-SP cells. It was recorded that there were 19 miRNAs showing difference among total 848 miRNAs of homo species, including 3 upregulated and 16 downregulated miRNAs in SP cells. Those low-expressed miRNAs were forecasted for their target genes using the soft ware TargetScan, it was found that majority genes focus on zinc finger protein, chromosome open reading frame, G-protein-coupled receptor and Dicer, and so on. Further more, it was noted that after cultured with serum medium, the sored SP cells could regenerated both SP and non-SP cells with a fraction size comparable with the original population, and the SP fraction was 4.3%, higher than unsorted cells. Through cloned cultured,3 subclone strains with different morphology were obtainned. The shape of one like a shuttle, the other were similar to square, and another showed to be leptosomatic and stagger named SPs, SPq, SPcm, respectively. The ultrastructure of one strain with leptosomatic and stagger shape indicated that the cellular differentiation were poorer than original popultion. Among these different cells derived from 3 subclones with different passages, the cellular double time, phase distribution of cell cycles, DNA ploid, CFE, invasioness, the expression of genes' mRNA and proteins associated with stem cell properties, the capabilities of cells to accumulate BrdU and tumorigenesis all revealed grand variances. Out of these subclones, a higher invasive cell strain named XLA-07/SPs has been obtained, which possessing of faster proliferation speed, higher CFE, more DNA content and amount of phase S in cell cycle, elevated expression of markers known as related to stem cell. More important was, it could reform tumor in Balb/c-nude mice, and the tumor tissue was proved to be adenocarcinoma similar to original tissue from patient by pathology. SP analysis of reformed tumor cells showed 8% fraction of SP in it, far higher than original population. Collectively, our data suggests that highly specific SP cells exist in Yunnan lung cancer cell lines. The SP population showed typical stem-like cancer cells characterizations.[Conclusions] SP cells were present in the four human lung cancer cell lines (XLS-06, XLA-07, GLC-82 and YTMLC-90) examined in this study. The sorted SP cells had high malignancy and properties of stem-like cancer cells compared with non SP cells. miRNA expression profiling was different contrasted SP cells with non-SP cells, there were 19 miRNAs showing difference among total 848 miRNAs of homo species, including 3 upregulated and 16 downregulated miRNAs in SP cells. SP population was heterogeneity which were composed of various cells possessing distinct biological characters. FCM could be used to demostrate the status of stem cells'accumulating BrdU to display the cellular asymmetry division, the technology was simple but reliable. Futher more, a potential human lung cancer stem cell line has been obtained, which will be further characterized.