Changes of lung cancer stem cells in patients with lung squamous carcinoma during primary culture

doi:10.3969/j.issn.2095-4344.2015.32.016

Abstract

Abstract:

BACKGROUND: Studies have shown that lung cancer stem cells can be isolated from the lung cancer cell lines, But there are few reports on in vitro isolation, culture and identification of lung cancer stem cells in patients with lung squamous carcinoma.
OBJECTIVE: To establish the feasible methods of harvesting lung cancer stem cells from fresh lung cancer tissues in patients with lung squamous carcinoma, and to investigate the alterations in cell number and function during primary culture.
METHODS: Side population cells were isolated by collagenase digestion, Ficoll density gradient centrifugation and Hoechst 33342 efflux properties. The isolated cells were isolated and cultured in conditioned medium. Flow cytometry method was used to detect lung cancer stem cells based on the cell surface markers CD133 and CD44, and the positive rates of CD133+, CD44+ and CD133+/CD44+ were recorded. The single cell clones assay, flat colony formation assay and the cell sphere formation assay were used to identify the stem-like characteristics of lung cancer stem cells between the first and fourth generations.
RESULTS AND CONCLUSION: The positive rates of CD133+, CD44+ and CD133+/CD44+ cells at the fourth generation were increased significantly, and the positive rates of CD133+ and CD133+/CD44+ cells at passage 4 were significantly higher than those at the first generation. The abilities of single cell clone formation, the flat colony formation and the cell sphere formation in the fourth-generation cells were greatly enhanced compared with the first-generation cells. Experimental findings showed that stem cell-like lung cancer cells were obtained from fresh lung cancer tissue in patients with lung squamous carcinoma, which stably and rapidly amplified in vitro, laying the foundation for the further study of the heterogeneity and drug resistance of lung cancer stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Lung Neoplasms, Neoplastic Stem Cells, Cells, Cultured

CLC Number:

R394.2

Liu Zhe-liang, Wu Jiao, Wang Lin-xian, Chen Yue-jun, Wu Guan-yu, Xiao Gao-ming. Changes of lung cancer stem cells in patients with lung squamous carcinoma during primary culture[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(32): 5172-5176.

Figures/Tables 2

References

[1] Proctor RN. Tobacco and the global lung cancer epidemic. Nat Rev Cancer. 2001;1(1):82-86.
[2] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62(1):10-29.
[3] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646-674.
[4] Jemal A, Thun MJ, Ries LA, et al. Annual report to the nation on the status of cancer, 1975-2005, featuring trends in lung cancer, tobacco use, and tobacco control. J Natl Cancer Inst. 2008;100(23):1672-1694.
[5] Gentry SN, Jackson TL. A mathematical model of cancer stem cell driven tumor initiation: implications of niche size and loss of homeostatic regulatory mechanisms. PLoS One. 2013; 8(8):e71128.
[6] Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer. 2008;8(10):755-768.
[7] Clarke MF, Dick JE, Dirks PB, et al. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006; 66(19):9339-9344.
[8] Tang DG. Understanding cancer stem cell heterogeneity and plasticity. Cell Res. 2012;22(3):457-472.
[9] Golebiewska A, Brons NH, Bjerkvig R, et al. Critical appraisal of the side population assay in stem cell and cancer stem cell research. Cell Stem Cell. 2011;8(2):136-147.
[10] Mizrak D, Brittan M, Alison M. CD133: molecule of the moment. J Pathol. 2008;214(1):3-9.
[11] Keysar SB, Jimeno A. More than markers: biological significance of cancer stem cell-defining molecules. Mol Cancer Ther. 2010;9(9):2450-2457.
[12] Chen YC, Hsu HS, Chen YW, et al. Oct-4 expression maintained cancer stem-like properties in lung cancer-derived CD133-positive cells. PLoS One. 2008;3(7):e2637.
[13] Hermann PC, Huber SL, Herrler T, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell. 2007; 1(3):313-323.
[14] Ma S, Chan KW, Lee TK, et al. Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations. Mol Cancer Res. 2008;6(7):1146-1153.
[15] Miki J, Furusato B, Li H, et al. Identification of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized primary nonmalignant and malignant tumor-derived human prostate epithelial cell lines and in prostate cancer specimens. Cancer Res. 2007;67(7):3153-3161.
[16] Zhang Q, Shi S, Yen Y, et al. A subpopulation of CD133(+) cancer stem-like cells characterized in human oral squamous cell carcinoma confer resistance to chemotherapy. Cancer Lett. 2010;289(2):151-160.
[17] Matsumoto K, Arao T, Tanaka K, et al. mTOR signal and hypoxia-inducible factor-1 alpha regulate CD133 expression in cancer cells. Cancer Res. 2009;69(18):7160-7164.
[18] Eramo A, Lotti F, Sette G, et al. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 2008;15(3):504-514.
[19] Du L, Wang H, He L, et al. CD44 is of functional importance for colorectal cancer stem cells. Clin Cancer Res. 2008; 14(21): 6751-6760.
[20] Shimada Y, Ishii G, Nagai K, et al. Expression of podoplanin, CD44, and p63 in squamous cell carcinoma of the lung. Cancer Sci. 2009;100(11):2054-2059.
[21] Leung EL, Fiscus RR, Tung JW, et al. Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS One. 2010;5(11):e14062.
[22] Schulenburg A, Blatt K, Cerny-Reiterer S, et al. Cancer stem cells in basic science and in translational oncology: can we translate into clinical application. J Hematol Oncol. 2015; 8(1):16.
[23] Lathia JD, Venere M, Rao MS, et al. Seeing is believing: are cancer stem cells the Loch Ness monster of tumor biology. Stem Cell Rev. 2011;7(2):227-237.
[24] Park TS, Donnenberg VS, Donnenberg AD, et al. Dynamic Interactions Between Cancer Stem Cells And Their Stromal Partners. Curr Pathobiol Rep. 2014;2(1):41-52.
[25] Wang P, Gao Q, Suo Z, et al. Identification and characterization of cells with cancer stem cell properties in human primary lung cancer cell lines. PLoS One. 2013;8(3): e57020.
[26] Pacini N, Borziani F. Cancer stem cell theory and the warburg effect, two sides of the same coin. Int J Mol Sci. 2014;15(5): 8893-8930.
[27] Ciurea ME, Georgescu AM, Purcaru SO, et al. Cancer stem cells: biological functions and therapeutically targeting. Int J Mol Sci. 2014;15(5):8169-8185.