Skip to main content
  • Research Article
  • Published:

CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549

Abstract

Cancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44+/24+ and CD44+/CD24−/low subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44+/CD24+ and CD44+/CD24−/low subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serumfree medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.

Abbreviations

ABC:

ATP-binding cassette

ADC:

adenocarcinoma

ALDH1:

aldehyde dehydrogenase 1

bFGF:

basic fibroblast growth factor

CFU:

colony-forming unit

CSCs:

cancer stem cells

DEAB:

diethylaminobenzaldehyde

EGF:

epidermal growth factor

GFs:

growth factors

LCC:

large-cell carcinoma

NSCLC:

non-small cell lung carcinoma

SCC:

squamous-cell carcinoma

SP:

side population

References

  1. Travis, W.D. Pathology of lung cancer. Clin. Chest. Med. 32 (2011) 669–692.

    Article  PubMed  Google Scholar 

  2. Boman, B.M. and Wicha, M.S. Cancer stem cells: a step toward the cure. J. Clin. Oncol. 26 (2008) 2795–2799.

    Article  PubMed  Google Scholar 

  3. Jordan, C.T., Guzman, M.L. and Noble, M. Cancer stem cells. N. Engl. J. Med. 355 (2006) 1253–1261.

    Article  PubMed  CAS  Google Scholar 

  4. Reya, T., Morrison, S.J., Clarke, M.F. and Weissman, I.L. Stem cells, cancer, and cancer stem cells. Nature 414 (2001) 105–111.

    Article  PubMed  CAS  Google Scholar 

  5. Rivera, C., Rivera, S., Loriot, Y., Vozenin, M.C. and Deutsch, E. Lung cancer stem cell: new insights on experimental models and preclinical data. J. Oncol. 2011 (2011) 549181.

    Article  PubMed Central  PubMed  Google Scholar 

  6. Sun, S., Schiller, J.H., Spinola, M. and Minna, J.D. New molecularly targeted therapies for lung cancer. J. Clin. Invest. 117 (2007) 2740–2750.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  7. Bertolini, G., Roz, L., Perego, P., Tortoreto, M., Fontanella, E., Gatti, L., Pratesi, G., Fabbri, A., Andriani, F., Tinelli, S., Roz, E., Caserini, R., Lo Vullo, S., Camerini, T., Mariani, L., Delia, D., Calabrò, E., Pastorino, U. and Sozzi, G. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc. Natl. Acad. Sci. USA 106 (2009)16281–16286.

    Article  PubMed Central  PubMed  Google Scholar 

  8. Eramo, A., Lotti, F., Sette, G., Pilozzi, E., Biffoni, M., Di Virgilio, A., Conticello, C., Ruco, L., Peschle, C. and De Maria, R. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 15 (2007) 504–514.

    Article  PubMed  CAS  Google Scholar 

  9. Piechaczek, C. CD133. J. Biol. Regul. Homeost. Agents 15 (2001) 101–102.

    PubMed  CAS  Google Scholar 

  10. Chen, Y.C., Hsu, H.S., Chen, Y.W., Tsai, T.H., How, C.K., Wang, C.Y., Hung, S.C., Chang, Y.L., Tsai, M.L., Lee, Y.Y., Ku, H.H. and Chiou, S.H. Oct-4 expression maintained cancer stem-like properties in lung cancerderived CD133-positive cells. PLoS One 3 (2008) e2637.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  11. Al-Hajj, M., Wicha, M.S., Benito-Hernandez, A., Morrison, S.J. and Clarke, M.F. Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA 100 (2003) 3983–3988.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  12. Leung, E.L., Fiscus, R.R., Tung, J.W., Tin, V.P., Cheng, L.C., Sihoe, A.D., Fink, L.M., Ma, Y. and Wong, M.P. Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS One 5 (2010) e14062.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  13. Naor, D., Wallach-Dayan, S.B., Zahalka, M.A. and Sionov, R.V. Involvement of CD44, a molecule with a thousand faces, in cancer dissemination. Semin. Cancer Biol. 18 (2008) 260–267.

    Article  PubMed  CAS  Google Scholar 

  14. Hurt, E.M., Kawasaki, B.T., Klarmann, G.J., Thomas, S.B. and Farrar, W.L. CD44+ CD24 — prostate cells are early cancer progenitor/stem cells that provide a model for patients with poor prognosis. Br. J. Cancer 98 (2008) 756–765.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  15. Yeung, T.M., Gandhi, S.C., Wilding, J.L., Muschel, R. and Bodmer, W.F. Cancer stem cells from colorectal cancer-derived cell lines Proc. Natl. Acad. Sci. USA 107 (2010) 3722–3727.

    Article  PubMed Central  PubMed  Google Scholar 

  16. Yoshida, A., Hsu, L. and Dave, V. Retinal oxidation activity and biological role of human cytosolic aldehyde dehydrogenase. Enzyme 46 (1992) 239–244.

    PubMed  CAS  Google Scholar 

  17. Jiang, F., Qiu, Q., Khanna, A., Todd, N.W., Deepak, J., Xing, L., Wang, H., Liu, Z., Su, Y., Stass, S.A. and Ktz, R.L. Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer. Mol. Cancer Res. 7 (2009) 330–338.

    Article  PubMed  CAS  Google Scholar 

  18. Ucar, D., Cogle, C.R., Zucali, J.R., Ostmark, B., Scott, E.W., Zori, R., Gray, B.A. and Moreb, J.S. Aldehyde dehydrogenase activity as a functional marker for lung cancer. Chem. Biol. Interact 178 (2009) 48–55.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  19. Ding, X.W., Wu, J.H. and Jiang, C.P. ABCG2: a potential marker of stem cells and novel target in stem cell and cancer therapy. Life Sci. 86 (2010) 631–637.

    Article  PubMed  CAS  Google Scholar 

  20. Ho, M.M., Ng, A.V., Lam, S. and Hung, J.Y. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res. 67 (2007) 4827–4833.

    Article  PubMed  CAS  Google Scholar 

  21. Charloux, A., Quoix, E., Wolkove, N., Small, D., Pauli, G. and Kreisman, H. The increasing incidence of lung adenocarcinoma: reality or artefact? A review of the epidemiology of lung adenocarcinoma. Int. J. Epidemiol. 26 (1997) 14–23.

    Article  PubMed  CAS  Google Scholar 

  22. Sung, J.M., Cho, H.J., Yi, H., Lee, C.H., Kim, H.S., Kim, D.K., Abd El-Aty, A.M., Kim, J.S., Landowski, C.P., Hediger, M.A. and Chin, H.C. Characterization of a stem cell population in lung cancer A549 cells. Biochem. Biophys. Res. Commun. 371 (2008) 163–167.

    Article  PubMed  CAS  Google Scholar 

  23. Barrandon, Y. and Green, H. Three clonal types of keratinocyte with different capacities for multiplication. Proc. Natl. Acad. Sci. USA 84 (1987) 2302–2306.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  24. Li, H., Chen, X., Calhoun-Davis, T., Claypool, K. and Tang, D.G. PC3 human prostate carcinoma cell holoclones contain self-renewing tumorinitiating cells. Cancer Res. 68 (2008) 1820–1825.

    Article  PubMed  CAS  Google Scholar 

  25. Zhou, Z.H., Ping, Y.F., Yu, S.C., Yi, L., Yao, X.H., Chen, J.H., Cui, Y.H. and Bian, X-W. A novel approach to the identification and enrichment of cancer stem cells from a cultured human glioma cell line. Cancer Lett. 281 (2009) 92–99.

    Article  PubMed  CAS  Google Scholar 

  26. Tan, L., Sui, X., Deng, H. and Ding, M. Holoclone forming cells from pancreatic cancer cells enrich tumor initiating cells and represent a novel model for study of cancer stem cells. PLoS One 6 (2011) e23383.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  27. Franken, N.A., Rodermond, H.M., Stap, J., Haveman, J. and Van Bree, C. Clonogenic assay of cells in vitro. Nat. Protoc. 1 (2006) 2315–2319.

    Article  PubMed  CAS  Google Scholar 

  28. Pastrana, E., Silva-Vargas, V. and Doetsch, F. Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell 8 (2011) 486–498.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  29. Friedrich, J., Seidel, C., Ebner, R. and Kunz-Schughart, L.A. Spheroid-based drug screen: considerations and practical approach. Nat. Protoc. 4 (2009) 309–324.

    Article  PubMed  CAS  Google Scholar 

  30. Hill, R.P. Identifying cancer stem cells in solid tumors: case not proven. Cancer Res. 66 (2006) 1891–1895.

    Article  PubMed  CAS  Google Scholar 

  31. Singh, S.K., Hawkins, C., Clarke, I.D., Squire, J.A., Bayani, J., Hide, T., Henkelman, R.M., Cusimano, M.D. and Dirks, P.B. Identification of human brain tumour initiating cells. Nature 432 (2004) 396–401.

    Article  PubMed  CAS  Google Scholar 

  32. Meng, X., Li, M., Wang, X., Wang, Y. and Ma, D. Both CD133+ and CD133 — subpopulations of A549 and H446 cells contain cancer-initiating cells. Cancer Sci. 100 (2009) 1040–1046.

    Article  PubMed  CAS  Google Scholar 

  33. Akunuru, S, Zhai, Q.J. and Zheng, Y. Non-small cell lung cancer stem/progenitor cells are enriched in multiple distinct phenotypic subpopulations and exhibit plasticity. Cell Death Dis. 3 (2012) e352.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  34. Stuelten, C.H., Mertins, S.D., Busch, J.I., Gowens, M., Scudiero, D.A., Burkett, M.W., Hite, K.M., Alley, M., Hollingshead, M., Shoemaker, R.H. and Niederhuber, J.E. Complex display of putative tumor stem cell markers in the NCI60 tumor cell line panel. Stem Cells 28 (2010) 649–660.

    Article  PubMed  CAS  Google Scholar 

  35. Levina, V., Marrangoni, A.M., DeMarco, R., Gorelik, E. and Lokshin, A.E. Drug-selected human lung cancer stem cells: cytokine network, tumorigenic and metastatic properties. PLoS One 3 (2008) e3077.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  36. Tirino, V., Camerlingo, R., Franco, R., Malanga, D., La Rocca, A., Viglietto, G., Rocco, G. and Pirozzi, G. The role of CD133 in the identification and characterisation of tumour-initiating cells in non-smallcell lung cancer. Eur. J. Cardiothorac. Surg. 36 (2009) 446–453.

    Article  PubMed  Google Scholar 

  37. Fargeas, C., Huttner, W. and Corbeil, D. Nomenclature of prominin-1 (CD133) splice variants-an update. Tissue Antigens 69 (2007) 602–606.

    Article  PubMed  CAS  Google Scholar 

  38. Shmelkov, S.V., St Clair, R., Lyden, D. and Rafii, S. AC133/CD133/Prominin-1. Int. J. Biochem. Cell Biol. 37 (2005) 715–719.

    Article  PubMed  CAS  Google Scholar 

  39. Wang, P., Gao, Q., Suo, Z., Munthe, E., Solberg, S., Ma, L., Wang, M., Westerdaal, N.A., Kvalheim, G. and Gaudernack, G. Identification and characterization of cells with cancer stem cell properties in human primary lung cancer cell lines. PLoS One 8 (2013) e57020.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  40. Kim, J., Jung, J., Lee S.J., Lee J.S. and Park M.J. Cancer stem-like cells persist in established cell lines through autocrine activation of EGFR signaling. Oncol. Lett. 3 (2012) 607–612.

    PubMed Central  PubMed  CAS  Google Scholar 

  41. Kelly, J.J., Stechishin, O., Chojnacki, A., Lun, X., Sun, B., Senger, D.L., Forsyth, P., Auer, R.N., Dunn, J.F., Cairncross, J.G., Parney I.F., and Weiss, S. Proliferation of human glioblastoma stem cells occurs independently of exogenous mitogens. Stem Cells 27 (2009) 1722–1733.

    Article  PubMed  CAS  Google Scholar 

  42. Li, G., Chen, Z., Hu, Y.D., Wei, H., Li, D., Ji, H. and Wang, D-L. Autocrine factors sustain glioblastoma stem cell self-renewal. Oncol. Rep. 21 (2009) 419–424.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Zahra Madjd or Marzieh Ebrahimi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Roudi, R., Madjd, Z., Ebrahimi, M. et al. CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549. Cell Mol Biol Lett 19, 23–36 (2014). https://doi.org/10.2478/s11658-013-0112-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-013-0112-1

Key words