Tumor Biology

, Volume 37, Issue 10, pp 14069–14081 | Cite as

Characterization of cancer stem cells from different grades of human colorectal cancer

  • J. Manhas
  • A. Bhattacharya
  • S. K. Agrawal
  • B. Gupta
  • P. Das
  • S. V. S. Deo
  • S. Pal
  • S. SenEmail author
Original Article


Colorectal cancer (CRC) is one of the most common solid tumors worldwide. Recent evidence suggests that a population of cancer cells, called cancer stem cells (CSCs), is responsible for tumor heterogeneity, invasion, metastasis, therapeutic resistance, and recurrence of CRC. The isolation and characterization of CSCs using cell surface markers have been reported previously with varying results. In this study, we investigated a panel of four putative CSC markers, CD44, CD24, CD166, and EpCAM, to define CRC-CSC. Paraffin embedded tissue samples from different grades of primary, untreated CRC were analyzed for the expression of four CSC markers CD44, CD326, CD24, and CD166, using immunohistochemistry. Flow cytometric analysis of CRC-CSC from HT29 (low grade) and HCT116 (high grade) human colorectal cancer cell lines was done. Marker-based isolation of CSC and non-CSC-bulk-tumor cells from HT29 was done using FACS, and tumor sphere assay was performed. There was a statistically significant difference (p < 0.05) in the expression of CD44, CD326, and CD166 between cases and controls. A novel cutoff distribution of CD44 and CD166 was suggested to help for better immunohistochemical analysis of CRC. Higher prevalence of CSC was seen in high-grade CRC as compared to low-grade CRC. Sorted and cultured CD44 + CD166+ cells formed tumor spheres, suggesting that these cells, having properties of self renewal and anchorage independent proliferation, were in fact CSC. Hence, CD44 and CD166 may serve as good CRC-CSC markers when used together with novel cutoff immunohistochemistry (IHC) expression levels.


Colorectal cancer Cancer stem cells CD44 CD166 



The authors are grateful to the Department of Biotechnology (DBT), Govt. of India, for the Cancer Biology Grant; BD Jamia Hamdard FACS Academy, New Delhi, for the FACS training; and Dr. Jayanth Kumar Palanichamy for his guidance and support.

Compliance with ethical standards

Conflicts of interest


Supplementary material

13277_2016_5232_MOESM1_ESM.doc (84 kb)
ESM 1 (DOC 84 kb)


  1. 1.
    Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010;60(5):277–300. doi: 10.3322/caac.20073.CrossRefPubMedGoogle Scholar
  2. 2.
    Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–86. doi: 10.1002/ijc.29210.CrossRefPubMedGoogle Scholar
  3. 3.
    Larsson SC, Wolk A. Meat consumption and risk of colorectal cancer: a meta-analysis of prospective studies. Int J Cancer. 2006;119(11):2657–64. doi: 10.1002/ijc.22170.CrossRefPubMedGoogle Scholar
  4. 4.
    Larsson SC, Orsini N, Wolk A. Vitamin B6 and risk of colorectal cancer: a meta-analysis of prospective studies. Jama. 2010;303(11):1077–83. doi: 10.1001/jama.2010.263.CrossRefPubMedGoogle Scholar
  5. 5.
    Park S, Kim Y, Shin HR, Lee B, Shin A, Jung KW, et al. Population-attributable causes of cancer in Korea: obesity and physical inactivity. PLoS One. 2014;9(4), e90871. doi: 10.1371/journal.pone.0090871.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Peppone LJ, Mahoney MC, Cummings KM, Michalek AM, Reid ME, Moysich KB, et al. Colorectal cancer occurs earlier in those exposed to tobacco smoke: implications for screening. J Cancer Res Clin Oncol. 2008;134(7):743–51. doi: 10.1007/s00432-007-0332-8.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Van Cutsem E, Dicato M, Arber N, Berlin J, Cervantes A, Ciardiello F, et al. Molecular markers and biological targeted therapies in metastatic colorectal cancer: expert opinion and recommendations derived from the 11th ESMO/World Congress on Gastrointestinal Cancer, Barcelona, 2009. Ann Oncol. 2010;21 Suppl 6:vi1–vi10. doi: 10.1093/annonc/mdq273.PubMedGoogle Scholar
  8. 8.
    Van Cutsem E, Nordlinger B, Cervantes A, Group EGW. Advanced colorectal cancer: ESMO Clinical Practice Guidelines for treatment. Ann Oncol. 2010;21 Suppl 5:v93–7. doi: 10.1093/annonc/mdq222.CrossRefPubMedGoogle Scholar
  9. 9.
    Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A. 2007;104(3):973–8. doi: 10.1073/pnas.0610117104.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med. 2007;58:267–84. doi: 10.1146/ Scholar
  11. 11.
    Chandler JM, Lagasse E. Cancerous stem cells: deviant stem cells with cancer-causing misbehavior. Stem Cell Res Ther. 2010;1(2):13. doi: 10.1186/scrt13.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133(4):704–15. doi: 10.1016/j.cell.2008.03.027.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Kong D, Li Y, Wang Z, Sarkar FH. Cancer stem cells and epithelial-to-mesenchymal transition (EMT)-phenotypic cells: are they cousins or twins? Cancers. 2011;3(1):716–29. doi: 10.3390/cancers30100716.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S, et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res. 2005;11(18):6520–7. doi: 10.1158/1078-0432.CCR-05-0468.CrossRefPubMedGoogle Scholar
  15. 15.
    Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature. 2004;432(7015):396–401. doi: 10.1038/nature03128.CrossRefPubMedGoogle Scholar
  16. 16.
    Al-Hajj M, Becker MW, Wicha M, Weissman I, Clarke MF. Therapeutic implications of cancer stem cells. Curr Opin Genet Dev. 2004;14(1):43–7. doi: 10.1016/j.gde.2003.11.007.CrossRefPubMedGoogle Scholar
  17. 17.
    Aguirre-Ghiso JA. Models, mechanisms and clinical evidence for cancer dormancy. Nat Rev Cancer. 2007;7(11):834–46. doi: 10.1038/nrc2256.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Pantel K, Alix-Panabieres C, Riethdorf S. Cancer micrometastases. Nat Rev Clin Oncol. 2009;6(6):339–51. doi: 10.1038/nrclinonc.2009.44.CrossRefPubMedGoogle Scholar
  19. 19.
    Cohen E, Ophir I, Shaul YB. Induced differentiation in HT29, a human colon adenocarcinoma cell line. J Cell Sci. 1999;112(Pt 16):2657–66.PubMedGoogle Scholar
  20. 20.
    Nautiyal J, Kanwar SS, Yu Y, Majumdar AP. Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells. J Mol Signal. 2011;6:7. doi: 10.1186/1750-2187-6-7.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Zhan J, Niu M, Wang P, Zhu X, Li S, Song J, et al. Elevated HOXB9 expression promotes differentiation and predicts a favourable outcome in colon adenocarcinoma patients. Br J Cancer. 2014;111(5):883–93. doi: 10.1038/bjc.2014.387.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Yeung TM, Gandhi SC, Wilding JL, Muschel R, Bodmer WF. Cancer stem cells from colorectal cancer-derived cell lines. Proc Natl Acad Sci U S A. 2010;107(8):3722–7. doi: 10.1073/pnas.0915135107.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Yeoman LC, Wan CW, Zorbas MA. Transferrin and insulin enhance human colon tumor cell growth by differentiation class specific mechanisms. Oncol Res. 1996;8(7-8):273–9.PubMedGoogle Scholar
  24. 24.
    Ozawa M, Ichikawa Y, Zheng YW, Oshima T, Miyata H, Nakazawa K, et al. Prognostic significance of CD44 variant 2 upregulation in colorectal cancer. Br J Cancer. 2014;111(2):365–74. doi: 10.1038/bjc.2014.253.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Pitule P, Cedikova M, Daum O, Vojtisek J, Vycital O, Hosek P, et al. Immunohistochemical detection of cancer stem cell related markers CD44 and CD133 in metastatic colorectal cancer patients. Biomed Res Int. 2014;2014:432139. doi: 10.1155/2014/432139.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer. 2008;8(10):755–68. doi: 10.1038/nrc2499.CrossRefPubMedGoogle Scholar
  27. 27.
    Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci U S A. 2007;104(24):10158–63. doi: 10.1073/pnas.0703478104.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, et al. Identification and expansion of human colon-cancer-initiating cells. Nature. 2007;445(7123):111–5. doi: 10.1038/nature05384.CrossRefPubMedGoogle Scholar
  29. 29.
    Li L, Neaves WB. Normal stem cells and cancer stem cells: the niche matters. Cancer Res. 2006;66(9):4553–7. doi: 10.1158/0008-5472.CAN-05-3986.CrossRefPubMedGoogle Scholar
  30. 30.
    Du L, Wang H, He L, Zhang J, Ni B, Wang X, et al. CD44 is of functional importance for colorectal cancer stem cells. Clin Cancer Res. 2008;14(21):6751–60. doi: 10.1158/1078-0432.CCR-08-1034.CrossRefPubMedGoogle Scholar
  31. 31.
    Napier SL, Healy ZR, Schnaar RL, Konstantopoulos K. Selectin ligand expression regulates the initial vascular interactions of colon carcinoma cells: the roles of CD44v and alternative sialofucosylated selectin ligands. J Biol Chem. 2007;282(6):3433–41. doi: 10.1074/jbc.M607219200.CrossRefPubMedGoogle Scholar
  32. 32.
    Patrawala L, Calhoun-Davis T, Schneider-Broussard R, Tang DG. Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44 + alpha2beta1+ cell population is enriched in tumor-initiating cells. Cancer Res. 2007;67(14):6796–805. doi: 10.1158/0008-5472.CAN-07-0490.CrossRefPubMedGoogle Scholar
  33. 33.
    Ozawa H, Kotake K, Kobayashi H, Kobayashi H, Sugihara K. Prognostic factors for peritoneal carcinomatosis originating from colorectal cancer: an analysis of 921 patients from a multi-institutional database. Surg Today. 2014;44(9):1643–50. doi: 10.1007/s00595-013-0735-x.CrossRefPubMedGoogle Scholar
  34. 34.
    Aigner S, Ramos CL, Hafezi-Moghadam A, Lawrence MB, Friederichs J, Altevogt P, et al. CD24 mediates rolling of breast carcinoma cells on P-selectin. FASEB J. 1998;12(12):1241–51.PubMedGoogle Scholar
  35. 35.
    Zheng J, Li Y, Yang J, Liu Q, Shi M, Zhang R, et al. NDRG2 inhibits hepatocellular carcinoma adhesion, migration and invasion by regulating CD24 expression. BMC Cancer. 2011;11(251):1–9. doi: 10.1186/1471-2407-11-251.Google Scholar
  36. 36.
    van Kempen LC, van den Oord JJ, van Muijen GN, Weidle UH, Bloemers HP, Swart GW. Activated leukocyte cell adhesion molecule/CD166, a marker of tumor progression in primary malignant melanoma of the skin. Am J Pathol. 2000;156(3):769–74. doi: 10.1016/S0002-9440(10)64943-7.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Klein WM, Wu BP, Zhao S, Wu H, Klein-Szanto AJ, Tahan SR. Increased expression of stem cell markers in malignant melanoma. Mod Pathol. 2007;20(1):102–7. doi: 10.1038/modpathol.3800720.CrossRefPubMedGoogle Scholar
  38. 38.
    Kristiansen G, Pilarsky C, Wissmann C, Stephan C, Weissbach L, Loy V, et al. ALCAM/CD166 is up-regulated in low-grade prostate cancer and progressively lost in high-grade lesions. Prostate. 2003;54(1):34–43. doi: 10.1002/pros.10161.CrossRefPubMedGoogle Scholar
  39. 39.
    Burkhardt M, Mayordomo E, Winzer KJ, Fritzsche F, Gansukh T, Pahl S, et al. Cytoplasmic overexpression of ALCAM is prognostic of disease progression in breast cancer. J Clin Pathol. 2006;59(4):403–9. doi: 10.1136/jcp.2005.028209.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Mezzanzanica D, Fabbi M, Bagnoli M, Staurengo S, Losa M, Balladore E, et al. Subcellular localization of activated leukocyte cell adhesion molecule is a molecular predictor of survival in ovarian carcinoma patients. Clin Cancer Res. 2008;14(6):1726–33. doi: 10.1158/1078-0432.CCR-07-0428.CrossRefPubMedGoogle Scholar
  41. 41.
    Verma A, Shukla NK, Deo SV, Gupta SD, Ralhan R. MEMD/ALCAM: a potential marker for tumor invasion and nodal metastasis in esophageal squamous cell carcinoma. Oncology. 2005;68(4-6):462–70. doi: 10.1159/000086989.CrossRefPubMedGoogle Scholar
  42. 42.
    Weichert W, Knosel T, Bellach J, Dietel M, Kristiansen G. ALCAM/CD166 is overexpressed in colorectal carcinoma and correlates with shortened patient survival. J Clin Pathol. 2004;57(11):1160–4. doi: 10.1136/jcp.2004.016238.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Munz M, Baeuerle PA, Gires O. The emerging role of EpCAM in cancer and stem cell signaling. Cancer Res. 2009;69(14):5627–9. doi: 10.1158/0008-5472.CAN-09-0654.CrossRefPubMedGoogle Scholar
  44. 44.
    Fong D, Moser P, Kasal A, Seeber A, Gastl G, Martowicz A, et al. Loss of membranous expression of the intracellular domain of EpCAM is a frequent event and predicts poor survival in patients with pancreatic cancer. Histopathology. 2014;64(5):683–92. doi: 10.1111/his.12307.CrossRefPubMedGoogle Scholar
  45. 45.
    O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445(7123):106–10. doi: 10.1038/nature05372.CrossRefPubMedGoogle Scholar
  46. 46.
    Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, et al. Identification of pancreatic cancer stem cells. Cancer Res. 2007;67(3):1030–7. doi: 10.1158/0008-5472.CAN-06-2030.CrossRefPubMedGoogle Scholar
  47. 47.
    Yamashita T, Honda M, Nakamoto Y, Baba M, Nio K, Hara Y, et al. Discrete nature of EpCAM+ and CD90+ cancer stem cells in human hepatocellular carcinoma. Hepatology. 2013;57(4):1484–97. doi: 10.1002/hep.26168.CrossRefPubMedGoogle Scholar
  48. 48.
    Sukach AN, Ivanov EN. Formation of spherical colonies as a property of stem cells. Tsitologiia. 2007;49(11):916–22.PubMedGoogle Scholar
  49. 49.
    Vermeulen L, Todaro M, de Sousa MF, Sprick MR, Kemper K, Perez Alea M, et al. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci U S A. 2008;105(36):13427–32. doi: 10.1073/pnas.0805706105.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Ahmed MA, Al-Attar A, Kim J, Watson NF, Scholefield JH, Durrant LG, et al. CD24 shows early upregulation and nuclear expression but is not a prognostic marker in colorectal cancer. J Clin Pathol. 2009;62(12):1117–22. doi: 10.1136/jcp.2009.069310.CrossRefPubMedGoogle Scholar
  51. 51.
    Carulli AJ, Samuelson LC, Schnell S. Unraveling intestinal stem cell behavior with models of crypt dynamics. Integr Biol. 2014;6(3):243–57. doi: 10.1039/c3ib40163d.CrossRefGoogle Scholar
  52. 52.
    Merlos-Suarez A, Barriga FM, Jung P, Iglesias M, Cespedes MV, Rossell D, et al. The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell. 2011;8(5):511–24. doi: 10.1016/j.stem.2011.02.020.CrossRefPubMedGoogle Scholar
  53. 53.
    Shay JW, Wright WE. Telomeres and telomerase in normal and cancer stem cells. FEBS Lett. 2010;584(17):3819–25. doi: 10.1016/j.febslet.2010.05.026.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Braakhuis BJ, Tabor MP, Kummer JA, Leemans CR, Brakenhoff RH. A genetic explanation of Slaughter’s concept of field cancerization: evidence and clinical implications. Cancer Res. 2003;63(8):1727–30.PubMedGoogle Scholar
  55. 55.
    Bendardaf R, Algars A, Elzagheid A, Korkeila E, Ristamaki R, Lamlum H, et al. Comparison of CD44 expression in primary tumours and metastases of colorectal cancer. Oncol Rep. 2006;16(4):741–6.PubMedGoogle Scholar
  56. 56.
    Tachezy M, Zander H, Gebauer F, Marx A, Kaifi JT, Izbicki JR, et al. Activated leukocyte cell adhesion molecule (CD166)—its prognostic power for colorectal cancer patients. J Surg Res. 2012;177(1):e15–20. doi: 10.1016/j.jss.2012.02.013.CrossRefPubMedGoogle Scholar
  57. 57.
    Bjerkvig R, Tysnes BB, Aboody KS, Najbauer J, Terzis AJ. Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat Rev Cancer. 2005;5(11):899–904. doi: 10.1038/nrc1740.CrossRefPubMedGoogle Scholar
  58. 58.
    Amsterdam A, Raanan C, Schreiber L, Freyhan O, Fabrikant Y, Melzer E, et al. Differential localization of LGR5 and Nanog in clusters of colon cancer stem cells. Acta Histochem. 2013;115(4):320–9. doi: 10.1016/j.acthis.2012.09.003.CrossRefPubMedGoogle Scholar
  59. 59.
    Amsterdam A, Shezen E, Raanan C, Schreiber L, Slilat Y, Fabrikant Y, et al. Differential staining of gamma synuclein in poorly differentiated compared to highly differentiated colon cancer cells. Oncol Rep. 2012;27(5):1451–4. doi: 10.3892/or.2012.1658.PubMedGoogle Scholar
  60. 60.
    Bapat SA. Human ovarian cancer stem cells. Reproduction. 2010;140(1):33–41. doi: 10.1530/REP-09-0389.CrossRefPubMedGoogle Scholar
  61. 61.
    Dangles-Marie V, Pocard M, Richon S, Weiswald LB, Assayag F, Saulnier P, et al. Establishment of human colon cancer cell lines from fresh tumors versus xenografts: comparison of success rate and cell line features. Cancer Res. 2007;67(1):398–407. doi: 10.1158/0008-5472.CAN-06-0594.CrossRefPubMedGoogle Scholar
  62. 62.
    Weiswald LB, Richon S, Massonnet G, Guinebretiere JM, Vacher S, Laurendeau I, et al. A short-term colorectal cancer sphere culture as a relevant tool for human cancer biology investigation. Br J Cancer. 2013;108(8):1720–31. doi: 10.1038/bjc.2013.132.CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Weiswald LB, Richon S, Validire P, Briffod M, Lai-Kuen R, Cordelieres FP, et al. Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness. Br J Cancer. 2009;101(3):473–82. doi: 10.1038/sj.bjc.6605173.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • J. Manhas
    • 1
  • A. Bhattacharya
    • 1
  • S. K. Agrawal
    • 1
  • B. Gupta
    • 2
  • P. Das
    • 2
  • S. V. S. Deo
    • 3
  • S. Pal
    • 4
  • S. Sen
    • 1
    Email author
  1. 1.Department of BiochemistryAll India Institute of Medical SciencesNew DelhiIndia
  2. 2.Department of PathologyAll India Institute of Medical SciencesNew DelhiIndia
  3. 3.Department of Surgical OncologyAll India Institute of Medical SciencesNew DelhiIndia
  4. 4.Department of Gastrointestinal SurgeryAll India Institute of Medical SciencesNew DelhiIndia

Personalised recommendations