Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research

  • Myoung-Eun Han
  • Tae-Yong Jeon
  • Sun-Hwi Hwang
  • Young-Suk Lee
  • Hyun-Jung Kim
  • Hye-Eun Shim
  • Sik Yoon
  • Sun-Yong Baek
  • Bong-Seon Kim
  • Chi-Dug Kang
  • Sae-Ock Oh
Research article

Abstract

Cancer stem cells have been hypothesized to drive the growth and metastasis of tumors. Because they need to be targeted for cancer treatment, they have been isolated from many solid cancers. However, cancer stem cells from primary human gastric cancer tissues have not been isolated as yet. For the isolation, we used two cell surface markers: the epithelial cell adhesion molecule (EpCAM) and CD44. When analyzed by flow cytometry, the EpCAM+/CD44+ population accounts for 4.5% of tumor cells. EpCAM+/CD44+ gastric cancer cells formed tumors in immunocompromised mice; however, EpCAM/CD44, EpCAM+/CD44 and EpCAM/CD44+ cells failed to do so. Xenografts of EpCAM+/CD44+ gastric cancer cells maintained a differentiated phenotype and reproduced the morphological and phenotypical heterogeneity of the original gastric tumor tissues. The tumorigenic subpopulation was serially passaged for several generations without significant phenotypic alterations. Moreover, EpCAM+/CD44+, but not EpCAM/CD44, EpCAM+/CD44 or EpCAM/CD44+ cells grew exponentially in vitro as cancer spheres in serum-free medium, maintaining the tumorigenicity. Interestingly, a single cancer stem cell generated a cancer sphere that contained various differentiated cells, supporting multi-potency and self-renewal of a cancer stem cell. EpCAM+/CD44+ cells had greater resistance to anti-cancer drugs than other subpopulation cells. The above in vivo and in vitro results suggest that cancer stem cells, which are enriched in the EpCAM+/CD44+ subpopulation of gastric cancer cells, provide an ideal model system for cancer stem cell research.

Keywords

Cancer stem cells Gastric cancer CD44 EpCAM Cancer sphere 

Notes

Acknowledgments

This work was supported by the medical research centre program of the Ministry of Education, Science and Technology/Korea Science and Engineering Foundation (2011-0006190), the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. 2009-0076704) and a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs, Republic of Korea (0920050).

Conflict of interest

There are no conflicts of interest.

References

  1. 1.
    Han ME, Lee YS, Baek SY, Kim BS, Kim JB, Oh SO (2009) Hedgehog signaling regulates the survival of gastric cancer cells by regulating the expression of bcl-2. Int J Mol Sci 10(7):3033–3043PubMedCrossRefGoogle Scholar
  2. 2.
    Yuasa Y (2003) Control of gut differentiation and intestinal-type gastric carcinogenesis. Nat Rev Cancer 3(8):592–600PubMedCrossRefGoogle Scholar
  3. 3.
    Correia M, Machado JC, Ristimaki A (2009) Basic aspects of gastric cancer. Helicobacter 14(Suppl 1):36–40PubMedCrossRefGoogle Scholar
  4. 4.
    Vogiatzi P, Vindigni C, Roviello F, Renieri A, Giordano A (2007) Deciphering the underlying genetic and epigenetic events leading to gastric carcinogenesis. J Cell Physiol 211(2):287–295PubMedCrossRefGoogle Scholar
  5. 5.
    Wu CW, Hsieh MC, Lo SS, Lui WY, P’Eng FK (1996) Results of curative gastrectomy for carcinoma of the distal third of the stomach. J Am Coll Surg 183(3):201–207PubMedGoogle Scholar
  6. 6.
    Wu CW, Hsieh MC, Lo SS, Tsay SH, Li AF, Lui WY, P’Eng FK (1997) Prognostic indicators for survival after curative resection for patients with carcinoma of the stomach. Dig Dis Sci 42(6):1265–1269PubMedCrossRefGoogle Scholar
  7. 7.
    Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM (2006) Cancer stem cells–perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res 66(19):9339–9344PubMedCrossRefGoogle Scholar
  8. 8.
    Brabletz T, Hlubek F, Spaderna S, Schmalhofer O, Hiendlmeyer E, Jung A, Kirchner T (2005) Invasion and metastasis in colorectal cancer: epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and beta-catenin. Cells tissues organs 179(1–2):56–65PubMedCrossRefGoogle Scholar
  9. 9.
    Brabletz T, Jung A, Spaderna S, Hlubek F, Kirchner T (2005) Opinion: migrating cancer stem cells-an integrated concept of malignant tumour progression. Nat Rev Cancer 5(9):744–749PubMedCrossRefGoogle Scholar
  10. 10.
    Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133(4):704–715PubMedCrossRefGoogle Scholar
  11. 11.
    Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, Bruns CJ, Heeschen C (2007) Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell stem cell 1(3):313–323PubMedCrossRefGoogle Scholar
  12. 12.
    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100(7):3983–3988PubMedCrossRefGoogle Scholar
  13. 13.
    Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63(18):5821–5828PubMedGoogle Scholar
  14. 14.
    Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65(23):10946–10951PubMedCrossRefGoogle Scholar
  15. 15.
    Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S, Van Belle PA, Xu X, Elder DE, Herlyn M (2005) A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 65(20):9328–9337PubMedCrossRefGoogle Scholar
  16. 16.
    O’Brien CA, Pollett A, Gallinger S, Dick JE (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445(7123):106–110PubMedCrossRefGoogle Scholar
  17. 17.
    Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445(7123):111–115PubMedCrossRefGoogle Scholar
  18. 18.
    Takaishi S, Okumura T, Tu S, Wang SS, Shibata W, Vigneshwaran R, Gordon SA, Shimada Y, Wang TC (2009) Identification of gastric cancer stem cells using the cell surface marker CD44. Stem cells 27(5):1006–1020PubMedCrossRefGoogle Scholar
  19. 19.
    Nishii T, Yashiro M, Shinto O, Sawada T, Ohira M, Hirakawa K (2009) Cancer stem cell-like SP cells have a high adhesion ability to the peritoneum in gastric carcinoma. Cancer Sci 100(8):1397–1402PubMedCrossRefGoogle Scholar
  20. 20.
    Fukuda K, Saikawa Y, Ohashi M, Kumagai K, Kitajima M, Okano H, Matsuzaki Y, Kitagawa Y (2009) Tumor initiating potential of side population cells in human gastric cancer. Int J Oncol 34(5):1201–1207PubMedGoogle Scholar
  21. 21.
    Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG (2005) Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2 + and ABCG2 − cancer cells are similarly tumorigenic. Cancer Res 65(14):6207–6219PubMedCrossRefGoogle Scholar
  22. 22.
    Burkert J, Otto WR, Wright NA (2008) Side populations of gastrointestinal cancers are not enriched in stem cells. J Pathol 214(5):564–573PubMedCrossRefGoogle Scholar
  23. 23.
    Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, Hoey T, Gurney A, Huang EH, Simeone DM, Shelton AA, Parmiani G, Castelli C, Clarke MF (2007) Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA 104(24):10158–10163PubMedCrossRefGoogle Scholar
  24. 24.
    Vescovi AL, Parati EA, Gritti A, Poulin P, Ferrario M, Wanke E, Frolichsthal-Schoeller P, Cova L, Arcellana-Panlilio M, Colombo A, Galli R (1999) Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 156(1):71–83PubMedCrossRefGoogle Scholar
  25. 25.
    Haraguchi N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, Akita H, Takiuchi D, Hatano H, Nagano H, Barnard GF, Doki Y, Mori M (2010) CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest 120(9):3326–3339Google Scholar
  26. 26.
    Lobo NA, Shimono Y, Qian D, Clarke MF (2007) The biology of cancer stem cells. Annu Rev Cell Dev Biol 23:675–699PubMedCrossRefGoogle Scholar
  27. 27.
    Smith MG, Hold GL, Tahara E, El-Omar EM (2006) Cellular and molecular aspects of gastric cancer. World J Gastroenterol 12(19):2979–2990PubMedGoogle Scholar
  28. 28.
    Liu YJ, Yan PS, Li J, Jia JF (2005) Expression and significance of CD44 s, CD44v6, and nm23 mRNA in human cancer. World J Gastroenterol 11(42):6601–6606PubMedGoogle Scholar
  29. 29.
    Yoo CH, Noh SH, Kim H, Lee HY, Min JS (1999) Prognostic significance of CD44 and nm23 expression in patients with stage II and stage IIIA gastric carcinoma. J Surg Oncol 71(1):22–28PubMedCrossRefGoogle Scholar
  30. 30.
    Winder T, Ning Y, Yang D, Zhang W, Power DG, Bohanes P, Gerger A, Wilson PM, Lurje G, Tang LH, Shah M, Lenz HJ (2010) Germline polymorphisms in genes involved in the CD44 signaling pathway are associated with clinical outcome in localized gastric adenocarcinoma (GA). Int J Cancer [Epub ahead of print]Google Scholar
  31. 31.
    Du W, Ji H, Cao S, Wang L, Bai F, Liu J, Fan D (2010) EpCAM: a potential antimetastatic target for gastric cancer. Dig Dis Sci 55 (8):2165–2171Google Scholar
  32. 32.
    Ponta H, Sherman L, Herrlich PA (2003) CD44: from adhesion molecules to signalling regulators. Nature Rev 4(1):33–45CrossRefGoogle Scholar
  33. 33.
    Barbour AP, Reeder JA, Walsh MD, Fawcett J, Antalis TM, Gotley DC (2003) Expression of the CD44v2–10 isoform confers a metastatic phenotype: importance of the heparan sulfate attachment site CD44v3. Cancer Res 63(4):887–892PubMedGoogle Scholar
  34. 34.
    Gunthert U, Hofmann M, Rudy W, Reber S, Zoller M, Haussmann I, Matzku S, Wenzel A, Ponta H, Herrlich P (1991) A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65(1):13–24PubMedCrossRefGoogle Scholar
  35. 35.
    Heider KH, Dammrich J, Skroch-Angel P, Muller-Hermelink HK, Vollmers HP, Herrlich P, Ponta H (1993) Differential expression of CD44 splice variants in intestinal- and diffuse-type human gastric carcinomas and normal gastric mucosa. Cancer Res 53(18):4197–4203PubMedGoogle Scholar
  36. 36.
    Klingbeil P, Marhaba R, Jung T, Kirmse R, Ludwig T, Zoller M (2009) CD44 variant isoforms promote metastasis formation by a tumor cell-matrix cross-talk that supports adhesion and apoptosis resistance. Mol Cancer Res 7(2):168–179PubMedCrossRefGoogle Scholar
  37. 37.
    Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367(6464):645–648PubMedCrossRefGoogle Scholar
  38. 38.
    Ponti D, Zaffaroni N, Capelli C, Daidone MG (2006) Breast cancer stem cells: an overview. Eur J Cancer 42(9):1219–1224PubMedCrossRefGoogle Scholar
  39. 39.
    Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM (2007) Identification of pancreatic cancer stem cells. Cancer Res 67(3):1030–1037PubMedCrossRefGoogle Scholar
  40. 40.
    Du L, Wang H, He L, Zhang J, Ni B, Wang X, Jin H, Cahuzac N, Mehrpour M, Lu Y, Chen Q (2008) CD44 is of functional importance for colorectal cancer stem cells. Clin Cancer Res 14(21):6751–6760PubMedCrossRefGoogle Scholar
  41. 41.
    Matzke A, Sargsyan V, Holtmann B, Aramuni G, Asan E, Sendtner M, Pace G, Howells N, Zhang W, Ponta H, Orian-Rousseau V (2007) Haploinsufficiency of c-Met in cd44-/- mice identifies a collaboration of CD44 and c-Met in vivo. Mol Cell Biol 27(24):8797–8806PubMedCrossRefGoogle Scholar
  42. 42.
    Guo YJ, Liu G, Wang X, Jin D, Wu M, Ma J, Sy MS (1994) Potential use of soluble CD44 in serum as indicator of tumor burden and metastasis in patients with gastric or colon cancer. Cancer Res 54(2):422–426PubMedGoogle Scholar
  43. 43.
    Munz M, Baeuerle PA, Gires O (2009) The emerging role of EpCAM in cancer and stem cell signaling. Cancer Res 69(14):5627–5629PubMedCrossRefGoogle Scholar
  44. 44.
    Baeuerle PA, Gires O (2007) EpCAM (CD326) finding its role in cancer. Br J Cancer 96(3):417–423PubMedCrossRefGoogle Scholar
  45. 45.
    Trzpis M, McLaughlin PM, de Leij LM, Harmsen MC (2007) Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule. Am J Pathol 171(2):386–395PubMedCrossRefGoogle Scholar
  46. 46.
    Gonzalez B, Denzel S, Mack B, Conrad M, Gires O (2009) EpCAM is involved in maintenance of the murine embryonic stem cell phenotype. Stem cells 27(8):1782–1791PubMedCrossRefGoogle Scholar
  47. 47.
    Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, Schlimok G, Diel IJ, Gerber B, Gebauer G, Pierga JY, Marth C, Oruzio D, Wiedswang G, Solomayer EF, Kundt G, Strobl B, Fehm T, Wong GY, Bliss J, Vincent-Salomon A, Pantel K (2005) A pooled analysis of bone marrow micrometastasis in breast cancer. N Engl J Med 353(8):793–802PubMedCrossRefGoogle Scholar
  48. 48.
    Pantel K, Woelfle U (2005) Detection and molecular characterisation of disseminated tumour cells: implications for anti-cancer therapy. Biochim Biophys Acta 1756(1):53–64PubMedGoogle Scholar
  49. 49.
    Balzar M, Winter MJ, de Boer CJ, Litvinov SV (1999) The biology of the 17–1A antigen (Ep-CAM). J Mol Med 77(10):699–712PubMedCrossRefGoogle Scholar
  50. 50.
    Osta WA, Chen Y, Mikhitarian K, Mitas M, Salem M, Hannun YA, Cole DJ, Gillanders WE (2004) EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res 64(16):5818–5824PubMedCrossRefGoogle Scholar
  51. 51.
    Maetzel D, Denzel S, Mack B, Canis M, Went P, Benk M, Kieu C, Papior P, Baeuerle PA, Munz M, Gires O (2009) Nuclear signalling by tumour-associated antigen EpCAM. Nat Cell Biol 11(2):162–171PubMedCrossRefGoogle Scholar
  52. 52.
    Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, Jia H, Ye Q, Qin LX, Wauthier E, Reid LM, Minato H, Honda M, Kaneko S, Tang ZY, Wang XW (2009) EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology 136(3):1012–1024PubMedCrossRefGoogle Scholar
  53. 53.
    Kimura O, Takahashi T, Ishii N, Inoue Y, Ueno Y, Kogure T, Fukushima K, Shiina M, Yamagiwa Y, Kondo Y, Inoue J, Kakazu E, Iwasaki T, Kawagishi N, Shimosegawa T, Sugamura K (2010) Characterization of the epithelial cell adhesion molecule (EpCAM) + cell population in hepatocellular carcinoma cell lines. Cancer sci 101(10):2145–2155Google Scholar
  54. 54.
    Becker KF, Atkinson MJ, Reich U, Becker I, Nekarda H, Siewert JR, Hofler H (1994) E-cadherin gene mutations provide clues to diffuse type gastric carcinomas. Cancer Res 54(14):3845–3852PubMedGoogle Scholar
  55. 55.
    Yokota J, Yamamoto T, Miyajima N, Toyoshima K, Nomura N, Sakamoto H, Yoshida T, Terada M, Sugimura T (1988) Genetic alterations of the c-erbB-2 oncogene occur frequently in tubular adenocarcinoma of the stomach and are often accompanied by amplification of the v-erbA homologue. Oncogene 2(3):283–287PubMedGoogle Scholar
  56. 56.
    Kuhn S, Koch M, Nubel T, Ladwein M, Antolovic D, Klingbeil P, Hildebrand D, Moldenhauer G, Langbein L, Franke WW, Weitz J, Zoller M (2007) A complex of EpCAM, claudin-7, CD44 variant isoforms, and tetraspanins promotes colorectal cancer progression. Mol Cancer Res 5(6):553–567PubMedCrossRefGoogle Scholar
  57. 57.
    Schmidt DS, Klingbeil P, Schnolzer M, Zoller M (2004) CD44 variant isoforms associate with tetraspanins and EpCAM. Exp Cell Res 297(2):329–347PubMedCrossRefGoogle Scholar
  58. 58.
    Grimshaw MJ, Cooper L, Papazisis K, Coleman JA, Bohnenkamp HR, Chiapero-Stanke L, Taylor-Papadimitriou J, Burchell JM (2008) Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res 10(3):R52PubMedCrossRefGoogle Scholar
  59. 59.
    Scopelliti A, Cammareri P, Catalano V, Saladino V, Todaro M, Stassi G (2009) Therapeutic implications of cancer initiating cells. Expert Opin Biol Ther 9(8):1005–1016PubMedCrossRefGoogle Scholar
  60. 60.
    Lakshman M, Subramaniam V, Wong S, Jothy S (2005) CD44 promotes resistance to apoptosis in murine colonic epithelium. J Cell Physiol 203(3):583–588PubMedCrossRefGoogle Scholar
  61. 61.
    Ohashi R, Takahashi F, Cui R, Yoshioka M, Gu T, Sasaki S, Tominaga S, Nishio K, Tanabe KK, Takahashi K (2007) Interaction between CD44 and hyaluronate induces chemoresistance in non-small cell lung cancer cell. Cancer Lett 252(2):225–234PubMedCrossRefGoogle Scholar
  62. 62.
    Yaqin M, Runhua L, Fuxi Z (2007) Analyses of Bcl-2, Survivin, and CD44v6 expressions and human papillomavirus infection in cervical carcinomas. Scand J Infect Dis 39(5):441–448PubMedCrossRefGoogle Scholar
  63. 63.
    Cordo Russo RI, Garcia MG, Alaniz L, Blanco G, Alvarez E, Hajos SE (2008) Hyaluronan oligosaccharides sensitize lymphoma resistant cell lines to vincristine by modulating P-glycoprotein activity and PI3 K/Akt pathway. Int J Cancer 122(5):1012–1018PubMedCrossRefGoogle Scholar
  64. 64.
    Lin YH, Yang-Yen HF (2001) The osteopontin-CD44 survival signal involves activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. J Biol Chem 276(49):46024–46030PubMedCrossRefGoogle Scholar
  65. 65.
    Nubel T, Preobraschenski J, Tuncay H, Weiss T, Kuhn S, Ladwein M, Langbein L, Zoller M (2009) Claudin-7 regulates EpCAM-mediated functions in tumor progression. Mol Cancer Res 7(3):285–299PubMedCrossRefGoogle Scholar
  66. 66.
    Vazquez A, Grochola LF, Bond EE, Levine AJ, Taubert H, Muller TH, Wurl P, Bond GL (2010) Chemosensitivity profiles identify polymorphisms in the p53 network genes 14-3-3tau and CD44 that affect sarcoma incidence and survival. Cancer Res 70(1):172–180Google Scholar
  67. 67.
    Marx J (2007) Molecular biology. Cancer’s perpetual source? Science 317(5841):1029–1031PubMedCrossRefGoogle Scholar
  68. 68.
    Kelly PN, Dakic A, Adams JM, Nutt SL, Strasser A (2007) Tumor growth need not be driven by rare cancer stem cells. Science 317(5836):337PubMedCrossRefGoogle Scholar
  69. 69.
    Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morrison SJ (2008) Efficient tumour formation by single human melanoma cells. Nature 456(7222):593–598PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel AG 2011

Authors and Affiliations

  • Myoung-Eun Han
    • 1
    • 2
  • Tae-Yong Jeon
    • 3
  • Sun-Hwi Hwang
    • 3
  • Young-Suk Lee
    • 1
  • Hyun-Jung Kim
    • 1
    • 2
  • Hye-Eun Shim
    • 1
    • 2
  • Sik Yoon
    • 1
  • Sun-Yong Baek
    • 1
  • Bong-Seon Kim
    • 1
  • Chi-Dug Kang
    • 4
  • Sae-Ock Oh
    • 1
    • 2
  1. 1.Department of AnatomySchool of Medicine, Pusan National UniversityYangsan-siRepublic of Korea
  2. 2.Medical Research Center for Ischemic Tissue RegenerationPusan National UniversityYangsanRepublic of Korea
  3. 3.Department of SurgerySchool of Medicine, Pusan National UniversityYangsanRepublic of Korea
  4. 4.Department of BiochemistrySchool of Medicine, Pusan National UniversityYangsanRepublic of Korea

Personalised recommendations