Gastric Cancer

, Volume 22, Issue 5, pp 967–979 | Cite as

Identification of different gene expressions between diffuse- and intestinal-type spheroid-forming gastric cancer cells

  • Jong Won Lee
  • Jae Sook Sung
  • Young Soo Park
  • Seok Chung
  • Yeul Hong KimEmail author
Original Article



Three-dimensional in vitro spheroid models are unique because they are considered for enrichment of specific cell populations with self-renewal ability. In this study, we explored the different mechanisms of gastric cancer spheroid-forming cells according to the Lauren classification.


We isolated and enriched cells with self-renewal ability using spheroid-forming methods from gastric cancer cell lines. The expression of candidate target genes was investigated using western blot and qRT-PCR analysis. Lentiviral shRNA knockdown of target gene expression was performed and the effects on spheroid, colony forming, and tumorigenic ability were analyzed.


The SNU-638, SNU-484, MKN-28, and NCI-N87 successfully formed spheroid from single cell and enriched for self-renewal ability from 11 gastric cancer cell lines, including diffuse and intestinal types. The expression of SOX2 and E-cadherin increased in spheroid-forming cells in a diffuse-type cell line (SNU-638 and SNU-484), but not in the intestinal type (MKN-28 and NCI-N87). In contrast, ERBB3 expression was only increased in intestinal-type spheroid cells. The depletion of each candidate target gene expression suppressed self-renewal ability to grow as spheroids and colonies in a soft agar assay. In particular, down-regulated ERBB3 in the intestinal-type cell lines inhibited tumor growth in a mouse xenograft model. We found that high ERBB3 gene expression correlates with decreased survival in the intestinal type of gastric cancer.


Our results suggest that diffuse- and intestinal-type spheroid-forming cells express genes differently. Our data suggest that these candidate genes from spheroid-forming cells can be used in applications in targeted therapy.


Gastric cancer Cellular spheroid Lauren classification Diffuse type Intestinal type 



This study was supported by a Grant from the National Research Foundation of Korea (NRF), which is funded by the Korean government (MEST) (No. 2010-0020986).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Animal studies

All institutional and national guidelines for the care and use of laboratory animals were followed.


  1. 1.
    Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108.CrossRefGoogle Scholar
  2. 2.
    Jung KW, Won YJ, Kong HJ, Lee ES. Cancer statistics in Korea: incidence, mortality, survival, and prevalence in 2015. Cancer Res Treat. 2018;50:303–16.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hohenberger P, Gretschel S. Gastric cancer. Lancet. 2003;362:305–15.CrossRefPubMedGoogle Scholar
  4. 4.
    Visvader JE, Lindeman GJ. Cancer stem cells: current status and evolving complexities. Cell Stem Cell. 2012;10:717–28.CrossRefPubMedGoogle Scholar
  5. 5.
    Magee JA, Piskounova E, Morrison SJ. Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell. 2012;21:283–96.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ivanov DP, Grabowska AM. Spheroid arrays for high-throughput single-cell analysis of spatial patterns and biomarker expression in 3D. Sci Rep. 2017;7:41160.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Weiswald LB, Bellet D, Dangles-Marie V. Spherical cancer models in tumor biology. Neoplasia. 2015;17:1–15.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S, et al. A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res. 2005;65:9328–37.CrossRefPubMedGoogle Scholar
  9. 9.
    Wright MH, Calcagno AM, Salcido CD, Carlson MD, Ambudkar SV, Varticovski L. Brca1 breast tumors contain distinct CD44+/CD24− and CD133+ cells with cancer stem cell characteristics. Breast Cancer Res. 2008;10:R10.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Takaishi S, Okumura T, Tu S, Wang SS, Shibata W, Vigneshwaran R, et al. Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells. 2009;27:1006–20.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Todaro M, Alea MP, Di Stefano AB, Cammareri P, Vermeulen L, Iovino F, et al. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell. 2007;1:389–402.CrossRefGoogle Scholar
  12. 12.
    Qureshi-Baig K, Ullmann P, Rodriguez F, Frasquilho S, Nazarov PV, Haan S, et al. What do we learn from spheroid culture systems? Insights from tumorspheres derived from primary colon cancer tissue. PLoS One. 2016;11:e0146052.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Song Z, Yue W, Wei B, Wang N, Li T, Guan L, et al. Sonic hedgehog pathway is essential for maintenance of cancer stem-like cells in human gastric cancer. PLoS One. 2011;6:e17687.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Fennema E, Rivron N, Rouwkema J, van Blitterswijk C, de Boer J. Spheroid culture as a tool for creating 3D complex tissues. Trends Biotechnol. 2013;31:108–15.CrossRefPubMedGoogle Scholar
  15. 15.
    Han XY, Wei B, Fang JF, Zhang S, Zhang FC, Zhang HB, et al. Epithelial-mesenchymal transition associates with maintenance of stemness in spheroid-derived stem-like colon cancer cells. PLoS One. 2013;8:e73341.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Davidowitz RA, Selfors LM, Iwanicki MP, Elias KM, Karst A, Piao H, et al. Mesenchymal gene program-expressing ovarian cancer spheroids exhibit enhanced mesothelial clearance. J Clin Investig. 2014;124:2611–25.CrossRefPubMedGoogle Scholar
  17. 17.
    Lauren P. The two histological main types of gastric. Carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification. Acta Pathol Microbiol Scand. 1965;64:31–49.CrossRefPubMedGoogle Scholar
  18. 18.
    Lynch HT, Grady W, Suriano G, Huntsman D. Gastric cancer: new genetic developments. J Surg Oncol. 2005;90:114–33 (discussion 33).CrossRefPubMedGoogle Scholar
  19. 19.
    Hu B, El Hajj N, Sittler S, Lammert N, Barnes R, Meloni-Ehrig A. Gastric cancer: classification, histology and application of molecular pathology. J Gastrointest Oncol. 2012;3:251–61.PubMedPubMedCentralGoogle Scholar
  20. 20.
    TCGA. Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 2014;513:202–9.CrossRefGoogle Scholar
  21. 21.
    Wang M, Busuttil RA, Pattison S, Neeson PJ, Boussioutas A. Immunological battlefield in gastric cancer and role of immunotherapies. World J Gastroenterol. 2016;22:6373–84.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kim ST, Cristescu R, Bass AJ, Kim KM, Odegaard JI, Kim K, et al. Comprehensive molecular characterization of clinical responses to PD-1 inhibition in metastatic gastric cancer. Nat Med. 2018;24:1449–58.CrossRefPubMedGoogle Scholar
  23. 23.
    Kankeu Fonkoua L, Yee NS. Molecular characterization of gastric carcinoma: therapeutic implications for biomarkers and targets. Biomedicines. 2018;6:32.CrossRefPubMedCentralGoogle Scholar
  24. 24.
    Tan IB, Ivanova T, Lim KH, Ong CW, Deng N, Lee J, et al. Intrinsic subtypes of gastric cancer, based on gene expression pattern, predict survival and respond differently to chemotherapy. Gastroenterology. 2011;141:476–85 (85.e1–11).CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Lee JW, Sung JS, Park YS, Chung S, Kim YH. Isolation of spheroid-forming single cells from gastric cancer cell lines: enrichment of cancer stem-like cells. Biotechniques. 2018;65:197–203.CrossRefPubMedGoogle Scholar
  26. 26.
    Zhi QM, Chen XH, Ji J, Zhang JN, Li JF, Cai Q, et al. Salinomycin can effectively kill ALDH(high) stem-like cells on gastric cancer. Biomed Pharmacother. 2011;65:509–15.CrossRefPubMedGoogle Scholar
  27. 27.
    Lee JS, Hmama Z, Mui A, Reiner NE. Stable gene silencing in human monocytic cell lines using lentiviral-delivered small interference RNA. Silencing of the p110alpha isoform of phosphoinositide 3-kinase reveals differential regulation of adherence induced by 1alpha,25-dihydroxycholecalciferol and bacterial lipopolysaccharide. J Biol Chem. 2004;279:9379–88.CrossRefPubMedGoogle Scholar
  28. 28.
    Losman JA, Looper RE, Koivunen P, Lee S, Schneider RK, McMahon C, et al. (R)-2-hydroxyglutarate is sufficient to promote leukemogenesis and its effects are reversible. Science. 2013;339:1621–5.CrossRefPubMedGoogle Scholar
  29. 29.
    Lanczky A, Nagy A, Bottai G, Munkacsy G, Szabo A, Santarpia L, et al. miRpower: a web-tool to validate survival-associated miRNAs utilizing expression data from 2178 breast cancer patients. Breast Cancer Res Treat. 2016;160:439–46.CrossRefPubMedGoogle Scholar
  30. 30.
    Liu J, Ma L, Xu J, Liu C, Zhang J, Liu J, et al. Spheroid body-forming cells in the human gastric cancer cell line MKN-45 possess cancer stem cell properties. Int J Oncol. 2013;42:453–9.CrossRefPubMedGoogle Scholar
  31. 31.
    Wang AM, Huang TT, Hsu KW, Huang KH, Fang WL, Yang MH, et al. Yin Yang 1 is a target of microRNA-34 family and contributes to gastric carcinogenesis. Oncotarget. 2014;5:5002–16.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Lau WM, Teng E, Chong HS, Lopez KA, Tay AY, Salto-Tellez M, et al. CD44v8-10 is a cancer-specific marker for gastric cancer stem cells. Cancer Res. 2014;74:2630–41.CrossRefPubMedGoogle Scholar
  33. 33.
    Chang HR, Park HS, Ahn YZ, Nam S, Jung HR, Park S, et al. Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems. BMC Cancer. 2016;9:200.CrossRefGoogle Scholar
  34. 34.
    Rotem A, Janzer A, Izar B, Ji Z, Doench JG, Garraway LA, et al. Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation. Proc Natl Acad Sci USA. 2015;112:5708–13.CrossRefPubMedGoogle Scholar
  35. 35.
    Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal. 2013;6:pl1.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Shihab HA, Rogers MF, Gough J, Mort M, Cooper DN, Day IN, et al. An integrative approach to predicting the functional effects of non-coding and coding sequence variation. Bioinformatics. 2015;31:1536–43.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell population by fluorescence activated cell sorting (FACS). J Vis Exp. 2010. Scholar
  38. 38.
    Leis O, Eguiara A, Lopez-Arribillaga E, Alberdi MJ, Hernandez-Garcia S, Elorriaga K, et al. Sox2 expression in breast tumours and activation in breast cancer stem cells. Oncogene. 2012;31:1354–65.CrossRefPubMedGoogle Scholar
  39. 39.
    Boumahdi S, Driessens G, Lapouge G, Rorive S, Nassar D, Le Mercier M, et al. SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma. Nature. 2014;511:246–50.CrossRefPubMedGoogle Scholar
  40. 40.
    Hutz K, Mejias-Luque R, Farsakova K, Ogris M, Krebs S, Anton M, et al. The stem cell factor SOX2 regulates the tumorigenic potential in human gastric cancer cells. Carcinogenesis. 2014;35:942–50.CrossRefPubMedGoogle Scholar
  41. 41.
    van Roy F, Berx G. The cell-cell adhesion molecule E-cadherin. Cell Mol Life Sci. 2008;65:3756–88.CrossRefGoogle Scholar
  42. 42.
    Zhang J, Chen XY, Huang KJ, Wu WD, Jiang T, Cao J, et al. Expression of FoxM1 and the EMT-associated protein E-cadherin in gastric cancer and its clinical significance. Oncol Lett. 2016;12:2445–50.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Becker KF, Hofler H. Frequent somatic allelic inactivation of the E-cadherin gene in gastric carcinomas. J Natl Cancer Inst. 1995;87:1082–4.CrossRefPubMedGoogle Scholar
  44. 44.
    Manuel Iglesias J, Beloqui I, Garcia-Garcia F, Leis O, Vazquez-Martin A, Eguiara A, et al. Mammosphere formation in breast carcinoma cell lines depends upon expression of E-cadherin. PLoS One. 2013;8:e77281.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Mujoo K, Choi BK, Huang Z, Zhang N, An Z. Regulation of ERBB3/HER3 signaling in cancer. Oncotarget. 2014;5:10222–36.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Pinkas-Kramarski R, Soussan L, Waterman H, Levkowitz G, Alroy I, Klapper L, et al. Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J. 1996;15:2452–67.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Hayashi M, Inokuchi M, Takagi Y, Yamada H, Kojima K, Kumagai J, et al. High expression of HER3 is associated with a decreased survival in gastric cancer. Clin Cancer Res. 2008;14:7843–9.CrossRefPubMedGoogle Scholar
  48. 48.
    He XX, Ding L, Lin Y, Shu M, Wen JM, Xue L. Protein expression of HER2, 3, 4 in gastric cancer: correlation with clinical features and survival. J Clin Pathol. 2015;68:374–80.CrossRefPubMedGoogle Scholar

Copyright information

© The International Gastric Cancer Association and The Japanese Gastric Cancer Association 2019

Authors and Affiliations

  • Jong Won Lee
    • 1
    • 3
  • Jae Sook Sung
    • 2
  • Young Soo Park
    • 2
  • Seok Chung
    • 4
  • Yeul Hong Kim
    • 1
    • 2
    • 3
    Email author
  1. 1.Brain Korea 21 Plus Project for Biomedical ScienceKorea University College of MedicineSeoulRepublic of Korea
  2. 2.Cancer Research InstituteKorea University College of MedicineSeoulRepublic of Korea
  3. 3.Division of Oncology/Hematology, Department of MedicineKorea University Anam Hospital, Korea University College of MedicineSeoulRepublic of Korea
  4. 4.School of Mechanical EngineeringKorea UniversitySeoulRepublic of Korea

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