Pediatric Surgery International

, 24:1229

Three-dimensional neuroblastoma cell culture: proteomic analysis between monolayer and multicellular tumor spheroids

  • Hari R. Kumar
  • Xiaoling Zhong
  • Derek J. Hoelz
  • Frederick J. Rescorla
  • Robert J. Hickey
  • Linda H. Malkas
  • John A. Sandoval
Original Article

Abstract

Introduction

Solid tumors, such as neuroblastoma (NB), are associated with a heterogeneous cell environment. Multicellular tumor spheroid (MCTS) cultures have been shown to better mimic growth characteristics of in vivo solid tumors. Because tumor spheroid growth patterns may be quite different from standard two-dimensional culture systems, we sought to compare the protein expression profiles of two- and three-dimensional in vitro NB cultures, i.e., monolayers and MCTS.

Materials and methods

Human NB cells were grown as both monolayers and spheres. Nuclear and cytosolic proteins were analyzed for differentially secreted proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and selected polypeptides were identified by mass spectrometry (LC-MS/MS).

Results

Several metabolic (transketolase, triosephosphate isomerase, pyruvate kinase M1/M2, alpha enolase, and phosphoglycerate mutase-1), cell stress response (heat shock proteins (HSP) 90, 70, and 60; antioxidant, thioredoxin), cell structure (septin 2, adenyl cyclase-associated protein-1), tubulin β-2 chain, actin, translationally controlled tumor protein and cofilin), signal transduction (peptidyl prolyl cis/trans isomerase A), biosynthetic (phosphoserine aminotransferase) and transport (cellular retinoic acid binding protein 1) polypeptides were overexpressed in spheroids. Several protein groups were differentially expressed between NB monolayers and spheroids.

Conclusion

The altered proteins among NB spheroids may represent an important link between monolayer cell cultures and in vivo experiments and thus a more ideal in vitro culture system for determining the precise threedimensional microenvironment of NB.

Keywords

Neuroblastoma Monolayers 3-D Culture Multicellular spheroid Proteomics Tumor microenvironment 

References

  1. 1.
    Vaupel P, Kallinowski F, Okunieff P (1989) Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 49:6449–6465PubMedGoogle Scholar
  2. 2.
    Thomlinson RH, Gray LH (1955) The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer 9:539–549PubMedGoogle Scholar
  3. 3.
    Teicher BA (1994) Hypoxia and drug resistance. Cancer Metastasis Rev 13:139–168. doi:10.1007/BF00689633 PubMedCrossRefGoogle Scholar
  4. 4.
    Subarsky P, Hill RP (2003) The hypoxic tumour microenvironment and metastatic progression. Clin Exp Metastasis 20:237–250. doi:10.1023/A:1022939318102 PubMedCrossRefGoogle Scholar
  5. 5.
    Hockel M et al (1996) Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res 56:4509–4515PubMedGoogle Scholar
  6. 6.
    Sutherland RM, McCredie JA, Inch WR (1971) Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. J Natl Cancer Inst 46:113–120PubMedGoogle Scholar
  7. 7.
    Sutherland RM (1988) Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240:177–184. doi:10.1126/science.2451290 PubMedCrossRefGoogle Scholar
  8. 8.
    Genc M et al (2004) Enhancement of effects of irradiation by gemcitabine in a glioblastoma cell line and cell line spheroids. J Cancer Res Clin Oncol 130:45–51. doi:10.1007/s00432-003-0506-y PubMedCrossRefGoogle Scholar
  9. 9.
    Xing H et al (2005) Effect of the cyclin-dependent kinases inhibitor p27 on resistance of ovarian cancer multicellular spheroids to anticancer chemotherapy. J Cancer Res Clin Oncol 131:511–519. doi:10.1007/s00432-005-0677-9 PubMedCrossRefGoogle Scholar
  10. 10.
    McDevitt MR et al (2000) An alpha-particle emitting antibody ([213Bi]J591) for radioimmunotherapy of prostate cancer. Cancer Res 60:6095–6100PubMedGoogle Scholar
  11. 11.
    Senekowitsch-Schmidtke R (1999) Binding of EGF peptide and EGF receptor antibodies and its fragments in different tumor models. Hybridoma 18:29–35PubMedCrossRefGoogle Scholar
  12. 12.
    Fullerton NE et al (2005) Application of targeted radiotherapy/gene therapy to bladder cancer cell lines. Eur Urol 47:250–256. doi:10.1016/j.eururo.2004.09.009 PubMedCrossRefGoogle Scholar
  13. 13.
    Boyd M et al (2002) Transfectant mosaic spheroids: a new model for evaluation of tumour cell killing in targeted radiotherapy and experimental gene therapy. J Gene Med 4:567–576. doi:10.1002/jgm.293 PubMedCrossRefGoogle Scholar
  14. 14.
    Sutherland R, Macfarlane W (1978) Cytotoxicity of radiosensitizers in multicell spheroids: combination treatment with hyperthermia. Br J Cancer Suppl 37:168–172Google Scholar
  15. 15.
    Dubessy C et al (2000) Spheroids in radiobiology and photodynamic therapy. Crit Rev Oncol Hematol 36:179–192. doi:10.1016/S1040-8428(00)00085-8 PubMedCrossRefGoogle Scholar
  16. 16.
    Freyer JP, Sutherland RM (1980) Selective dissociation and characterization of cells from different regions of multicell tumor spheroids. Cancer Res 40:3956–3965PubMedGoogle Scholar
  17. 17.
    Sutherland RM, Durand RE (1984) Growth and cellular characteristics of multicell spheroids. Recent Results Cancer Res 95:24–49PubMedGoogle Scholar
  18. 18.
    Knuchel R et al (1988) Interactions between bladder tumor cells as tumor spheroids from the cell line J82 and human endothelial cells in vitro. J Urol 139:640–645PubMedGoogle Scholar
  19. 19.
    Sminia P et al (2003) Oxygenation and response to irradiation of organotypic multicellular spheroids of human glioma. Anticancer Res 23:1461–1466PubMedGoogle Scholar
  20. 20.
    McLoughlin P et al (2004) Transcriptional responses to epigallocatechin-3 gallate in HT 29 colon carcinoma spheroids. Genes Cells 9:661–669. doi:10.1111/j.1356-9597.2004.00754.x PubMedCrossRefGoogle Scholar
  21. 21.
    Rofstad EK et al (1996) Apoptosis, energy metabolism, and fraction of radiobiologically hypoxic cells: a study of human melanoma multicellular spheroids. Int J Radiat Biol 70:241–249. doi:10.1080/095530096144978 PubMedCrossRefGoogle Scholar
  22. 22.
    Griffon-Etienne G, Merlin JL, Marchal C (1997) Evaluation of Taxol in head and neck squamous carcinoma multicellular tumor spheroids. Anticancer Drugs 8:48–55. doi:10.1097/00001813-199701000-00006 PubMedCrossRefGoogle Scholar
  23. 23.
    Dunkern TR, Mueller-Klieser W (1999) Quantification of apoptosis induction by doxorubicin in three types of human mammary carcinoma spheroids. Anticancer Res 19:3141–3146PubMedGoogle Scholar
  24. 24.
    Minard KR, Guo X, Wind RA (1998) Quantitative 1H MRI and MRS microscopy of individual V79 lung tumor spheroids. J Magn Reson 133:368–373. doi:10.1006/jmre.1998.1493 PubMedCrossRefGoogle Scholar
  25. 25.
    Cunningham S et al (2000) A gene therapy approach to enhance the targeted radiotherapy of neuroblastoma. Med Pediatr Oncol 35:708–711. doi :10.1002/1096-911X(20001201)35:6<708::AID-MPO49>3.0.CO;2-FPubMedCrossRefGoogle Scholar
  26. 26.
    Weber W, Weber J, Senekowitsch-Schmidtke R (1996) Therapeutic effect of m-[131I]- and m-[125I]iodobenzylguanidine on neuroblastoma multicellular tumor spheroids of different sizes. Cancer Res 56:5428–5434PubMedGoogle Scholar
  27. 27.
    Horan Hand P et al (1985) Influence of spatial configuration of carcinoma cell populations on the expression of a tumor-associated glycoprotein. Cancer Res 45:833–840PubMedGoogle Scholar
  28. 28.
    Desoize B, Jardillier J (2000) Multicellular resistance: a paradigm for clinical resistance? Crit Rev Oncol Hematol 36:193–207. doi:10.1016/S1040-8428(00)00086-X PubMedCrossRefGoogle Scholar
  29. 29.
    Mueller-Klieser W (2000) Tumor biology and experimental therapeutics. Crit Rev Oncol Hematol 36:123–139. doi:10.1016/S1040-8428(00)00082-2 PubMedCrossRefGoogle Scholar
  30. 30.
    Mueller-Klieser W (1997) Three-dimensional cell cultures: from molecular mechanisms to clinical applications. Am J Physiol 273:1109–1123Google Scholar
  31. 31.
    Folkman J, Moscona A (1978) Role of cell shape in growth control. Nature 273:345–349. doi:10.1038/273345a0 PubMedCrossRefGoogle Scholar
  32. 32.
    Svendsen CN et al (1998) A new method for the rapid and long term growth of human neural precursor cells. J Neurosci Methods 85:141–152. doi:10.1016/S0165-0270(98)00126-5 PubMedCrossRefGoogle Scholar
  33. 33.
    Sandoval JA, Hickey RJ, Malkas LH (2005) Isolation and characterization of a DNA synthesome from a neuroblastoma cell line. J Pediatr Surg 40:1070–1077. doi:10.1016/j.jpedsurg.2005.03.054 PubMedCrossRefGoogle Scholar
  34. 34.
    Sandoval JA et al (2006) Novel peptides secreted from human neuroblastoma: useful clinical tools? J Pediatr Surg 41:245–251. doi:10.1016/j.jpedsurg.2005.10.048 PubMedCrossRefGoogle Scholar
  35. 35.
    Warburg O (1930) The metabolism of tumours. Constable Press, LondonGoogle Scholar
  36. 36.
    Calderwood SK et al (2006) Heat shock proteins in cancer: chaperones of tumorigenesis. Trends Biochem Sci 31:164–172. doi:10.1016/j.tibs.2006.01.006 PubMedCrossRefGoogle Scholar
  37. 37.
    Snell K (1985) Enzymes of serine metabolism in normal and neoplastic rat tissues. Biochim Biophys Acta 843:276–281PubMedGoogle Scholar
  38. 38.
    Backman E et al (2007) Thioredoxin, produced by stromal cells retrieved from the lymph node microenvironment, rescues chronic lymphocytic leukemia cells from apoptosis in vitro. Haematologica 92:1495–1504. doi:10.3324/haematol.11448 PubMedCrossRefGoogle Scholar
  39. 39.
    Lu KP et al (2006) Targeting carcinogenesis: a role for the prolyl isomerase Pin1? Mol Carcinog 45:397–402. doi:10.1002/mc.20216 PubMedCrossRefGoogle Scholar
  40. 40.
    Sani BP et al (1980) Retinoic acid binding protein in experimental and human colon tumor. Cancer 190:60–61Google Scholar
  41. 41.
    Friedrich J, Ebner R, Kunz-Schughart LA (2007) Experimental anti-tumor therapy in 3-D: spheroids-old hat or new challenge? Int J Radiat Biol 83:849–871. doi:10.1080/09553000701727531 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Hari R. Kumar
    • 1
  • Xiaoling Zhong
    • 1
  • Derek J. Hoelz
    • 3
  • Frederick J. Rescorla
    • 1
    • 2
  • Robert J. Hickey
    • 3
  • Linda H. Malkas
    • 3
  • John A. Sandoval
    • 2
  1. 1.Section of Pediatric SurgeryIndiana University School of Medicine and Riley Children’s HospitalIndianapolisUSA
  2. 2.Department of SurgeryIndiana University School of Medicine and Riley Children’s HospitalIndianapolisUSA
  3. 3.Division of Hematology/OncologyIndiana University School of Medicine and Riley Children’s HospitalIndianapolisUSA

Personalised recommendations