Development of an innovative 3D cell culture system to study tumour--stroma interactions in non-small cell lung cancer cells

PLoS One. 2014 Mar 24;9(3):e92511. doi: 10.1371/journal.pone.0092511. eCollection 2014.

Abstract

Introduction: We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model.

Methods: Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC.

Results: Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype.

Conclusion: We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma, Non-Small-Cell Lung / pathology*
  • Cell Communication*
  • Cell Culture Techniques / methods*
  • Cell Line, Tumor
  • Cell Proliferation
  • Coculture Techniques
  • Drug Discovery
  • Fibroblasts / cytology
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Lung Neoplasms / pathology*
  • Stromal Cells / cytology

Grant support

This work was funded by means of a Ph.D grant of the Austrian Society of Haematology and Oncology (OeGHO) and further financially supported by the Verein für Tumorfoschung. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.