title: Development of an innovative 3D cell culture system to study tumour--stroma interactions in non-small cell lung cancer cells.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Internal Medicine V, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Arno
authors.initials: A
authors.lastname: Amann
authors.affiliation: Department of Anatomy, Histology and Embryology, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Marit
authors.initials: M
authors.lastname: Zwierzina
authors.affiliation: Department of Internal Medicine V, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Gamerith
authors.affiliation: Department of Anatomy, Histology and Embryology, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Mario
authors.initials: M
authors.lastname: Bitsche
authors.affiliation: Department of Internal Medicine V, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Huber
authors.affiliation: Department of Anatomy, Histology and Embryology, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Georg F
authors.initials: GF
authors.lastname: Vogel
authors.affiliation: Department of Anatomy, Histology and Embryology, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Blumer
authors.affiliation: Department of Internal Medicine V, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Koeck
authors.affiliation: Department of Anatomy, Histology and Embryology, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Elisabeth J
authors.initials: EJ
authors.lastname: Pechriggl
authors.affiliation: InSphero AG, Schlieren, Canton of Zürich, Switzerland.
authors.forename: Jens M
authors.initials: JM
authors.lastname: Kelm
authors.affiliation: Department of Internal Medicine V, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hilbe
authors.affiliation: Department of Internal Medicine V, Medical University Innsbruck, Innsbruck, Tyrol, Austria.
authors.forename: Heinz
authors.initials: H
authors.lastname: Zwierzina
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000473:pathology*; D002450:Cell Communication; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018920:Coculture Techniques; D055808:Drug Discovery; D005347:Fibroblasts / Q000166:cytology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology*; D017154:Stromal Cells / Q000166:cytology