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

Introduction: We describe a novel 3D co-culture method using non-small cell lung cancer (NSCLC) cell lines in combination with a lung fibroblast cell line in the hanging drop technology. Compared to other 3D models, it better reflects tumour growth in its microenvironment since no addition of growth factors or artificial extracellular matrix (ECM) substitutes are required.

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 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. Furthermore, an increase of vimentin expression 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 hanging drops technology 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.