3D Cultivation of Lung Cancer Cell Lines

Most of our knowledge regarding our cells' function was gained by two dimensional (2D) in vitro experiments. For this, cells were cultured and manipulated on plastic dishes which is a fast and cost-effective method. However, a lot of these results could not be directly transferred to human tissue. The cultivation of cells in a third dimension is now an important tool in cell biology and tissue engineering since it mimics the cell cluster and the microenvironment of natural tissue more accurately. There are already many data that were gained by three dimensional (3D) culturing of tumor cells in a laminin-rich extracellular matrix, especially for breast cancer cell lines.

In this study, we implemented the 3D cell culture for the five lung cancer cell lines H358, H1299, A427, H23 and Colo-699 in a collagen I or matrigel matrix. For each cell line, we did not find any organoid structures but observed a different ability to form either compact, round spheroids or loose cell aggregates when seeded into either extracellular matrix. Lung cancer cells that were grown in a matrigel matrix showed an upregulation of many proteins that are involved in the formation of adherens junctions (e.g. E-cadherin, α-catenin, β-catenin) or focal adhesions (e.g. Paxillin, Vinculin). Interestingly, for cells that were cultivated in a collagen I matrix, most of these proteins were completely undetectable. One protein that showed a strong upregulation in this experimental design is the transcription factor Sox9. This protein is overexpressed in lung adenocarcinomas and can be activated by TGF-β, Notch and Hedgehog signaling. Preliminary results showed that TGF-β signaling seems not to be responsible for the enhanced expression of Sox9.

The cultivation of floating spheroids in a matrix-free environment also revealed an upregulation of cell contact proteins like E-cadherin, α-catenin, paxillin and vimentin in H358 cells. These first results are comparable to the matrigel-cultivated spheroids pointing towards a 3D cultivation effect rather than a matrix effect. Thus, this method has the advantage of studying the cell cluster alone without the influence of the surrounding microenvironment.

In summary, we established the 3D cell culture for several lung cancer cell lines in two different matrices or in a matrix-free environment in order to analyze the 3D cell cluster with and without environmental effects. Depending on the matrix, the cells showed major differences in protein expression compared to the two dimensional cell culture, but also the environment itself led to a different expression profile that needs further investigation.