Imaging and profiling lung fibroblasts in 3D cell culture models

Introduction: In conventional 2D monolayer cell culture systems cell-matrix adhesions are well known and studied protein aggregations with functions in cell-adhesion, mechanotransduction, cell-signaling, invasion and migration. However, to date little is known about focal adhesion formation in mesenchymal cells growing in 3D cell culture environments and in vivo, where cells are completely embedded in an extracellular matrix. 3D cell culture models more closely resemble the physiological in vivo condition. Currently, it is a hot debate and ongoing controversy whether cells in such a 3D matrix would form adhesions at all. Attempts to visualize subcellular structures (like focal adhesions) in 3D cell culture models bear several microscopic limitations such as low working distances, lack of high resolution, high fluorescent background and weak antibody stainings. Therefore, we are interested in establishing 3D cell culture models and improving microscopic techniques for studying subcellular structures such as focal adhesions and cytoskeletal proteins in lung fibroblasts and their impacts on 3D migration and invasion. New findings will help to understand the underlying mechanisms of chronic lung diseases like fibrosis or pulmonary metastases.

Methods: We developed and characterized several 3D collagen based cell culture models which are apt for imaging of live and fixed lung fibroblasts (primary and CCL206 cell-line) with low and high-magnification objectives. These models were extensively used for live imaging on our high-speed live cell imaging system as well as on a confocal scanning system (LSM710). Additionally, we employed qRT-PCR for transcriptional profiling of focal adhesion components comparing lung fibroblasts (primary and CCL206 cell-line) cultured upon conventional 2D plastic surfaces with those embedded in 3D collagen matrices.

Results: With our established 3D cell culture collagen models we investigated single cell migration as well as dynamics of subcellular structures. By using CCL206 lung fibroblasts cultured in 3D collagen we demonstrated an altered morphology and mode of migration when compared to their 2D cultured counterparts. Furthermore, we observed a highly significant transcriptional deregulation of various genes involved in migration in 3D collagen embedded CCL206 fibroblasts.

Outlook: In order to get a deeper insight into the molecular mechanisms and signaling pathways involved in migration and invasion in 3D, we plan to investigate differenzial expression of the lung fibroblast transcriptome by microarray analysis and substantiate our findings on protein levels.