Three-Dimensional Cell Culture Conditions Affect the Proteome of Cancer-Associated Fibroblasts.

In vitro cell culture systems are an invaluable tool for cell biological research to study molecular pathways and to characterize processes critical in human pathophysiology. However, the experimental conditions in two-dimensional (2D) cell cultures often differ substantially from the in vivo situation, which continuously raises concerns about the reliability and conferrability of the obtained results. Three-dimensional (3D) cell cultures have been shown to closer mimic in vivo conditions and are commonly employed, for example, in pharmacological screens. Here, we introduce a 3D cell culture system based on a mixture of collagen I and matrigel amenable to stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry-based proteomics analyses. We study the extra- and intracellular proteomic response of skin fibroblast isolated from healthy volunteers in comparison to cancer-associated fibroblasts (CAF) on 3D culture conditions. Both, control cells and CAF, change their proteomic composition based on the culture conditions. Critically, cell type differences observed in 2D are often not preserved in 3D, which commonly closer resemble phenotypes observed in vivo. Especially, extracellular matrix and plasma membrane proteins are differentially regulated in 2D versus 3D.