Three-dimensional culture suppresses pancreatic fibroblast activation and limits paracrine signaling between pancreatic cancer cells and fibroblasts

Background Pancreatic fibroblasts play a significant role in the development, progression, and treatment limitations of pancreatic ductal adenocarcinoma (PDAC), making them an essential area of research. Traditional 2D in vitro models fail to fully capture complexities of these stromal cells in PDAC such as interactions, morphology, and response to stimuli present in in vivo environments, making 3D culture systems a promising platform for further studies. Methods We compared the behavior of pancreatic fibroblasts in 2D and 3D culture systems, by analyzing the effects of different cultures on transcriptomics. Primary murine pancreatic fibroblasts were cultured in monolayers and 3D spheroids. An indirect co-culture system between fibroblasts and KC murine pancreatic cancer cells (KCs) served to investigate paracrine effects of cancer cells. RNA sequencing and RT-PCR were utilized for data analysis, supported by Oil Red O staining. Results Inactivation of fibroblasts in the 3D magnetic levitation model was noted by downregulation of activation markers Acta2, Fap, and Ctgf , and upregulation of a lipid-droplet stabilizer Plin2 . The HIF-1 signaling pathway was enriched in the 3D model compared to 2D. Oil Red O staining demonstrated upregulation of cytoplasmic lipid droplets, in 3D vs. 2D culture models. Activation marker downregulation and lipid accumulation seen in both monocultures and co-cultures in 3D indicated a cell-specific behavior rather than tumor cell influence. Conclusion The 3D magnetic levitation culture could serve as a model to study fibroblasts in an inactivated state, while co-culture with KC tumor cells could be helpful for cancer-fibroblast paracrine interaction studies.