RNA-seq of CAFs grown on different substrates

Almost all solid tumors become stiff with progression of cancer. Cancer Associated Fibroblasts (CAFs), most abundant stromal cells in the tumor microenvironment (TME), are known to mediate the stiffening. While the biochemical crosstalk between CAFs and cancer cells have been widely investigated, it is yet not clear whether CAFs in stiffer TME promote metastatic progression, and if so, how. To address this question, here we explore the role of mechanical stiffness and cell traction force in regulating human colorectal CAF (CAF05 cell line) gene expressions that are relevant to metastatic progression. We cultured CAFs on 2D polyacrylamide hydrogels with increasing elastic modulus (E) of 1, 10 and 40 kPa, and quantified corresponding cell spreading area and cytoskeletal force. We performed genome-wide transcriptome analyses in these cells to identify differentially expressed genes on 40 KPa compared to those on 1 kPa substrate. The latter represents normal tissue stiffness of colon. CAFs were grown on polyacrylamide hydrogel (PA gel) substrates of 1 kPa, 10 kPa and 40 kPa elastic modulus. Fibronectin coating were applied on the substrates. Cell were also grown on plastic dish that has an elastic modulus of 3.4 GPa. After 48 hrs of culture, RNA was extracted using Trizol reagent and then processed for RNAseq.