3D Mini-Organotypic Model of PDAC

Human PDAC cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, USA). PS1 cell line (immortalized non-tumoural PSC) was developed and characterized previously in our laboratory^{39 (link)}. The mini-organotypic model^{37 (link)}, was used to examine the effect of genetic manipulation or ATRA treatment on PDAC cells (MiaPaCa2 and CaPan1) and PSC (PS1) co-cultured in 3D, physio-mimetic environment. Mini-organotypic cultures were incubated for 7 days before being harvested, fixed in 10% neutral buffered formalin (BAF‐0010‐03 A; CellPath Ltd), submerged in 70% ethanol, embedded in paraffin and cut into 4 µm sections. Treatment of organotypic cultures was performed daily with 1 µM ATRA (Cat No. R2625; Sigma) in 100% ethanol or vehicle control. For organotypic cultures using PTX3 siRNA, stellate cells were first transfected with PTX3 siRNA or non-targeting (NT) control, harvested after 24 h and then plated into organotypic gels, and incubated for 5 days. Cancer cell lines (MiaPaCa2 or AsPC1) and stellate cell line (PS1: with NT siRNA or PTX3 siRNA) were used. Medium below the inserts were replaced daily and collected (undernatant) at the end of the cultures for further analysis. Each experiment had three technical replicates with three biological repeats.