Chromatin accessibility differentiates cancer associated fibroblast subtypes in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) remains the deadliest cancer with only 13% of five-year survival rate in patients. However, effective drug delivery and administration of immunotherapy have been limited due to the dense fibrotic stroma in PDAC tumor microenvironment, majorly contributed by cancer-associated fibroblasts (CAFs). Unfortunately, targeting CAFs to enhance PDAC treatment has not yet been successful due to the lack of understanding in CAF activation, heterogeneity, and plasticity. Previous scRNA-seq studies revealed different CAF subtypes with diverse phenotypes and functions but the underlying mechanisms of CAF activation remain unclear. Of note, CAFs do not genetically differ from their origins, and they are interconvertible depending on external factors and signaling cues. This plasticity feature suggests that CAFs are characterized by their cellular states rather than an end-of-point differentiation. Therefore, we hypothesized that CAFs activation is regulated by epigenetic reprogramming. Using ATAC-seq and innovative PDAC organoid-CAF co-culture models, we revealed distinct chromatin accessibility profiles of two major CAF subtypes, myofibroblasts and inflammatory fibroblasts in vitro . Gained differentially accessible regions were identified across genomes, located not only at promoters but also at enhancers, and they were highly associated with transcriptional signatures of each subtype. In line with this, analysis on snATAC-seq of the fibroblast population within PDAC tumor tissues of both human patients and mouse models also confirmed the distinct chromatin accessibility profiles of different CAF subtypes. Interestingly, by overlaying with snRNA-seq, we discovered that chromatin structure of CAF was primed to be accessible towards late-stage myofibroblast subtype during tumorigenesis. Overall, our study demonstrates the epigenetic basis of CAF plasticity both in vitro and in vivo , suggesting a novel therapeutic strategy of epigenetically targeting CAF to remodel the stroma and improve treatment outcomes in PDAC patients.