Deciphering Stroma-Cancer Cell Circuits Regulated by Transcription Factor EB (TFEB) in Pancreatic Ductal Adenocarcinoma (PDAC)

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent and aggressive form of pancreatic cancer, characterized by late detection and resistance to treatment, leading to high mortality rates. The dense fibrotic stroma characteristic of PDAC, mainly produced by activated pancreatic stellate cells (PSCs), hinders drug delivery and promotes tumor progression. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy. Recent studies have demonstrated that high levels of TEFB expression are involved in fibroblasts activation through TGF-β pathway. Based on these studies, we hypothesized that overexpression of TFEB in PSCs would reduce their activation and tumor stroma formation, improving chemotherapy delivery. In 2D cultures, qPCR analysis of PSCs co-culture with HPAF II cells revealed an increase in fibronectin and α-SMA expression, indicating stromal activation, while PSCs co-culture with BxPC-3 cells induced a more inflammatory profile. TFEB overexpression decreased the expression of stromal and inflammatory markers. These results were confirmed by immunofluorescence analysis. Bulk RNA sequencing analysis also demonstrated that TFEB overexpression reduced the fibrotic response produced by CAFs and negatively regulated TGF-β signaling. In 3D cultures, immunofluorescence analysis of spheroids produced from mixed PDAC cells and PSCs cells, revealed that TFEB overexpression in PSCs reduced fibroblast cluster formation and fibronectin expression, suggesting a decrease in fibroblast activation. These findings suggest TFEB as a potential therapeutic target to reduce fibrosis and inflammation in PDAC, enhancing treatment efficacy and reducing tumor aggressiveness.