Targeting cysteine cathepsins to boost gemcitabine in PDAC

Cysteine cathepsins are increasingly recognized as important regulators of lysosomal function and autophagy, two processes that help cancer cells survive under treatment-induced stress. In pancreatic ductal adenocarcinoma (PDAC), where gemcitabine is still widely used as first-line chemotherapy, growing evidence indicates that drug-induced autophagy contributes to the development of resistance. In this study, we asked whether autophagy triggered by gemcitabine depends on cysteine cathepsins and whether blocking these proteases can improve gemcitabine's antitumor activity. We focused on cathepsins L, B, and V, major lysosomal proteases with established roles in proteolysis and emerging functions in survival signalling. Using three PDAC cell lines with distinct genetic backgrounds and chemosensitivity (PANC1, Capan2, BxPC3), we treated cells with sublethal LC30 and LC60 concentrations of gemcitabine to trigger stress responses without inducing extensive cell death, thereby allowing us to dissect adaptive autophagy from direct cytotoxicity. Using western blot analysis and immunocytochemistry, we examined expression, processing, and subcellular localization of cathepsins and key autophagy markers, and we quantified autophagic flux and lysosomal function in the presence or absence of selective inhibitors of cathepsins V, L, and B. In parallel, cell viability and apoptosis assays were used to assess treatment responses, and 3D patient-derived organoids and NK cell co-culture systems were employed to validate our findings in more physiologically relevant models. Our results show that gemcitabine-induced autophagy is at least partly dependent on cysteine cathepsin activity, and that pharmacologic inhibition of these enzymes interferes with autophagic processing and makes PDAC cells more sensitive to gemcitabine. We confirmed these findings in patient-derived organoids, where the combination of gemcitabine and cathepsin inhibition reduced tumour cell viability more effectively than either treatment alone. In addition, we tested how this combination affects immune recognition and found that natural killer (NK) cell-mediated killing was increased in gemcitabine- and cathepsin inhibitor-treated tumour cells. Overall, our data identify cathepsins L, B, and V as key modulators of gemcitabine-induced autophagy and immune susceptibility in PDAC and support their further exploration as therapeutic targets to overcome chemoresistance and improve clinical responses.