Placental mesenchymal stem cells: A promising platform for advancing gene therapy in pancreatic ductal adenocarcinoma.

The extreme lethality and limited treatment options for pancreatic ductal adenocarcinoma (PDAC) underscore the urgent need for innovative therapeutic strategies. This study presents the first preclinical investigation of a cell-free gene-directed enzyme prodrug therapy (GDEPT) based on conditioned medium (CM) from placenta-derived mesenchymal stem cells (PlacMSCs) engineered to express the yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT) fusion enzyme. The CM was concentrated tenfold (cCM) and characterized by proteomics, transmission electron microscopy, and Western blotting, confirming extracellular vesicle (EV) enrichment and UPRT expression. Therapeutic efficacy was evaluated in coculture models comprising PDAC cell lines (BxPC-3, MIA PaCa-2, SU.86.86), patient-derived xenograft organoids (PDXOs), and cancer-associated fibroblasts (PCAFs). Immunocytochemistry and Western blot analyses revealed a predominant myofibroblastic CAF phenotype, characterized by strong alpha-smooth muscle actin (αSMA) expression and low interleukin-6 levels. Treatment with yCD::UPRT-PlacMSC-cCM in the presence of 5-fluorocytosine (5-FC) enabled efficient enzymatic conversion to 5-fluorouracil (5-FU), yielding 10 μg/mL from an initial 100 μg/mL of 5-FC. This resulted in robust, dose-dependent cytotoxicity (50 % to 80 % reduction in viability) across monocultures and stromal-rich cocultures, effectively overcoming PCAF-mediated drug resistance. Therapeutic response was governed primarily by tumor cell characteristics rather than PCAF heterogeneity. In PDXOs derived from two early-stage (IA, IIB) primary tumors and one metastatic lesion, 100 µL of yCD::UPRT-PlacMSC-cCM induced cytotoxicity comparable to 1 μg/mL of 5-FU, while 25 µL was insufficient to significantly reduce viability. Collectively, these findings demonstrate that yCD::UPRT-PlacMSC-cCM delivers potent, stromal-bypassing cytotoxicity in PDAC models and represents a promising cell-free therapeutic approach for this treatment-refractory cancer.