Unveiling the Interplay between Mitochondrial Dysfunction and Mito EVs Secretion in Pancreatic Ductal Adenocarcinoma (PDAC)

The extracellular trafficking of mitochondrial com ponents has been increasingly linked to tumor survival and chemoresistance across various human malignancies (1,2). Furthermore, recent evidence highlights a notable enrichment of circulating extracellular vesicles (EVs) carrying specific mitochondrial DNA (mtDNA) muta tions in the serum of patients with pancreatic ductal adenocarcinoma (PDAC) (3). Building on our prior find ings that demonstrated the exocytosis of fragmented mitochondria by an aggressive PDAC histotype (4), this study aims to correlate the metabolic reprogramming of PDAC cells, specifically involving mitochondrial impair ment, with the EV-mediated intercellular shuttle of mito chondrial cargoes. The study was initiated using human pancreatic cancer cell lines and subsequently translated into 3D patient-derived organoids (PDOs). Interestingly, distinct mitochondrial alterations in terms of function and morphology were confirmed in human pancreatic cancer cell lines (PANC-1, BXPC-3) compared to healthy controls (HPDE), which correlated with an increased release of mitochondrial cargoes into the supernatant. In the 3D models, initial PDO stratification using Gene Set Enrichment Analysis (GSEA), combined with subsequent functional assays, revealed that advanced tumors with suppressed mitophagy adapt to mitochondrial dysfunc tion by actively releasing defective mitochondrial car goes, specifically marked by TOMM20 and NDUFA4L2. Furthermore, preliminary treatment with the inhibitor MRTX 1133 suggests that the KRAS G12D mutation plays a key regulatory role in maintaining mitochondri al homeostasis through mitophagy. In conclusion, this study highlights the potential of mitochondria-derived vesicle markers for PDAC diagnosis, identifying NDU FA4L2 as a highly specific candidate due to its link to active tumor reprogramming. Future research should explore how NDUFA4L2-enriched vesicles alter sur rounding stromal cells and assess whether this mecha nism correlates with tumor aggressiveness to enhance patient stratification and personalized therapy.