Collagen nanofiber alignment attenuates leader-follower energetic and metabolic differences during collective...

The extracellular matrix (ECM) is a dynamic microenvironment that influences cell behavior and fate, with changes in its architecture linked to processes such as differentiation and disease progression in cancer. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer associated with a dense ECM composed of collagen I fibers. While considerable research on PDAC metastasis has focused on single-cell migration, recent studies suggest PDAC cells undergo a process called "collective migration" as groups. This study investigates PDAC migration and metabolism on electrospun collagen I nanofiber meshes. A 3D-printed, removable insert is used to create a gap-like a scratch-that allows for analysis of cell migration on intact fibers. By tuning nanofiber orientation, this study replicates the PDAC ECM during stromal remodeling to assess cellular metabolism and the energetic state of leader and follower cells during migration. Interestingly, the data suggests that nanofiber architecture strongly modulates differences between PDAC leader and follower cells during collective migration. While leader cells require more ATP and rely more on oxidative phosphorylation in both conditions, this reliance is particularly pronounced in the random condition. These findings underscore the interplay between collective migration, ECM architecture, and metabolism in PDAC migration. STATEMENT OF SIGNIFICANCE: The role of the stromal extracellular matrix (ECM) in metastasis and migration is well studied, yet its impact on metabolic processes underlying metastasis, especially in pancreatic ductal adenocarcinoma (PDAC), remains unclear. PDAC is marked by high metastasis rates and a densely fibrotic ECM, with increased collagen alignment correlating to poor prognosis. In this study, we explore how ECM architecture, represented by near-native electrospun collagen nanofibers, influences collective migration and metabolism in PDAC. Our findings show that increased collagen alignment reduces metabolic differences, including ATP/ADP ratio, gene expression, and mitochondrial membrane potential, between leader and follower cells during migration. This is the first study to investigate leader-follower dynamics in PDAC collective migration using tunable, stromal-mimicking, fibrous substrates.