Differential Effects\nof Confinement on the Dynamics\nof Normal and Tumor-Derived Pancreatic Ductal Organoids

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of\nthe epithelia\ncomprising the ductal network of the pancreas. During disease progression,\nPDAC tumors recruit fibroblasts that promote fibrosis, increasing\nlocal tissue stiffness and subjecting epithelial cells to increased\ncompressive forces. Previous in vitro studies have documented cytoskeletal\nand nuclear adaptation following compressive stresses in two-dimensional\n(2D) and three-dimensional (3D) environments. However, a comparison\nof the responses of normal and tumor-derived ductal epithelia to physiologically\nrelevant confinement remains underexplored, especially in 3D organoids.\nHere we control confinement with an engineered 3D microenvironment\ncomposed of Matrigel mixed with a low yield stress granular microgel.\nNormal and tumor-derived murine pancreas organoids (normal and tumor)\nwere cultured for 48 h within this composite 3D environment or in\npure Matrigel to investigate the effects of confinement on morphogenesis\nand lumen expansion. In confinement, tumor organoids (mT) formed a\nlumen that expanded rapidly, whereas normal organoids (mN) expanded\nmore slowly. Moreover, a majority of normal organoids in more-confined\nconditions exhibited an inverted apicobasal polarity compared to those\nin less-confined conditions. Tumor organoids exhibited a collective\n“pulsing” behavior that increased in confinement. These\npulses generated forces sufficient to locally overcome the yield stress\nof the microgels in the direction of organoid expansion. Normal organoids\nmore commonly exhibit unidirectional rotation. Our in vitro microgel\nconfinement platform enabled the discovery of two distinct modes of\ncollective force generation in organoids that may shed light on the\nmutual interactions between tumors and the microenvironment. These\ninsights into in vitro dynamics may deepen our understanding of how\nthe confinement of healthy cells within a fibrotic tumor niche disrupts\ntissue organization and function in vivo.