In situ tailored confining microenvironment for lung cancer spheroids.

The mechanical properties and physical confinement of the extracellular matrix (ECM) are crucial roles in regulating tumor growth and progression. Extensive efforts have been dedicated to replicating the physical characteristics of tumor tissue by developing two-dimensional (2D) and three-dimensional (3D) in vitro models. However, it remains a significant challenge to modulate the local microenvironment around the specific cells according to the culture progress. In this study, we develop a 3D culture platform for multicellular lung cancer spheroids using a gelatin-based hydrogel with adjustable density and stiffness. Then, by utilizing a two-photon mediated bioprinting technique, we construct 3D confining microstructures with micrometer accuracy to enclose the selected spheroids within the hydrogel matrix. Diverse transcriptional profilings of cells are observed in response to the increased ECM density and stiffness compared to the additional confining stress. In addition, changed confining stress can regulate the tumor cells with contrary impacts on the cell cycle-related pathways. Our model not only allows for modifications to the mechanical microenvironment of the overall matrix but also facilitates localized adjustments throughout the culture evolution. This approach serves as a valuable tool for investigating tumor progression and understanding cell-ECM interactions.