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Aquaporin 5 expression regulates MDA-MB-231 spheroid multicellular invasion | American Journal of Physiology-Cell Physiology | American Physiological Society Aquaporins are water transport proteins that regulate pro-metastatic behaviors in cancer, including cell invasion, proliferation, and epithelial-to-mesenchymal transition. Aquaporin 5, an isoform virtually absent from healthy tissues, is overexpressed in numerous cancer types. Studies have connected Aquaporin 5 to the migratory and invasive properties of single cells in two-dimensional assays; however, the role of Aquaporin 5 in inducing invasive phenotypes in models that recapitulate the tumor microenvironment remains unknown. To address this gap, MDA-MB-231 cell lines were created with Aquaporin 5 overexpression and knockdown to identify the resulting single and collective cell motility in three-dimensional models. Upon validating the developed cell lines, Aquaporin 5 expression regulated cell motility and invasion in varied biaxial microenvironments. Additionally, Aquaporin 5 was found to play a unique role in regulating MDA-MB-231 spheroid development, influencing the formation, size, circularity, and adhesion, distinct from its function in two-dimensional models. Finally, increased Aquaporin 5 expression intensified the invasive capacity of spheroid multicellular protrusions by polarizing to their invasive front. In summary, this work expands upon the knowledge that Aquaporin 5 enhances cell motility, while elucidating the previously unreported adhesive and multicellular invasive effects of Aquaporin 5 in a three-dimensional model. https://doi.org/10.1152/ajpcell.00408.2025