Abstract
Aquaporins are water transport proteins that regulate prometastatic 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. In addition, 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.NEW & NOTEWORTHY We identify a role for aquaporin 5 (AQP5) in multicellular spheroid invasion-a collective behavior distinct from two-dimensional (2D) migration and implicated in cancer progression. Elevated AQP5 expression increased spheroid cohesion alongside increased cell-cell adhesion proteins, contrasting with earlier observations that AQP5 disrupts cell-cell junctions in 2D. These findings reveal that AQP5 regulates invasion through context-dependent mechanisms in three-dimensional (3D) environments, highlighting the importance of studying AQP5 in models that capture the complexity of tumor architecture and collective cell dynamics.