Effect of Ageratum fastigiatum on Viability, Migration and Proliferation of Breast Cancer Cells in 2D and 3D Culture Models

Triple-negative breast cancer (TNBC) is an aggressive subtype with limited therapeutic options, underscoring the need for novel antitumor agents and more physiologically relevant in vitro models. Although species of the Ageratum genus exhibit biological activity, the antitumor potential of Ageratum fastigiatum and its evaluation in three-dimensional (3D) microenvironment have not been previously investigated. This study evaluated the cytotoxic, antiproliferative, and antimigratory effects of the ethanolic extract of Ageratum. fastigiatum on TNBC cells MDA-MB-231 using conventional two-dimensional (2D) cultures and a 3D tumor-on-a-chip model. In 2D cultures, cell viability assessed by the MTT assay revealed an IC50 of 308 µg/mL, and approximately 80% reduction in viability at a concentration of 500 µg/mL. The extract significantly inhibited cell migration in scratch assays and reduced colony formation in clonogenic assays. Cell cycle analysis showed an increased sub-G0/G1 population at the IC50 concentration, indicating apoptosis-associated cell cycle disruption. In the 3D microfluidic model, tumor cells embedded in a collagen hydrogel exhibited reduced sensitivity to the extract compared to 2D cultures, with viable cell areas of approximately 15%, compared to 20% under control group , highlighting the influence of the extracellular matrix on treatment response. Additionally, the extract inhibited cell migration by approximately 59% in the 3D model at the IC25 concentration (289 µg/mL). In conclusion, the ethanolic extract of A. fastigiatum demonstrated antitumor activity against TNBC cells, and the incorporation of 3D microfluidic models highlights the importance of tumor microenvironment based platforms for a more physiologically relevant evaluation of therapeutic candidates.