Development of a reliable method for human triple-negative breast cancer organotypic culture: Improving imaging and genomic studies in 3D cultures.

Triple-negative breast cancer (TNBC) is highly aggressive and lacks targeted therapies, posing a major challenge in oncology. Traditional two-dimensional (2D) cell cultures fail to capture the tumor microenvironment's complexity, whereas three-dimensional (3D) cultures provide a more accurate model of tumor biology. We developed an advanced 3D culture system for TNBC cell lines BT-20 and MDA-MB-231, enhancing the hanging-drop method with Matrigel to restore essential extracellular matrix interactions. Confocal imaging showed MDA-MB-231 cells forming clusters typical of aggressive carcinoma, while BT-20 cells organized into duct-like structures. Molecular analysis of PI3K and β-catenin target genes revealed distinct expression patterns, with PI3K overexpressed and β-catenin downregulated in 3D cultures. Moreover, β-catenin distribution in the 3D cell culture closely resembles its pattern in tissue. These findings underscore the value of 3D models in understanding TNBC progression and in supporting the exploration of novel therapeutic strategies.