Mammosphere Assay Reveals Api5-Induced Stemness in Non-Tumorigenic Breast Epithelial Cell Lines

Breast cancer remains the most prevalent cancer among women globally, with the highest age-standardised incidence and mortality rates. Despite the advancements in therapies targeting breast cancer, 20% of the patients exhibit relapse of the disease, which may be attributed to a population of therapy-resistant cancer stem cells (CSCs). These CSCs can survive conventional treatments by remaining in a quiescent stage or persist due to incomplete resection of the tumor mass, resulting in recurrence at a later stage. Therefore, recent research has focused on identifying, isolating and targeting this resistant population of cells. In vitro, the mammosphere formation assay serves as a robust three-dimensional suspension culture system for enriching and propagating stem-like cells within breast epithelial populations. Here, we present an approach utilising the mammosphere assay to evaluate the cancer stem cell-like phenotype that is enabled in non-tumorigenic breast epithelial cells by the overexpression of Apoptosis Inhibitor 5 (Api5). The protocol involves propagating mammospheres through serial passages to assess mammosphere formation efficiency and self-renewal capacity. Additionally, protein lysates collected from mammospheres are probed for established stemness markers via immunoblotting. This assay enables the investigation of novel regulators, such as Api5, of cancer stem cell-like properties in breast epithelial cell populations. The approach described can be adapted for comparative studies of molecular interventions or for screening therapeutic agents targeting CSCs in vitro.