A simple and cost-effective method for generating spheroids from triple-negative breast cancer cell line (MDA-MB-231)

Abstract Breast cancer (BC) is the most frequently diagnosed malignancy in women and a leading cause of cancer-related mortality worldwide. Molecular classification based on estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 expression guides prognosis and therapy, with triple-negative breast cancer (TNBC)—lacking ER, PR, and HER2 representing 15–20% of cases. TNBC’s aggressive behavior, early recurrence, and limited treatment options underscore the need for improved models to develop targeted therapies. While monolayer (2D) cultures have advanced cancer research, they poorly replicate the three-dimensional (3D) tumor microenvironment (TME), leading to translational gaps. 3D spheroids address these limitations by recapitulating cell-cell/matrix interactions, metabolic gradients, and hypoxic cores, offering a physiologically relevant platform for studying metastasis, drug resistance, and therapeutic screening. Here, we present a simple, cost-effective method for generating spheroids. This protocol is applicable across different cell types, bridging the gap between traditional 2D cultures and in vivo studies. 3D cell culture opens the door to personalized medicine and drug discovery. Key features Employs a cost-effective, lab-made agarose coating to create ultra-low attachment surfaces in standard 96-well plates. Specifically optimized for generating consistent spheroids from the aggressive MDA-MB-231 triple-negative breast cancer cell line. Generates measurable spheroids within 96 hours using only basic cell culture equipment and an orbital shaker. Provides a clear workflow from spheroid formation to quantitative size analysis using freely available (ImageJ) and common (GraphPad Prism) software. Competing Interest Statement The authors have declared no competing interest.