Molecular Subtype-Specific Breast Cancer Organoids: Development of an FGF-Free Estradiol Valerate Culture System for Precision Drug Screening

Breast cancer, a prevalent malignancy among women, exhibits high heterogeneity, highlighting the critical need for personalized treatment strategies. Breast cancer organoids (BCOs), an emerging in vitro research tool, closely mimic the biological characteristics of tumors and demonstrate distinct differences in therapeutic response, prognosis, and biological features. However, the incomplete culture system of BCOs is still a challenging aspect of current research. In order to further optimize the existing culture formula of BCOs, we collected tumor tissue samples from breast cancer patients with various molecular subtypes, including Luminal A, Luminal B, HER2 overexpressing (HER2 ⁺ ), and triple-negative breast cancer (TNBC). The samples were extracted and cultured using commercially available media. We observed that some media failed to sustain the growth of breast cancer organoids, while others, although supporting organoid survival, led to the loss of parental molecular subtype characteristics in most cases. Through literature review and screening of key factors, we developed an FGF-free estradiol valerate (EV) culture system. Using triple-positive breast cancer samples, we optimized the concentration of EV. Our results indicated that 0, 10, and 20 µM EV maintained normal organoid growth, whereas 50 µM EV promoted adherent differentiation, which is detrimental to long-term organoid culture. Further analysis revealed that 20 µM EV not only preserved the organoid cellular morphology but also retained the parental molecular subtype features. Using this optimized culture system, we successfully cultivated breast cancer organoids from TNBC, Luminal, and HER2 ⁺ samples. We then developed a drug sensitivity prediction model using our established organoid culture system, which included triple-negative, Luminal, and HER2-positive breast cancer subtypes. By comparing imaging data before and after clinical treatment, we found a high concordance between drug sensitivity in the organoid model and clinical efficacy. This study provides an optimized culture medium system for the cultivation of breast cancer organoids and validates its potential application in precision drug screening. This model promises to deliver precise and effective treatment strategies for breast cancer patients, further improving their prognosis and quality of life, and ushering in a new chapter in personalized breast cancer treatment.