Abstract Background: Breast cancer remains a leading cause of cancer-related morbidity and mortality worldwide. Emerging evidence suggests that a subpopulation of circulating tumor cells—circulating cancer stem cells (cCSCs)—plays a pivotal role in metastasis, therapy resistance, and disease relapse. However, functional assessment of these cells in a minimally invasive manner poses a significant clinical challenge. Tumorsphere assays offer a unique in vitro platform to enrich and evaluate the self-renewal capacity of cCSCs, potentially reflecting the biological aggressiveness of the tumor. Establishing a reliable method for cultivating tumorspheres from peripheral blood may provide a novel biomarker for monitoring tumor progression and facilitating patient stratification. Materials and Methods: A total of 98 breast cancer patients, representing stages I to IV, were enrolled in this study. Peripheral blood samples were collected from each patient to detect cCSCs using the stemtrac® method, followed by a standardized tumorsphere-forming assay. Results: Tumorsphere count showed a significant correlation with disease stage and metastatic status. Patients with stage IV disease exhibited a higher median tumorsphere count compared to those with stage I (30 vs. 10; p 0.002). Metastatic patients had more tumorspheres than non-metastatic patients (30 vs. 15; p 0.005), with the highest counts observed in cases with multiple metastases (median 61; p 0.001). Additionally, higher tumorsphere counts were associated with features of tumor aggressiveness, including HER2/neu positivity (35 vs. 10; p 0.05), increased Ki-67 index, and higher histological grade Conclusion: These findings underscore the clinical relevance of tumorsphere assays as a minimally invasive method for monitoring tumor biology. The number of tumorspheres derived from peripheral blood may serve as a surrogate biomarker for disease progression, metastatic potential, and tumor aggressiveness, offering promising implications for real-time patient stratification and personalized treatment planning in breast cancer. Citation Format: M. PizonD. SchottK. Pachmann. Tumorsphere Formation as a Liquid Biopsy Biomarker of Tumor Aggressiveness and Metastatic Burden in Breast abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-03-01.
Pizon et al. (2026) studied this question.