Abstract Background: Cancer stem cells (CSCs) are the critical subpopulation of cancer cells involved in tumor formation, progression, metastasis, and drug resistance. CSCs also have been considered the main cause of cancer recurrence, and the identification of targets and biomarkers is still under development. In this study, primary cultures from peripheral blood were established, using sphere-forming assays for CSC enrichment, and correlated with clinicopathologic features. Methods: Circulating cancer stem cells were cultured under conditions favoring growth of tumorspheres from 72 patients with breast cancer, including a subpopulation of 23 patients with metastatic disease. Cell viability, stem cell marker expression, and ALDH 1 activity were evaluated by fluorescence scanning microscope (Olympus Scan®R). Results: Sphere formation was observed in 79% of patients with breast cancer. In the current study we found that the number of tumorspheres depended on stage of disease. Patients with metastatic disease had statistically significantly more tumorspheres compared to patients without metastasis (median 30 vs. 10; p=0.002). Furthermore, patients with multiple metastases had more tumorspheres as compared to patients with single metastasis (median 30 vs. 60; p<0.05). Analysis of surface marker expression profile of tumorspheres cultured from peripheral blood showed that spheres had typical phenotype of cancer stem cells with a high enzymatic activity for ALDH 1. There was no sphere formation in a control group with 20 healthy donors. Conclusion: Identifying the circulating cancer stem cell may provide more clinically useful prognostic information and can be the basis for developing novel therapies. Citation Format: Monika Pizon, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann. The number of tumorspheres cultured from peripheral blood is directly related to presence of metastasis in breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B34.