Abstract 1938: Immune regulatory molecule expression on circulating tumor cells and circulating cancer stem cells in breast cancer patients

Abstract Background: Circulating Epithelial Tumor Cells (CETCs/CTCs) are known to evade host immune surveillance by modulating the expression of immune regulatory molecules such as PD-L1 and CTLA-4 in cancer patients. Circulating Cancer Stem Cells (cCSCs) play a critical role in cancer recurrence and metastasis. Although the exact mechanisms underlying immune evasion by cCSCs remain unclear, recent studies suggest that the increased expression of specific immune checkpoint molecules on these cells may facilitate immune evasion and enhance their self-renewal capacity. In this study, we analyzed CETCs/CTCs and cCSCs for the expression of immune checkpoint molecules, particularly PD-L1 and CTLA-4, as non-invasive biomarkers of immune evasion and potential predictors of immunotherapy efficacy. Methods: The study included 80 patients with breast cancer at various stages of the disease. CETCs/CTCs and cCSCs determination was performed using maintrac and the sphere formation assay, respectively. Additional staining with anti-PD-L1 and anti-CTLA-4 antibodies was used to assess the expression of PD-L1 and CTLA-4 on both CETC/CTCs and breast tumorspheres. Results: CETCs/CTCs were detected in all patients, with counts ranging from 100 to 1150/ml of peripheral blood. The proportion of PD-L1-positive CETCs/CTCs ranged from 25% to 89%, with significantly higher PD-L1 expression observed in patients with triple-negative breast cancer (TNBC) compared to those with other biological subtypes. The proportion of CTLA-4-positive CETCs/CTCs ranged from 10% to 66%, which was significantly lower than PD-L1 expression levels. The expression of immune checkpoint molecules on CETCs/CTCs exhibited heterogeneity both within and between patients. The number of tumorspheres increased significantly with tumor progression and the aggressiveness of the primary tumor. All tumorspheres in all patients expressed both immunoregulatory molecules, PD-L1 and CTLA-4, with substantial heterogeneity in their expression levels. Conclusion: The findings indicate that evaluating PD-L1 and CTLA-4 expression on CETCs/CTCs and cCSCs from breast cancer patients is both feasible and informative. The quantity of tumorspheres cultured from peripheral blood serves as a direct marker of the primary tumor's aggressiveness and proliferative capacity. Tumorspheres with elevated PD-L1 and CTLA-4 expression may play a key role in immune regulation. Gaining deeper insights into the interplay between cCSCs and tumor immunology could pave the way for strategies to target this small, therapy-resistant subpopulation more effectively. Citation Format: Monika Pizon, Dorothea Schott, Erika Schill, Ulrich Pachmann, Katharina pachmann. Immune regulatory molecule expression on circulating tumor cells and circulating cancer stem cells in breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1938.