The expression of CD47 on tumor cells can act as a "don't eat me signal" against phagocytosis by macrophages and dendritic cells. Moreover, PD-L1-expressing tumor cells inhibit the anti-tumor activity of cytotoxic T cells. Circulating tumor cells (CTCs) expressing these molecules could overcome elimination by the immune system. In the current study, we evaluated for the first time the co-expression of CD47 and PD-L1 on single CTCs from patients with metastatic breast cancer (mBC).


Triple immunofluorescence staining was performed on peripheral blood mononuclear cells (PBMC) cytospin preparations from 18 CTC-positive patients with mBC, using antibodies against cytokeratins (for CTC detection), CD47 and PD-L1. Blood samples were obtained before the initiation of first-line chemotherapy. A total of 1*106 PBMCs were analyzed per patientusing the Ariol microscopy system. The expression levels of CD47 and PD-L1 were characterized as high or low/-, after quantification by the Ariol system, using the MDA.MB.231 breast cancer cell line as positive control.


A total of 23 CTCs (median: 1, range: 1-4) were identified. CD47-expressing CTCs were detected in 94.4% of patients and represented 91.3% of total CTCs. However, high CD47 expression was confirmed in 38.9% and 43.5% of patients and CTCs, respectively. PD-L1 expression was evident in 27.8% of patients and in 21.7% of CTCs, whereas CTCs expressing high levels of PD-L1 were identified in 16.% of patients and represented 13% of total CTCs. Co-expression of CD47 and PD-L1 (CD47+/PD-L1+) was observed in 21.7% of CTCs, whereas 69.6% of CTCs expressed CD47 only (CD47+/PD-L1-). No CTCs expressing only PD-L1 (CD47-/PD-L1+) were detected and 2 of 23 cells were negative for both markers (CD47-/PD-L1- ). Regarding the differential expression levels of CD47 and PD-L1, the phenotype CD47low/-/PD-L1low/- was the most abundant both at the patient (61.1%) and the CTC level (52.2%). CD47high/PD-L1low/- CTCswere observed in 33.3% and 34.8% of patients and CTCs, respectively whereas only 1 CD47low/-/PD-L1high CTC was detected in one patient. Interestingly, CD47high/PD-L1high CTCs were identified in only 8.7% of CTCs.


CD47 expression is identified in the great majority of CTCs in mBC and could represent a potent signal to facilitate the escape from innate immune response. PD-L1 expression on CTCs is less commonly observed and could serve for the attenuation of an adaptive anti-tumor immune response. Interestingly, CD47 and PD-L1 are co-expressed in a subset of CTCs, whereas simultaneous high expression on single CTCs is even less common. The expression of these molecules is currently further investigated in a larger cohort of patients with mBC and in patients with early disease, in order to evaluate their differential distribution that potentially reflects the equilibrium and/or escape from the immune surveillance.

Citation Format: Mavroudis D, Papadaki MA, Tsoulfas PG, Troullinou K, Apostolopoulou CA, Papadaki C, Agelaki S. Co-expression of molecules associated with innate and adaptive immune response on single CTCs of patients with metastatic breast cancer (mBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-13.