The role of CD47 and PD-L1 expression on circulating tumor cells (CTCs) remains unclear, and it is currently unknown whether their distribution varies between the blood and tumor tissue in breast cancer (BC). In this study, CD47 and PD-L1 expression was investigated a) on peripheral blood mononuclear cell (PBMC) cytospins from early (n = 100) and metastatic (n = 98) BC patients, by triple immunofluorescence for CD47/PD-L1/Cytokeratins, and b) on matched primary and/or metastatic tumor tissue from CTC-positive patients using immunohistochemistry. CD47+and/orPD-L1+ CTCs were detected in 11%, 16.9%, and 29.6% of early, recurrent, and de novo metastatic patients (p = 0.016). In metastatic disease, CD47highand/orPD-L1high CTCs were associated with disease progression (p = 0.005) and shorter progression-free survival (PFS) (p = 0.010), and independently predicted for an increased risk of relapse (HR: 2.719; p = 0.008) and death (HR: 2.398; p = 0.034). PD-L1 expression rates differed between CTCs and tissue tumor cells and between peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) (positive concordance of 3.8% and 4%, respectively). CD47 expression also differed between CTCs and tumor cells (positive concordance of 11.5%). In conclusion, CTCs expressing CD47 and PD-L1 have independent poor prognostic implications in metastatic BC, indicating a potential role of innate and adaptive immune evasion mechanisms in their metastatic potential. The clinical value of the parallel assessment of the peripheral and local immune response merits further evaluation in BC.

We herein aimed to assess the effect of eribulin mesylate on the cancer stem cell (CSC)/EMT-like phenotype of CTCs, and to investigate the prognostic role of CTC detection and monitoring for eribulin-treated BC patients. Peripheral blood was obtained at baseline (n = 42 patients) and 8 days after treatment initiation (C1D8: n = 22), and on disease progression (PD: n = 26). PBMCs cytospins were immunofluorescently stained for Cytokeratins/ALDH1/TWIST1/DAPI and analyzed via Ariol microscopy. CTCs were detected in 33.3%, 27.3%, and 23.1% of patients at baseline, C1D8, and PD, respectively. Accordingly, partial-EMT+ CTCs represented 61.3%, 0%, and 37.5% of total CTCs, whereas the CSC-like phenotype was consistently expressed by 87.5%, 75%, and 91.7% of CTCs at the respective time points. Interestingly, the CSC+/partial-EMT+ subset prevailed at baseline, but it was eradicated on C1D8 and resurged again during PD. CTC detection at baseline was associated with reduced PFS (p = 0.007) and OS (p = 0.005), and was an independent risk factor for death (HR: 3.779, p = 0.001; multivariate analysis). The CSC+/partial-EMT+ CTCs emerged as the only subset with adverse prognostic significance, while CTC monitoring during eribulin therapy improved the prediction of disease progression. These results indicate that resistant CTC subsets persevere eribulin treatment and highlight the prognostic implications of CTC analyses for eribulin-treated BC patients.