Epithelial-to-mesenchymal Transition Heterogeneity of Circulating Tumor Cells and Their Correlation With MDSCs and Tregs in HER2-negative Metastatic Breast Cancer Patients.

BACKGROUND:To investigate the correlation between circulating tumor cells (CTCs) bearing cancer stem cell (CSC) and epithelial-to-mesenchymal (EMT) phenotypes and the different immunosuppressive cells in peripheral blood of patients with metastatic breast cancer (mBC). MATERIALS AND METHODS:Blood was obtained from 38 pre-treated patients with mBC before a new line of treatment. CTC detection and characterization was performed by triple immunofluorescent staining, while Myeloid-derived Suppressor Cells (MDSCs) and T regulatory cells (Tregs) were analyzed by multi-flow cytometry. RESULTS:CTCs were detected in 16 (42.1%) of patients. Based on the co-expression of ALDH1, TWIST and CK, CTCs revealed an important heterogeneity: CTCs with a CSC/partial-EMT, CSC/Epithelial-like, non-CSC/partial-EMT and non-CSC/Epithelial-like phenotype were detected in 7 (18.4%), 7 (18.4%), 1 (1.4%) and 9 (23.7%) of patients, respectively. Immunophenotyping of MDSCs identified 2 monocytic [M-MDSCs; CD14+CD15+CD11b+CD33+HLA-DR-Lin- (CD14+CD15+) and CD14+CD15-CD11b+CD33+ HLA-DR-Lin- (CD14+CD15-)] and one granulocytic [G-MDSCs; CD14-CD15+CD11b+CD33+HLA-DR-Lin- (CD14- CD15+)] subpopulations, expressing inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS), respectively. Patients with detectable CTCs had a higher frequency of Tregs (CD3+CD4+CD25high; p=0.022) whereas a positive correlation was found between CTC counts and the percentage of Tregs (p=0.005) and CD14+CD15+ M-MDSCs (p=0.024). Patients with a partial-EMT phenotype had a higher frequency of CD14+CD15+ M-MDSCs (p=0.023). Patients harboring the non-CSC/epithelial-like CTC subpopulation had an increased frequency of CD14-CD15+ G-MDSCs (p=0.020), along with decreased levels of CD3+CD4+CD25high FoxP3+ Tregs (p=0.020). CONCLUSION:These findings provide evidence that CTCs in ER+/HER2- mBC patients may be under the control of the immune system and various immune escape mechanisms might be involved during the different stages of their biological evolution.